Phil Mitchells lectures. Flashcards
1
Q
What is the definition of transcription?
A
‘Recognition of DNA sequences by DNABP which allows targeting of RNAP to specific genes.’
2
Q
What is an example of a ‘high rate’ gene?
A
Pol I transcriptional unit.
3
Q
How can you visualise transcription?
A
Miller spreads.
4
Q
What can you see on Miller Spreads?
A
The density of polymerases.
5
Q
On a miller spread where are the RNA molecules that have been expressed more?
A
On the right.
6
Q
If genes have the same copy number they are expressed at the same level. True or false?
A
False.
7
Q
What is the definition of a transcription factor?
A
Proteins that recognise certain DNA sequences.
8
Q
Is the promoter region large in Euks and Proks?
A
Euks.
9
Q
In what two ways can TF regulate gene expression?
A
- Stimulate RNAP.
2. Alter the structure of chromatin.
10
Q
When the chromatin is looser is it transcribed more or less?
A
More.
11
Q
What are Barr Bodies?
A
The condensed form of the X chromosome.
12
Q
What are receptors in the cytoplasm called?
A
Nucleoreceptors.
13
Q
Name one type of pathway that is only activated for limited periods?
A
Cell growth and divison.
14
Q
What type of receptor is the Her2 receptor?
A
Surface.
15
Q
What drug can block the Her2 receptor?
A
Herceptin.
16
Q
What level are most genes regulated at?
A
Transcriptional. (Euk and Prok.)
17
Q
Do post transcriptional events matter more in proks or elks?
A
Euks.
18
Q
Most human genes contain introns with one main pattern of DNA splicing. True or false?
A
False, most have multiple patterns of splicing.
19
Q
What do multiple splicing patterns of eukaryotic genes result in?
A
More than one protein per gene.
20
Q
What percentage of inherited genetic disorders in humans are a result of pre-mRNA splicing?
A
30%.
21
Q
In humans what is the most common genetic disorder caused by pre-MRNA splicing?
A
Spinal Muscular Atrophy- causes lack of lung function an death at 2-3 years.
22
Q
Gene expression can be regulated post-transcriptionally in what two ways?
A
- Level of translation.
2. mRNA stability.
23
Q
Why are some transcripts localised to specific regions of the cell?
A
Ensures targeted expression.
24
Q
Ribonucleases provide activities that are central to ________, _______ ________ and _________.
A
- RNA processing.
- Quality control.
- Degradation.
25
What is the expression called when the gene can be switched on?
Inducible expression.
26
What is a model of a inducible genetic control system in budding yeast?
GAL switch.
27
What is the major degradation machinery in eukaryotic cells?
Exosome.
28
What are the four major steps in the general pathway of eukaryotic gene expression?
1. Chromatin remodelling.
2. Processing of nuc. MRNP.
3. Translation.
4. mRNA turnover.
29
When does GAL induce a signal?
When galactose is in a medium.
30
How are most genes expressed?
Constitutively.
31
Eukaryotic transcriptional control helps the cell respond to the environment, like in prokaryotes. Why else is it important (3 reasons)?
1. Celluar differentiation.
2. Development.
3. Cell signalling.
32
What can lose to control of regulated gene expression in multicellular organisms result in?
Developmental defects and cancer.
33
Apart from RNAP directed area being much larger than the directed are in proks, what else is it?
Much more dispersed.
34
Many genes can be influenced by a larger number of factors and sites in proks than in euks. True or false?
False, this is true for euks.
35
Can a gene be regulated by different factors acting on different promoter regions at different times?
Yes.
36
Transcription can be subject to control by multiple sets of regulatory factors as a result of more than one signal. What can this result in?
The generation of RNA transcripts with distinct 5' ends from differently regulated promoters.
37
What does the sex lethal gene control?
Sex determination in fruit flies.
38
In what gender of fruit fly does the PE (young) promoter express the sex lethal gene?
Female.
39
In what gender of fruit fly does the PL (adult) promoter express the sex lethal gene?
Male and female.
40
What sort of transcripts do the PL and PE promoters generate in fruit flies?
Distinct but overlapping.
41
Is the PL or the PE promoter further upstream?
PL.
42
What is a Cis- regulatory element?
Regions of non coding DNA that regulate the transcription of nearby genes.
43
What do highly inducible genes contain?
A TATA box.
44
Where is the TATA box found?
30 nucleotides upstream of the transcription start site.
45
Highly inducible genes are highly expressed. True or false?
False.
46
What is the 'real' meaning of the promoter?
The region in which RNAP binds to upstream of gene.
47
Only some genes have an initiator. True or false?
True.
48
What are initiator sequences?
Highly degenerate sequences around the transcription start site.
49
Why are CpG islands formed?
As CG tends to be under represented and all the genes cluster together.
50
What is a CpG island?
Clusters of CG bp in the promoters of some genes.
51
What part of the CpG island is methylated?
m5C.
52
Where are CpG islands methylated?
Within the genome.
53
When are CpG islands unmethylated?
In transcribed genes.
54
Genes from CpG islands can be transcribed from multiple sites. True or false?
True.
55
What is the purpose of deletion analysis?
To find regulatory elements within a promoter region.
56
Describe deletion analysis.
Deletion analysis is used to find regulatory elements within a promoter region. Short sections of the promoter regions are removed from either end (or both) and the sequence is inserted into a vector and cloned upstream of a suitable reporter gene. Can then assy the extracts for the level of BG or luciferase protein.
57
Does linker scanning mutagenesis or deletion analysis allow you to see regulatory elements within in a promoter region?
Deletion analysis.
58
Does linker scanning mutagenesis or deletion analysis allow you to determine short regions/ sequences and their requirement in transcriptional control?
Linker scanning mutagenesis.
59
Describe linker scanning mutagenesis.
Short regions within the complete promoter region are assayed for their requirement in transcriptional control. Short, overlapping sequences within the promoter region are mutated to generate a series of constructs, each containing randomised nucleotides within a specific region of the same length DNA.
60
What gene was linker scanning mutagenesis first carried out on?
Thymidine kinase gene.
61
What three sequences were found in the thymine kinase gene via linker scanning mutagenesis?
TATA box, and two promoter proximal elements (PE1 and PE2.)
62
What are enhancer elements?
DNA sequences found long distances away from the transcription start site which can increase transcription levels.
63
Although most enhancers are site specific, what enhancer works on most mammalian genes?
SV40.
64
What is the size of the SV40 enhancer?
366bp (small).
65
What direction does the SV40 enhancer work in?
Both.
66
Enhancer elements are found close to where SV40 binds. True or false?
False.
67
Where are enhancers often found?
Expression plasmids.
68
What is SV40 enhancer composed of?
Multiple elements that collectively have maximum activity..
69
Describe an RNA detection assay.
Hybridise each RNA to one labelled DNA molecule. Add S1 nuclease which degrades ss RNA but not RNA bound to DNA. Analyse amount of detected fragment to determine RNA level.
70
What is found in the promoter region of a typical mammalian gene?
TATA box elements and a number of promoter proximal elements.
71
Where are promoter proximal elements often found?
200 nucleotides upstream from the transcription start site.
72
What regulatory element can be found in introns?
Enhancer elements.
73
How far away from the transcription start site can enhancer regions be found (in both directions)?
10s of kilobases in both directions.
74
Yeast genes are smaller than mammalian genes. Where are the TATA box elements located?
90 nucleotides upstream of the transcription start site.
75
Yeast has less regulatory elements. What are the two options it can have?
1. Upstream activating sequence (UAS).
| 2. Upstream regulatory sequence ( URS).
76
What do yeast genes tend not to have?
CPG islands.
77
What do most yeast genes not have?
Introns.
78
Specific transcription factors are found upstream of every gene. True or False?
False.
79
How do specific transcription factors alter gene expression?
1. Indirectly interact with polymerases.
| 2. Interact with the chromatin.
80
Why do specific transcription factors tend to function in groups?
Allows a wider diversity of complexes.
81
What type of structure do transcription factors have?
Modular domain.
82
What are general transcription factors required for?
Assembly of RNA polymerase molecules at the beginning of genes.
83
Explain the process in which general transcription factors act.
The TATA box binding protein (TBP) in TFIID binds to DNA. This recruits TFIIB. RNAP can then bind with TFIIF. TFIIE and TFIIH then recruited.
84
In what groove does the TATA box bind?
Minor.
85
What does TFIIH do?
Unwinds double stranded DNA for transcription.
86
What is needed in addition to general transcription factors for activity at the RNA pol2 promoters?
Mediator complex.
87
What is the mediator complex made form?
Over 30 different proteins.
88
The mediator complex is very conserved. True or false?
True.
89
What are specific transcription factors?
Regulatory proteins that stimulate or repress the basel level of transcription by RNA polymerase II.
90
What does basel mean in regards to transcription?
Non stimulated, non repressed level of transcription that is independent of additional factors.
91
What makes up basel transcription?
The mediator complex and general transcription factors.
92
In prokaryotes transcription factors interact with RNA polymerase directly. What happens in eukaryotes?
Polymerase is regulated indirectly through interactions with the mediator complex or by changing the structure of chromatin.
93
What percentage of human genes encode for transcription factors?
10%.
94
Is the mediator complex always bound to pol2?
Yes.
95
How many specific transcription factors are found in yeast?
Over 150.
96
How many specific transcription factors are found in humans?
Over 3000.
97
What does transcriptional regulation involve?
Combinational control by multiple STFs.
98
What two domains do STFs have?
DNA binding domain and an activation/repression domain.
99
What do STfs bind to?
Coactivators and corepressors.
100
What separates the two domains in a STF?
Flexible linker region.
101
What does the regulatory activation/ the regulatory repression domain bind to?
Other proteins that are involved in transcriptional regulation.
102
What does the Gal4 activator do?
Induces expression of genes responsive to galactose.
103
What is a UAS?
Upstream activating sequence. It is found in the promoter region in response to galactose.
104
What are the DNA binding domain and the activation domain of Gal4 fused separately to experimentally?
'Bait' and 'Prey' proteins.
105
When can the Gal4 protein promote transcription of the reporter gene containing a UAS for gal?
When the bait and prey proteins interact.
106
Deletions of proteins such as Gal4 in the N and C terminal showed the modular structure of STFs. A small deletion in the DNA binding domain showed both domains lost their binding ability. What terminal was this found at?
N.
107
What three reporter genes were use in the yeast two-hybrid analysis (bait and prey experiment.)?
His3, ADE2, LacZ.
108
Is the bait or the prey protein the protein of interest in the yeast two-hybrid analysis?
Bait.
109
What can be used to make a series prey proteins?
A library of plasmids.
110
The yeast two-hybrid analysis assay is used to look at interactions between yeast proteins. True or false?
False, it can look at interactions between any proteins.
111
What will yeast mutants that have mutated ADE be?
Red.
112
What do enhancer regions consists of?
Multiple regulatory elements/ control elements that bind to transcription factors in a cooperative manner.
113
DNA binding proteins can bind to the minor groove in the DNA within the enhancer region causing it to bend. What is an example of such a DNA binding proteins?
HMG1.
114
How is an enhancer complex containing multiple subunits assembled?
Through a large number of protein-protein and protein-DNA interactions.
115
What is the best characterised enhancer?
B- Interferon complex.
116
What sort of dimers can transcription factors function as?
Homo or hetero dimers.
117
What type of dimers is the enhancer complex B-Interferon made from?
Heterodimers.
118
When will the heterodimers found in the enhancer complex B-interferon bind to adjacent regulatory elements?
When HMG1 is present.
119
How can multiple weak interactions between STFs be increased?
Through interactions within the DNA.
120
Using genetics, where can TF's be isolated from?
Yeast, flies and worms.
121
What is needed for galactose utilisation?
Gal2 permase, and the enzymes Gal1, Gal7 and Gal10.
122
What regulates the response to galactose?
1. Gal80 regulator protein.
| 2. Galactose sensor G3.
123
How have TF from mammalian cells been isolated?
Biochemical approaches.
124
What transcription factor regulates the GAL switch in yeast?
Gal4.
125
Gal80 allows the genes for galactose utilisation to be transcribed. When is its nuclear pool depleted?
When Galactose binds to Gal3, meaning Gal80 can bind to Gal3 in the cytoplasm.
126
What can be used to isolate transcription factors that interact with a regulatory element with a known sequence?
Gel-shift assays.
127
In gel-shift assays what is incubated together?
Short double-stranded DNA molecule with the regulatory element is incubated with a fractionated nuclear extract.
128
Why can gel-shift assays recognise fractions with the cognate-specific transcription factor?
Because DNA complexed to proteins moves slower than normal DNA.
129
Multiple cycles of a gel-shift assay allow purification of DNA binding proteins. Are these always transcription factors?
No.
130
Explain the in vitro assay for transcription factors.
Purified DNA binding protein is incubated with a sample containing the cognate promoter region and a sample that does not. Can they see if the protein stimulates or inhibits transcription. IN VITRO.
131
Explain the in vivo assay for transcription factors.
Two plasmids are put into host, one with the suspected TF and one with the reporter gene under control by the cognate regulatory element. Measure amount of rMRNA produced. If the DNABP is actually a tf expect the amount of rMRNA to be increased.
132
In vivo assays of transcription factors allows you to see if the isolated DNABP from gel-shift assays is actually a TF. What else can it help you do?
Mutational analysis of the STF and the specific promoter region to see which parts are the necessary parts.
133
Loss of function of repressor proteins can cause increase gene expression. What is an example of a protein this affects?
Wilms Tumour Protein (WT-1).
134
What can mutations in the Wilms Tumour Protein (WT-1) cause?
Nephroblastoma.
135
Is more known about activation domains or DNA binding domains?
DNA binding domains as they are less varied.
136
Are there a group of transcription factors with highly acidic or basic activation domains?
Acidic- Gal4 is included in this.
137
When are acidic activation domains poorly structured?
When they are not associated with any other proteins.
138
When are acidic activation domains well structured?
When they are associated with co-activators.
139
Can the activator/ co-activator interaction be regulated?
Yes.
140
What is CREB?
cAMP response element binding protein.
141
What is CREB an example of?
A transcriptional activator.
142
What is CREB's co-activator called?
CBP binding protein.
143
When can the transcriptional activator CREB interact with the co-activator CBP?
When it is phosphorylated.
144
For CREB to interact with CBP it has to be phosphorylated. What does this phosphorylation?
PKA- protein kinase A.
145
What regulates PKA, the kinase needed to phosphorylate CREB?
cAMP.
146
What does the transcriptional activator CREB bind to?
cAMP response elements- CREs.
147
CREB, the transcriptional activator, can bind to CREs in the genome. Where are CREs found?
Upstream of genes transcriptionally activated by the cAMP-dependant signalling pathway.
148
Acidic activation domains are poorly structured when not bound to other proteins. How are domains of nuclear-receptors different to this?
They have structure domains.
149
When are the activation domains of nuclear receptors active?
When they are bound to their ligands.
150
What triggers the interaction between the transcriptional activator and the co activator in nuclear receptors?
A hormone which causes a conformational change.
151
What does estrogen bound to its receptor generate?
Hydrophobic pocket that binds an amphipathic helix.
152
Estrogen bound it its receptor generates a pocket. What can this do?
Bind a amphipathic helix from the coactivator.
153
What can bind to the hydrophobic pocket of the estrogen receptor causing it to be sterically blocked?
Tamoxifen.
154
How do transcription activators and repressors bind to regulatory elements in the DNA?
In a sequence specific manner.
155
What groove in the DNA do activators/repressors bind to?
Major.
156
Transcription activators/ repressors bind to regulator sequences in the DNA through a sequence specific manner. How does this actually happen?
An alpha recogintion helix from the protein inserts into the DNAs major groove.
157
How are contacts made between the transcription activator/repressor and the DNA? What type of contacts are made?
Between amino acid residues in the recognition helix and the edges of bases within the DNA. The contacts are normally both hydrophobic and ionic.
158
Where is the homeodomain fold only found?
In eukaryotes. It is very similar to the helix-turn-helix found in phage transcriptional repressors such as cro.
159
How many amino acids make up the a-helical homeodomain?
60
160
What encodes for the a-helical homeodomain?
Homeobox.
161
What percentage are nucleotides are conserved in the homeodomains of different eukaryotes?
75%.
162
What do homeodomain proteins play a key role in?
Embryogenesis, development and gene expression patterns.
163
How are the genes encoding many homeodomain proteins found?
In Hoax gene clusters.
164
What does the order of Hoax gene clusters correlate to?
Spatial expression within the embryos.
165
What is the homeodomain fold?
A transcription factor.
166
Whats the biggest family of transcription factors in mammalian cells?
Zinc finger proteins.
167
What are the two major forms of zinc finger proteins?
C2H2 zinc finger and the C4 zinc finger.
168
Zinc finger proteins domains are folded around a central zinc ion through 4 contacts with 4 amino acid residues. What two combinations of amino acids can this be?
4 cysteine (C4) or 2 cysteines/ 2 histidines (C2H2).
169
How are the residues involved in the zinc finger model found in the primary sequence and why?
Two closely separated pairs- because the introverting looped peptide sequence when the structure is drawn out.
170
Transcription factors contain zinc fingers in fours. True or false?
False, they can contain multiple though.
171
How do proteins with zinc fingers bind DNA?
As a monomer.
172
What type group of zinc finger proteins contain nuclear receptors?
C4.
173
What are nuclear receptors transcription factors for?
Steroid hormones.
174
How many zinc finger domains are found in C4 zinc finger proteins?
Two. DNA is subsequently bound as a hetero or homo dimer.
175
What are basic leucine zipper proteins?
Coiled coiled dimers linked through parallel amphipathic helices with leucine in every 7th position.
176
What type of dimer is a leucine zipper protein?
Homo or hetero dimer.
177
What does the coil coil structure of the basic leucine zipper protein refer to?
Extended alpha helix regions of the tow subunits coiled around each other.
178
What charge status are the a helices involved in the coil coil in the basic leucine zipper?
Amphipathic, with the hydrophobic side making contact with the other subunit.
179
What contributes to the strong interactions between the two helices of the leucine zipper proteins?
The fact that every seventh residue is a leucine residue, contributing massively towards the hydrophobic interactions. These represent the 'zip'.
180
Many transcription factors are leucine zipper proteins. How do these interact with the DNA?
The alpha helices of the zipper are extended at their N terminal and grip the DNA at adjacent major grooves.
181
What contacts are present between the leucine zipper protein and the DNA?
Base specific contacts and electrostatic interactions between the basic residues and the phosphodiester backbone.
182
What does the general term 'basic zipper' refer to? (bZIP)
The larger group of proteins that can have residues other than leucine along the dimerisation surface.
183
There is a domain similar to the bZIP domain where the extended alpha helices are interrupted with loop regions. What is this domain called?
Basic-helix-loop-helix. (bHLH)
184
What generates alternative heterodimers?
The combination of different transcription factors.
185
What is increased due to the formation of alternative heterodimers?
The diversity of DNA sequences that can be recognised/ expands the way the factors can be regulated.
186
Apart from the formation of alternative heterodimers, what else is another combination control mechanism of transcriptional control?
Binding of unrelated DNA binding proteins.
187
What may two proteins that bind weakly to adjacent sites within the DNA be able to form in isolation?
A stable transcription complex through intermolecular protein-protein interactions.
188
Two proteins that would normally bind to adjacent sites in the DNA may be able to bind to each other in isolation. What is this called?
Cooperative binding.
189
What is cooperative binding dependant on?
Relative position of the two recognition elements within the DNA.
190
Cooperative binding can happen between distinct DNA binding proteins. What is an example of this?
NFAT and API bind cooperatively to the IL2 promotor-proximal region.
191
What does cooperative binding between NFAT and API require?
Specific spacing of the recognition site within the DNA.
192
Some transcription factor dimers recognise the same DNA sequence. What does this result in?
Different activation domains together in different combinations at the same site.
193
Some transcription factor dimes recognise different DNA sequences. What does this result in?
An increased number of potential targets.
194
What type of DNA binding protein can bind to repressors?
bZIP.
195
The structure of eukaryotic chromosomes varies during the cell cycle and the chromosomes of interphase nuclei are less condensed meaning they are yet to be packaged into chromatin. True or false?
False. The chromosomes are still highly packaged into chromatin.
196
What is packaged into chromatin?
Chromosomes, RNA and protein.
197
Chromatin contains 50% of histone protein by mass. True or false?
False. It contains 50% of histone and non histone protein by mass.
198
What percentage of an average cell nucleus is taken up by DNA's double helix?
5%.
199
What percentage of a sperm nucleus is taken up by the DNA double helix?
60%.
200
DNA packaged within the chromatin of a somatic cell during interphase must remain accessible to proteins involved in what?
1. Transcription.
2. Replication.
3. DNA repair.
201
Is DNA always accessible?
No.
202
When chromatin is extracted from the nucleus under low salt concentrations and in the absence of mg2+ what is it said to resemble?
'Beads on a string.'
203
Chromatin extracted from the nucleus under low salt concentrations and in the absence of mg2+ is said to resemble 'beads on a string'. What are these beads/ whats the string?
The beads are the nucleosomes and the string is linker DNA.
204
What do nucleosomes consist of?
DNA would around a core of histone proteins.
205
How big are nucleosomes in diameter?
10nm.
206
When chromatin is extracted from the nucleus under physiological conditions (salt and mg2+ present) it does not resemble 'beads on a string'. What does it resemble instead?
More compact 30nm fibres.
207
Can you move between the differently compacted chromatin fibres?
Yes.
208
What is the most compact chromatin fibre you can isolate?
100nm.
209
What are the core histone proteins?
H2A, H2B, H3, H4.
210
How many of the each of the core histone proteins is the histone made up off?
2 of each, 8 in total.
211
What dimers are formed between the core histone proteins?
H2A/H2B H3/H4 (there are two of each.)
212
How do the dimers made from the core histone proteins interleave?
In a 'handshake like interaction.'
213
Are histone found in prokaryotes or eukaryotes?
Eukaryotes.
214
What can the core of a nucleosome be described as?
Octameric.
215
What way does the DNA wind around the histones?
Left handed.
216
What are the specifications of the DNA turn around a histone?
147bp makes a 1.7 left handed turn.
217
The linker region between the nucleosomes is a constant length. True or false?
False. The length can vary.
218
What can the length of the linker DNA vary between?
10 and 100 base pairs. IN MULTIPLES OF TENS.
219
Why are DNA linker regions always in multiples of tens?
As that is one helical turn.
220
What histone protein is bound to the DNA as it enters and exits the nucleosome?
H1.
221
What can histone proteins be described as?
Highly basic.
222
Are histone proteins globular?
Fairly, but they also have non globular tails.
223
What are three examples of some histone variants found in some nucleosomes?
H2AX, H3.3, and CENP-A.
224
What are the flexible domains of histones also called?
Histone tails.
225
What core histones have a globular tail only at their N terminus?
H3 and H4.
226
What core histone proteins have a globular tail at both termini?
H2A and H2B.
227
H2AX is a histone varient. What is its role?
Functions in DNA repair and is widely distributed throughout the genome.
228
CENP-A is a histone varient. What is its role?
Involved in attachment of the chromosomes to the microtubules during mitosis.
229
H3.3 is a histone varient. What is its role?
Found in actively transcribed genes.
230
What is most DNA that is packaged in sperm packaged with?
Arginine rich proteins called protamines.
231
What can post translational modifications of core histones lead to?
Changes in the chromatin.
232
What are the two forms of chromatin?
Heterochromatin and euchromatin.
233
Chromatin is now distinguished through the use of epigenetic markers, how was it originally determined?
Cytologically.
234
What type of chromatin is more densely packed ?
Heterochromatin.
235
What are the two types of heterochromatin?
Constitutive and facultative.
236
Where is constitutive chromatin found?
Repetitive DNA sequences
Satellite DNA
Centromeric DNA regions
Telomeric DNA regions.
237
What type of heterochromatin is stably inherited through cell division?
Constitutive heterochromatin.
238
What is the main difference between constitutive heterochromatin and facultative heterochromatin?
Facultative heterochromatin can become decondensed.
239
What type of chromatin is transcriptionally active?
Euchromatin.
240
What size fibres can euchromatin be found in?
10nm and 30nm.
241
In what cell type is heterochromatin densely packed?
Interphase cells.
242
What is heterochromatin used to refer to?
Transcriptionally inactive DNA.
243
What are two markers of actively transcribed DNA?
AcH3K9 and MeH3K4.
244
What are two markers of heterochromatin?
MeH3K9 and MeH3K27.
245
Explain the charge patch hypothesis.
When lysine residues are acetylated they loose their charge meaning there are less interactions between two nucleosomes / between the DNA and a nucleosome meaning the DNA is more loosely packed and can be transcribed.
246
What facilities the structural changing between the condensed state and the decondensed sate of chromatin?
Acetylation and methylation of core histones.
247
What specific interaction does AcHK16 inhibit?
The electrostatic interaction between the H2 and H4 core histone proteins in adjacent nucleosomes.
248
What is the histone code?
The cumulative collection of core histone modifications.
249
Histone modifications can affect chromatin structure in trans and cis interactions. What are cis and trans interactions?
Trans are interactions with factors. Cis are interactions with nucleosomes.
250
The markers for heterochromatin are MeH3K9 and MeH3K4. Why are these different to acetylated lysines?
Methylation does not effect the charge status.
251
MeH3K9 and MeH3K27 are both markers for heterochomaitn. What mark is required for heterochromatin formation?
MeH3K9.
252
What are H3K9 methylated nucleosomes recognised by?
Proteins containing a chromodomain.
253
What is the most abundant protein found in heterochromatin?
HP1.
254
What do many proteins containing a chromodomain also contain?
A structurally related chromoshadow domain.
255
What is the purpose of the chromoshadow domain?
Allows for protein protein interactions.
256
What does chromatin immunoprecipitation allow?
The analysis of protein/DNA interactions in vivo.
257
How are chromatin immunprecipitation interactions stabilised?
Chemical cross linking.
258
Why is the ChIP technique possible?
Because of the large availability of antibodies to specific histone modifications.
259
What are the steps for ChIP?
1. Proteins are chemically cross linked to DNA.
2. Cells lysed.
3. DNA fragmented.
4. DNA fragments bound tp a specific protein are purified with available antibodies.
5. DNA analysed by PCR/ microarray.
260
What is the difference between using PCR or a microarray in the last step of ChIP?
PCR determines wether the protein is bound to a specific gene whereas microarray studies the localisation of the protein in a genome wide scale.
261
What can be used as a chemical cross linker inChIP?
Thermaldyhyde.
262
What are antibodies specific to in ChIP?
Acetylated or methylated histones.
263
What do you need to read the histone code?
Proteins that specifically recognise modified nucleosomes.
264
What part of the histone does the chromodomain bind to?
The histone tail.
265
How does the chromodomain bind to on the histone tail?
Methylated lysines.
266
What core histone protein binds H1 as a missing strand?
H3.
267
What is the purpose of the chromoshadow domain?
The chromosome domain on that protein can recognise a chromosome domain on another protein. Allows recruitment of more factors.
268
How does heterochromatin spread?
From an initiation point until it reaches the boundary.
269
What is heterochromatin spreading depend on?
1. H3K9 trimentylation.
2. HP-1.
3. Histone methlytransferase Suv3-9/Clr4.
270
What methyltransferase involved in heterochromatin spreading also contains a chromodomain?
Suv3-9.
271
How is the HMT activity of Suv3-9 stimulated?
Interaction of its chromodomain to an adjacent H3K9me3 nucleosomes.
272
What does binding of the Suv3-9 chromodomain to H3K9me3 promote?
Methylation of the neighbouring nucleosomes.
273
H3 binds H1 as a missing strand. What structure does this make?
Beta fold.
274
When are Hox genes maintained in a transcriptionally state/ active state?
During late stages of embryogenesis and through adult life.
275
What protein complex regulates Hox genes in late embryogenesis?
Polycomb.
276
What protein complex regulates Hox genes in adult life?
Trithroax.
277
What are PRC1 and PRC2.
The two major complexes found in the Polycomb complex.
278
What major complex in the polycomb complex is recruited by transcriptional repressors early during embryogenesis?
PRC2.
279
What is found in the PRC2 complex of the polycomb complex?
H3K27 specific histone methyltransferase subunit, also called the enhancer of zest.
280
What is the enhancer of zest?
The H3K27 specific histone methyltransferase subunit found in PRC2 of the polycomb complex.
281
What does the polycomb complex associate with?
Transcriptional repressors during early embryogenesis.
282
What does the enhancer of zest do?
Methylate specific H3K27 nucleosomes around the repressor.
283
What is the purpose of the PRC1 subunit in the Polycomb complex?
Condenses the structure of the chromatin.
284
How does the PRC1 subunit condense the chromatin?
It contains the Pc subunit which can bind to MeH3K27 containing nucleosomes and condense their structure.
285
What is Pc in the PRC1 subunit?
A dimeric chromodomain subunit.
286
What is the purpose of the polycomb repressive complex?
Allows the repression of certain hox genes in certain regions of the organism throughout its lifetime.
287
What does the trithorax complex contain?
A H3K4 specific HMT.
288
How does the thithorax complex act as a repressor?
It contains a H3K4 specific HMT which bind relatively stably to Me3H3K4 keeping them methlayed.
289
What is Methylated H3K4 a marker for?
Transcriptionally active chromatin. ODD CASE.
290
What are the two mating types of bakers yeast?
Mata and Matalpha.
291
What happens when a yeast mother cell divides?
It switches mating type.
292
What chromosome in yeast are the mating type loci found?
3.
293
What are the three mating type loci found on chromosome 3 in yeast?
HMLalpha, HMRa and MatA.
294
What mating type locus is central in yeast/ where gene conversion occurs?
MatA.
295
Recombination of the mating type locus in cell division in yeast is dependant on what?
HO endonucelase.
296
Repression of the HMLalpha and HMRa is dependant on what?
Adjacent silencer regions.
297
In yeast where does heterochromatin silence gene expression?
rDNA locus, telomeres, centromeres and mating type loci.
298
What binds to yeast DNA in silencer regions?
Rap1.
299
What protein does Rap1 (bound to silencer regions in yeast) recruit?
Sir proteins.
300
What is Sir2?
A histone deactlyase (H3K9, H4K16).
301
What does histone hypoacetylation allow?
Chromosome decondensation.
302
If a gene is inserted close to a silencer region what happens?
Its expression is blocked.
303
Once Sir2 has removed the acetyl group what can bind?
Sir2/Sir3/Sir4.
304
Once the Sir2/Sir3/Sir4 complex has bound to a deactylated nucleosome what happens?
The adjacent nucleosome is deacetylated. This only stops once a boundary element is reached.
305
What does packaging of the 10nm fibre into the 30nm require?
Linker histone H1 and the tails of the core histones.
306
Two classes of models have been proposed for the structural organisation of the 30nm fibre. What are these?
The solenoid model and the zig-zag ribbon model.
307
What is the solenoid model?
Chain of nucleosomes on the 30nm fibre are wound into a single coil.
308
What is the zig-zag ribbon model?
Linker DNA of the 30nm fibre stretches across a two-stranded left-handed double helix of nucleosomes.
309
In vivo when can the 10nm fibre be seen as a 30nm fibre?
Physiological salt conditions.
310
The solenoid model is more accepted than the zig-zag ribbon model. True or false?
False. They are equally accepted.
311
What can the zig zag model for the 30nm structure be described as?
Spherical and slightly flat, like stacked coins.
312
Is the 30nm strand still transcriptionally active?
Yes, anything higher is too condensed though.
313
The core histones are subjected to extensive post-translational modification. What are examples of these?
1. Acetylation of lysine residues.
2. Mono/ di/ tri methylation of lysine residues.
3. Phosphorylation of serines.
4. Phosphorylation of threonines.
5. Ubiquination of lysine.
6. Symmetrically/ asymmetrically dimethylated arginines.
314
Modifications to the core histone proteins can be mutually exclusive or mutually dependant. Name an example of a mutually exclusive modification?
Methylation of lysine which blocks acetylation.
315
Modifications to the core histone proteins can be mutually exclusive or mutually dependant. Name an example of a mutually dependant modification?
Ubiquitination of H2B is required for methylation of H3K4.
316
Why can the tails of histones make interactions with DNA and other proteins?
They are non rigid.
317
What do HATs do?
Add acetyl groups to the epsilon amino group of lysine residues.
318
What do HDAC do?
Readily reverse the actions of HATs.
319
HMTs methylate lysine side chains. What does this do in turn?
Prevents acetylation.
320
Is acetylation or methylation more easily reversed?
Acetylation.
321
What reverses methylation?
Lysine-Specific demethylases.
322
How can the 'chromatin landscape' of genes be studied?
Nuclease sensitivity assays.
323
What type of genes are hypersensitive to digestion with DNase I?
Actively transcribed.
324
What does chromatin immunoprecipitation use to determine where nucleosomes are found in specific genes?
Histone specific antibodies.
325
Where are Nucleosome-free regions observed (NFR)?
Upstream of transcription start sites.
326
What profile shows peaks close to transcription start sites ?
AcH3K9.
327
What changes upon gene activation?
Chromatin changes.
328
What gene was used in DNA sensitivity assays to show that nucleosomes change in development?
B-globin gene (fetal/ adult hb).
329
What type of assay showed that NFR were found upstream of transcription start sites?
ChIP assays.
330
What are NFR enriched with?
Eurochromatin markers such as AcH3K9.
331
Explain how the position of the nucleosome on the Gal operon was determined.
5 DNA sequences of the operon (A, B, C, D and E) were tested with PCR to see if a nucleosome had associated. (ChIP). CDE have nucleosomes present. No signal was found from A meaning a nucleosome was never present (UAS) meaning this region was never actively transcribed. B was found when glucose was low meaning that this was the TATA box region and nucleosomes were depleted/ added depending on if they were needed.
332
What can acetylation profiles show?
The level in which a gene is transcribed.
333
What are many yeast genes activated by?
Histone acetyltransferases- HATs.
334
What is the SAGA complex an example of and where is it found?
It is found in yeast and it is an example of a HAT (histone acetyl transferase).
335
What does the SAGA complex interact DIRECTLY with?
Transcriptional activators.
336
What are Gcn and Gal4 examples of?
Transcriptional activators.
337
What do transcriptional activators bind to?
Recognition elements within the DNA of target genes.
338
What is CBP?
A transcriptional activator with histone acetylase activity found in humans.
339
SAGA is only found in yeast. True or false?
True. Similar complexes are found in other eukaryotes.
340
CBP is found in humans, what is its homologue found in other eukaryotes?
Gcn5.
341
How is the saga complex recruited to the UAS?
Through transcriptional co activators.
342
What does acetylation do?
Looses nuclease interactions providing recognition sites for proteins with bromodomains.
343
What are two examples of a protein with a bromodomains?
1. Chromatin remodelling machines.
| 2. TFIID.
344
What is the catalytic part of HAT?
Gnc5.
345
Where does hyperacetylation happen?
Around the promoter.
346
What complexes move nucleosomes around the DNA?
Chromatin remodelling complexes (CRMs).
347
Why do CRMs need to move nucleosomes?
Certain important sequences such as the TATA box may be masked.
348
What do CRMs contain?
ATP DNA helices.
349
How do CRMs modify DNA/RNA proteins?
Modifying nucleic interactions.
350
What CRM can push nucleosomes causing them to slide along the chromosome?
SWI/ SNF.
351
What do SWI/SNF contain?
A bromo-containing subunit. This means it can interact with hyperacetylated nucleosomes.
352
In what histones does Gcn5 trigger direct acetylation in?
H2B and H3.
353
IN what histone does Gcn5 indirectly trigger acetylation in?
H4.
354
Hyperacetylation leads to decondensation of the nucleosome structure at the level of nucleosome interactions. What specific electrostatic interaction can be blocked for this?
H4K16-H2.
355
How are CRM's recruited to the promoter regions?
Through interactions with activators or repressors (this means they act as co activators or corepressors.)
356
What do repressors direct?
Condensation of chromatin through the recruitment of histone deacetylase complexs.
357
What is a major transcriptional repressor in eukaryotes?
Sin3/ Rpd3 HDAC.
358
What is the role of Sin3 in the Sin3/Rpd3 HDAC?
Catalytic histone deacetylase subunit.
359
What is the role of Rpd3 in the Sin3/ Rpd3 HDAC?
Allows for interactions with the transcriptional repressor Ume6.
360
What can Ume6, controlled by the Sin3/Rpd3 HDAC complex bind to?
Upstream regulatory sequences in the DNA.
361
What leads to transcriptional repression?
Chromatin condensation.
362
In mammalian cells what can transcriptional repressors also include?
Histone methyltransferases.
363
What is Suv3-9 an example of?
A HMT.
364
What type of cells express C5 DNA methyltransferases?
Mammalian.
365
What sequence in human cells is normally methylated?
CG.
366
Where in the genome is the level of methylation less than normal?
Promoter regions of actively transcribed genes.
367
What can CpG islands be described as?
Hypomethylated.
368
Where are DNA methylation patterns normally arranged?
In the oocyte.
369
When are DNA methylation patterns reestablished?
After fertilisation- these are maintained after subsequent cell divisions and differentiations.
370
M5CG binding proteins can recognise these caps forming complexes with HDACs and HMTS leading to chromosome condensation. What is an example of a M5CG binding protein?
MeCP2.
371
In what two ways can TF modulate transcription?
1. Affecting chromatin structure.
| 2. Mediator complex used to stimulate/inhibit pol2.
372
How large is the mediator complex?
Over 1MDa.
373
The mediator complex is required for all transcription. True or false?
False. It is only required for the transcription of pol2 transcribed genes.
374
What is the structure of the mediator complex?
It is very large (over 1MDa) and wraps around the promoter-bound RNA polymerase. It then forms multiple contacts with general transcription factors and specific transcription factors at both proximal and distal sites.
375
What is the mediator complex sort of like?
A general transcription factor.
376
What is positional effect variegation?
The integration of a well expressed gene close to a heterochromatic region resulting in transcriptional repression.
377
Is portion effect variegation inherited?
Yes, stably.
378
What are three well studied examples of positional effect variegation?
1. Mottled allele of the white eye locus in fruit flies. (specific mutation causes translocation)
2. Telomere position effect in budding yeast.
3. Kernal coloration in maize.
379
What is the main example of RNA-mediated transcriptional repression in mammalian cells?
X chromosome inactivation.
380
What is dosage compensation?
The inactivation of one X chromosome in females to ensure the expression of X linked genes is the same in males and females.
381
What is a silenced X chromosome visualised as?
A barr body.
382
Not all organisms use dosage compensation of the x chromosome. What do they do?
They increase the expression of the X chromosome in males.
383
What controls X chromosome inactivation?
The X- inactivation centre.
384
What does the Xic encode?
A number of non coding RNAs, including the Xist transcript.
385
How long is the Xist transcript?
17kb.
386
What postranslational modifications have happen to the Xist transcript?
Spliced, capped and polyadenylated.
387
Xist is retained in the nucleus. What does it do here?
Coats the inactive X chromosome.
388
What does Xist recruit?
Polycomb repressor complex 2 (PRC2).
389
What does PRC2 contain?
H3K27 specific histone methyltransferase Ez.
390
What regulates Xist?
Other noncoding RNAs in the Xic.
391
What is the antisense transcript Tsix involved in?
Regulation of Xist.
392
What is the transcriptome?
Combination of all RNAs in the cell.
393
Why is the transcriptome more complex than originally thought?
Eukaryotic cells contain many low abundance RNAs that are very unstable.
394
Where are unstable low abundance RNAs generated?
Nucleosome free regions at the promoters of normally expressed genes.
395
At some promoters what can transcription be?
Bidirectional. This allows for a rapid response in regulatory signals.
396
In one cell transcription can only happen in on direction at a time. True or false?
True.
397
What gene in budding yeast exemplifies how RNA regulates transcription?
Ser3.
398
What is the Ser3 gene required for in budding yeast?
Seine biosynthesis.
399
When is the Ser3 gene repressed in yeast?
When grown in a rich medium.
400
Describe transcription interference in regards to the yeast Ser3 gene?
A transcript is generated from the Ser3 intergenic region upstream. This transcript is called SRG1 and is Jon coding. It's role is to block TF from the promoter region of the Ser3 gene.
401
What two gal genes are expressed from the same promoter in opposite directions?
Gal 1 and Gal 10.
402
Is Gal1 or Gal10 a non coding RNA?
Gal10.
403
In how many cells are you expected to find a Gal10 uncoded RNA molecule?
1 in 17-20 cells.
404
Is uncoding RNA stable?
No it is very unstable.
405
What does Gal10 uncoded RNA lead to?
The recruitment of the Rpd3 complex which deacetlyates DNA in that region.
406
Why are only low levels of uncoded RNA needed for sufficient expression?
Heterochromatin state is stably inherited.
407
What is the main purpose of RNA interference?
Depletes specific gene products.
408
What triggers RNAi?
Double stranded RNA. This is normally either viral, from endogenous transcripts or engineered RNAs.
409
What endonuclease triggers digestion of dsRNA?
Dicer.
410
The dicer endonuclease digests dsRNA. What size fragments does this generate?
25nt long fragments.
411
What type of cuts does dicer make?
Staggered.
412
Where does one strand of RNA produced by the digestion of a dsRNA by an endonuclease such as dicer end up?
An RNA induced silencing complex (RISC) or a RNA induced transcriptional silencing complex (RITS). This happens though base paring of specific genes.
413
What happens in a RISC complex?
mRNA degradation.
414
What happen in a RITS complex?
Transcription inhibition.
415
Is the RISC or RITS complex important in the establishment and maintenance of heterochromatin in fission yeast?
RITS.
416
RITS complexes are important in the maintenance and establishment of heterochromatin in fission yeast. They contain RNA from ______ regions which can associate with the ______ protein ____. This allows them to bind to nucleosomes containing methylated ______. This then recruits the histone methyltransferase ______. Once this is recruited the RNA substitutes for a _________ allowing specificity for the formation of heterochromatin.
Centromeric, chromodomain, Chp1, H3K9, Suv3-9, DNA binding protein.
Nb. This all allows silencing at a specific loci.
417
Are nuclear receptors the same as cell surface receptors?
No.
418
What binds to signalling molecules causing activation or repression of transcription?
Nuclear receptors.
419
What can nuclear receptors be classed as?
Transcription factors.
420
Nuclear receptors are named from the fact that they originate from the nucleus. True or false?
False, they do not necessarily originate in the nucleus.
421
What is the activation/ repression of a gene depend on?
The concentration of the cognate sTF in its active form.
422
In what 6 ways is the activity of a transcription factor regulated?
1. De novo synthesis.
2. Binding of a ligand molecule (e.g a nuclear receptor.)
3. Post translational modification eg phosphorylation of cAMP.
4. Formation of a protein complex e.g. dimers.
5. Release from an inhibitor molecule e.g. Gal4/Gal80.
6. Proteolytic activation.
423
What is the signal transduction pathway?
The activation of a sTF through the binding of an extracellular signal to to a transmembrane cell receptor which triggers a signal to be relayed through over molecules.
424
What are three examples of small lipid soluble hormones made of 6 carbon rings?
1. Steroid hormones.
2. Retinoid hormones.
3. Thyroid hormones.
425
What do small lipid soluble hormones bind to within a cell?
A receptor which is a specific transcription factor.
426
What is a nuclear receptor/ nuclear hormone receptor?
A receptor within a cell which is also a specific transcription factor.
427
Where can ligand binding to a nuclear receptor occur?
In the cytoplasm or in the nucleus.
428
What type of small lipid soluble hormone is vitamin A an example of?
A retinoid hormone.
429
What three domains is a nuclear receptor made up of?
1. Central C4 zinc finger DNA binding domain.
2. C-terminal ligand binding domain.
3. N terminal activation/ repression domain.
430
Which of the three domains in a nuclear receptor can have a variable structure?
The N terminal activation/repression domain.
431
What are hormone response elements (REs)?
Regulatory elements recognised by nuclear receptors.
432
What type of hormone response element is up of made short inverted repeats?
Hormones recognised by the glucacorticoid receptor or the oestrogen class I nuclear receptors.
433
What are receptors that bind to inverted repeats?
Symmetrical homodimers.
434
What do class II nuclear receptors contain and what are they?
Heterodimers that contain a common RXR (retinoid receptor) monomer.
435
What type of nuclear receptor can recognise different sequences?
Class II as they are heterodimers.
436
What has to be distinct for Class II nuclear receptors to be able to bind different sequences?
Spacing between the sequences.
437
Both classes of nuclear receptors use inverted repeats to bind. True or false?
False, only type I do.
438
What class of nuclear receptor is only localised in the nucleus?
Class II (hetero.)
439
Explain the process of Class II nuclear activation in steps.
1. When a ligand is absent the retinoid receptor is bound to the RXR corepressor.
2. The RXR compressor recruits HDACs, deacetylating the core histones in neighbouring nucleosomes blocking transcription.
3. When a ligand binds to the receptor the nucleoreceptor can form a heterodimer with the RXR RECEPTOR causing the RXR corepressor allowing it to displace.
4. The heterodimeric binds to HATS that hyperacetylate nucleosomes and interact with the mediator complex iniatiing transcription.
440
Where are homodimeric class I receptors localised in the absence of a ligand?
In the cytoplasm.
441
The homodimeric class I receptors are localised in the cytoplasm. How are they anchored?
Through heat shock proteins.
442
What allows class I nucleoreceptors to enter the nucleus?
Binding of a ligand to the receptor releases it form the cytoplasmic anchoring complex allowing it to enter the nucleus .
443
What 4 things can extracellular signals control?
1. Cell growth.
2. Differentiation.
3. Homeostasis.
4. Communication.
444
What three things can signalling defects lead to?
1. Cancer.
2. Diabetes.
3. Immunodeficiency.
445
What is a receptor complex made up of?
A ligand bound to its receptor.
446
What does the formation of a receptor complex normally involve?
Dimerisation of a receptor molecule.
447
The receptor complex activates one protein kinase. True to false?
False it can activate one or multiple.
448
What is an example of a pathway where the receptor molecule directly activates a transcription factor?
JAK/STAT pathway.
449
What is an example of a pathway where the receptor molecule indirectly activates a transcription factor?
Ras/Map kinase pathway.
450
What are two examples of secondary signalling molecules that can be made in the cytosol?
cAMP and inositol phosphate.
451
Transcription factors can be activ ated in the cytosol or in the nucleus. What happens when they are activated in the cytosol?
They are translocated into the nucleus.
452
Can receptor molecules activate more than one pathway?
Yes.
453
What is always involved in a signalling pathway?
Protein kinase.
454
The same signal general always brings about the same response. True or false?
Fasle, the same signal can bring about a different response in different cells.
455
How many different cell surfaces does a mammalian cell normally express?
Over 100 different receptors.
456
What are cytokines?
Small secreted polypeptides that control growth and differentiation of specific cells especially cells involved in the immune response.
457
What are three examples of cytokines?
1. Interleukins.
2. Interferons.
3. Erythropoietins.
458
What are ertyhroproteins?
Cytokines involved in the response to reduce blood oxygen levels. They are produced in the kidney and induce red blood cell proliferation by stimulating the expression of the anti-apoptotic factor Bxl-Xl.
459
What does binding of a cytokine to its receptor induce?
Dimerisation of the receptor.
460
What are cytokine receptors firmly associated with?
JAK2 kinases.
461
What happens in regards to JAK2 kinases when the cytokine receptor dimerises?
They phosphorylate each other at a critical tyrosine residue found in the activation lip.
462
What does the phosphorylation of JAK2 kinase lead to?
A reduction in the Km for ATP/ the substrate (substrate concentration in which the rate is half its maximum). This leads to increased kinase activity.
463
Apart from the other JAK2 kinase what else does does the JAK2 kinase phosphorylate?
Receptor bound STAT transcription factors. (STAT= signal transduction and activation of transcription).
464
When the STAT is phosphorylated from the JAK2 associated with cytokine receptors it dissociates from the receptor. Then what happens?
The STAT protein dimerises. FORMS HOMODIMER.
465
What is exposed when the STAT is dimerised (in the cytokine receptor pathway)?
The nuclear localisation signal (NLS). This allows the protein to be imported into the nucleus.
466
What there domains make up the kinase receptor?
Extracellular, transmembrane and cytoplasmic.
467
What are STAT proteins specific to?
The receptor.
468
JAK kinase phosphorylation reduces the Km for the substrate or ATP. What do they then phosphorylate?
Several residues on the receptor.
469
What are STATS?
(Signal transduction and activation of transcription) proteins.
470
What have the STAT proteins got homology to?
SH2 on the receptor.
471
What STAT is involved in the erythropoietin receptor?
STAT5.
472
What are receptor tyrosine kinases activated by?
1. Protein hormones eg insulin.
| 2. Growth factors.
473
What type of receptors have intristic protein kinase activity?
Tyrosine kinase receptors.
474
What are the three main events that happen in a tyrosine kinase receptor?
1. Ligand binds to the receptor triggering dimerisation.
2. Dimerised receptor autophosphorylates and its kinase activity is activated.
3. Additional residues are phosphorylated.
475
What pathway can almost all receptor tyrosine kinases stimulate?
Ras/MaP kinase pathway (nitrogen activated protein kinase pathway.)
476
How many receptor tyrosine kinases do human cells have for epidermal growth?
4. Her1- Her4.
477
What happens to most rtk's when they bind to ligands and how?
They form a dimer. Once the ligand binds a surface loop is extended providing the dimerisation interface.
478
What rtk does not interact directly with the ligand but instead interacts with the partner receptors increasing the cells ability to respond to change ?
Her2.
479
How much her2 does a cell normally have?
Low levels.
480
The her2 gene is amplified in 25% of breast cancers by 10 fold. What can the cells now be described as?
EGF hypersensitive.
481
When cells are EGF hypersensitive what happens?
They respond to low levels of growth factors that would not normally cause cell growth- described as constitutive growth.
482
What does Her2 form heterodimers with?
Her 1, 3 and 4.
483
What is herceptin and what does it do?
It is a humanised antibody for the Her2 receptor. It can bind to the extended loop conformation of the receptor blocking it dimerising- used to treat breast cancer.
484
What does the Ras/ map kinase pathway involve?
A membrane bound GTPase which can act as a molecular switch.
485
What activates GTPases?
Guanine nucleotide exchange factors.
486
What deactivates GTPases?
GTPase activating proteins.
487
What does the GTPase involved in the Ras/Map kinase pathway do?
Activates MAP kinases which translocate into he nucleus and activate different target proteins.
488
What activates the GEF in the Ras/map kinase pathway?
The phosphorylated receptor.
489
What does GEF bind to?
GTP.
490
What does the bas once gene block?
The ability to hydrolyse GTP due to a mutation in gly12 (in was). this causes the Ras/map kinase pathway to always be on.
491
What is the full Ras/Map kinase pathway?
GEF-RAS-RAF-MEK kinases- MAPK.
492
What activate the transcription factor CREB?
cAMP/ Protein kinase A pathway, induced by G-protein coupled receptors.
493
How many transmembrane receptors do G protein coupled receptors have?
7.
494
What are G protein coupled receptors associated to?
Cytosolic trimeric GTPase.
495
Describe the process of CREB activation by G protein coupled receptors (5 steps).
1. Ligand receptor binding activates membrane anchored adenyl cyclase through a trimeric G protein.
2. Binding of extracellular receptor causes the production of a second signalling molecule within the cell.
3. Increased levels of cAMP produced by adenyl cyclase causes release of the catalytic subunit of protein kinase A.
4. PKA moves into the nucleus where it phosphorylates CREB.
5. Phosphorylated CREB binds to CRES in target genes. This then binds to coactivator CBP.
496
Where is protein kinase A normally found?
Bound to an inhibitor in the cytoplasm.
497
What three different processes can be regulated though CREB activation?
Odours, light, neurotransmitters.
498
What do functional cellular RNAs associate with?
RNA- binding proteins in ribonuceloproteins.
499
What RNA binding proteins does the mRNA associate with during transcription?
heteronuclear RNP proteins (hnRNP) proteins.
500
RNA binding proteins effect all stages of gene expression.. What are these 6 steps?
1. Transcription.
2. RNA processing.
3. mRNA export.
4. mRNA localisation.
5. mRNA stabilisation/ degradation.
6. Translational regulation.
501
How do RNA proteins bind?
Specifically and non specifically.
502
How tightly do RNA proteins bind?
Some tightly, some with low affinity.
503
What does 'dynamic association' describe?
How the RNA proteins that bind change during its lifetime.
504
What RNA/ protein interaction is more static?
rRNA and the ribosome.
505
What percent of human proteins interact with RNA?
5%.
506
The RNA recognition motif is one of the most abundant eukaryotic protein domains. What percentage of human proteins is it found in?
2%.
507
The RNA recognition motif can be found associated with other RNA interacting domains such as _______.
The zinc finger domains.
508
Multiple copies of the RNA recognition motif are never found together. True or false?
False.
509
How many amino acids make up the RNA recognition motif??
90.
510
What does the RNA recognition motif consist of?
An antiparallel beta sheet supported by two alpha helices.
511
How do RRM domains bind to the ssRNA?
With high affinity in a sequence specific manner.
512
Individual RRM often bind in what manner?
Cooperative.
513
Some proteins have RRM domains that have evolved to function as a protein interaction domain. What is an example of one of these proteins?
Y14.
514
What is the binding of the RRM domain to the ssRNA often mediated by?
Specific sequences within the the sheets/ the loops of the sheets.
515
Where is the double stranded RNA binding domain found ? (3 points)
1. RNase III enzymes.
2. Protein kinase R (viral defence).
3. RNA dependant adenosine deaminases (Innate immunity.)
516
Can prokaryotic proteins or eukaryotic proteins contain multiple double stranded RNA binding domains?
Eukaryotic.
517
How many residues is the double stranded RNA binding domain made up of?
65.
518
What is the double stranded RNA binding domain made up of?
An alpha helix supported by an antiparallel beta sheet.
519
How does the double stranded RNA binding domain interact with the dsRNA?
Interacts with an 11 nucleotide long stretch of the dsRNA in a sequence independent manner through interactions with the ribose 2' hydroxyl group on the phosphate backbone.
520
What is RNAse III?
A double stranded RNA endonuclease.
521
What is RNAse III homologues in?
Bacteria, viruses and eukaryotes.
522
What is the function of RNAse III?
Releases RNA molecules from polycistronic transcripts, especially rRNAS and tRNAs.
523
What two RNAse III related enzymes function in micro array processing and RN interference?
Dicer and Drosha.
524
How often do RNAse III enzymes cleave?
Twice on each strand to leave 3' overhangs.
525
Where does RNAse III cleave?
Double stranded stem loop structures.
526
In what two ways can ribonuclease cleave the phosphodiester backbone?
1. Hydrolysis with a water molecule.
| 2. Phosphorolysis with a phosphate group.
527
What RNA products are generated from endoribonucleases?
RNA products with a 3' hydroxyl group and a 5' phosphate group.
528
What do hydrolytic exonucleases release?
5' nucleoside monophosphates.
529
What do phosphorolytic exonucleases release?
5' nucleoside diphosphates.
530
What type of ribonuclease remain bound to the substrate and degrade the RNA to completion without producing intermediates?
Processive exoribonucleases.
531
What is the opposite to a progressive exoribonuclease?
A disruptive exoribonuclease. Unlike progressive exoribonucleases these dissociate after removing single nucleotides from the transcript.
532
Do progressive or disruptive exonucleases generate intermediates?
Disruptive.
533
What mechanism do cellar RNases use?
A two metal ion catalytic mechanism where Mg2+ ions coordinate with the phosphor atom to promote attack by the hydroxyl ion and stabilise the leaving group.
534
What are found in all organisms which degrade RNA to its constituent nucleotides?
Highly processive exoribonucleses related to bacterial enzymes RNase II and polynucleotide phosphorylases (PNPase).
535
Some exoribonucleases are highly progressive and are able to unfold and degrade highly structured RNAs. Some require proteins to do this and two other things. What are these other things?
1. Remove bound proteins.
| 2. Extend the substrate to provide a single-stranded binding site for the exonuclease poly(A) or poly(U).
536
What endonucleases are distrubtive and are involved in limited trimming reactions?
RNase D related enzymes and deadenylases.
537
Do eukaryotic cells have more 5'-3' exoribonucleses or 3' exoribonucleases?
3' exoribonucleases. They only have one or more 5'-3' exoribonucleases.
538
What do restriction enzymes/ DNAses need to degrade DNA and why?
Mg2+. because the bonds between the carbon and the phosphate are very stable and cleavage will be too slow without it.
539
What type of enzyme is PNPase?
Trimeric.
540
What structure does the PNPase enzyme form?
Barrel.
541
Where is the RNA degraded in regards to the PNPase barrel structure?
In the inner surface of the barrel (the transcript is threaded through the barrel.)
542
What is the PNPase-related exosome?
A major RNase complex in eukaryotic cells that functions in the limited trimming of RNA substrates in the processing pathways and the complete degradation of other RNA substrates.
543
What has the PNPase related exosome lost through evolution?
The barrel subunits have lost their RNase activity during evolution. Instead, additional subunits have been recruited to the core complex.
544
What unfolds the substrates so they can enter the PNPAse related exosome?
The TRAMP complex.
545
What does the TRAMP complex contain?
RNA helices and poly(A) polymerase.
546
Are RNase 2 and the PNPase complexes progressive or distributive?
Highly progressive. They degrade their substrates to completion.
547
Some RNases can degrade structured RNAs such as stem loops which normally protect the 3' end from degradation. What can other RNases function with and why?
Helicases to unfold the stem loop or polymerases to allow for substrate binding.
548
What can polymerases generate to provide a 'landing pad' for the ribonuclease?
A single stranded stretch of DNA (a DNA tail.)
549
What does the Poly(A) binding protein do?
Increase the efficiency of translation.
550
What is the role of thePNPase related exosome?
Degrades RNA and allows 3' maturation of stable RNAs.
551
What two things an protect RNA from structural degradation?
Stem loops and associated proteins.
552
Where do LSM proteins bind?
To U rich sequences at the 3' ends of RNAs.
553
What does La bind to and why?
Newly synthesised RNAs. Required for the maturation of tRNAs and small RNAs.
554
What does the Lsm2-8 bind to?
U6snRNA.
555
What does Lsm1-7 bind to?
Deactlyated mRNA.
556
What is the structure of the Lsm complex?
Heptameric Beta sheet barrel.
557
How large is the splicesome in humans?
4.8 MDa.
558
What carries out pre MRNA splicing?
The splicesome.
559
What makes up the splicesome?
5 distinct small nuclear RNP complexes called snurps.
560
What 5 snurps make up the splicesome?
U1, U2, U4, U5, U6.
561
What do individual snurps contain?
A single snRNA and between 6-10 proteins.
562
Some proteins are specific to a given snurp, what proteins are found in all pol2 transcript snurps?
Sm proteins.
563
How can Sm proteins (found in snurps) be recognised ?
Autoimmune antibodies in some patients suffering from systemic lupus erythematous (SLE).
564
What is the U6 snRNA associated with?
Lsm proteins - these are similar to sm proteins.
565
Introns are not joined by ligation reactions. How are the joined?
2 Transesterfication reactions.
566
Does transesterfication require ATP hydrolysis?
No.
567
Although the intron lariat is normally has no use so is degraded by endo and eco nucleases. Some however do have coding potential. What do they code for?
Small nucleoar RNAs and some microRNAS.
568
What essentially happens in a transesterification reaction?
One ester is swapped for another.
569
What is key to the process of splicing?
Base pairing between snRNAs and pre-mRNA or between two snRNAs.
570
What distinct snRNA binds to the 5' splice site?
snRNA found in snRNP U1.
571
What distinct snRNA binds to the branchpoint adenosine?
snRNA found in snRNP U2.
572
What sequence does the U2 snRNA bind to at the branchpoint adenosine?
TACTAAC.
573
What happens when U2 snRNA binds to the branchpoint adenosine?
It base pairs with the conserved TACTAAC sequence kicking out the A residue from the pre-mana/U2 heteroduplex.
574
What there snurps associate as a trip-snurp?
U4/U5/U6.
575
After U1 and U2 have bound to the pre-mrna and U4/U5/U6 have formed a tri-snurp what snurps are subsequently released?
U1 and U4.
576
What three snurps make up the catalytic splicesome and out of these three which two are catalytic?
U5/U6/U2 with U6 and U2 being catalytic.
577
What do many of the subunits of the splicesome have?
RNA helicases activity.
578
The RNA helicases found in the splicesome often do not unwind RNA, what do they function as instead?
Chaperons by facilitating structural changes instead.
579
Although the tranesterfication reactions involved in splicing do not require ATP hydrolysis what related process does?
RNA helicases found in the splicesome need ATP during splicesome assembly, catalysis and disassembly.
580
What is Prp16?
It is a RNA dependant ATPase.
581
What is the role of Prp16 in regards to the splicesome?
It mediates a structural rearrangement of allows the second catalytic step of splicing to happen.
582
What can the intron lariat be degraded by?
Debranching enzymes and RNAses.
583
Where is the splicesome assembled?
On the preMRNA.
584
U3 is not part of the splicesome- what is its function?
It has a critical function in ribosome biogenesis.
585
Sm proteins are common to all snurps and allow them to bind to U rich sequences. Why?
They have a specialised ring structure.
586
What snurp is found at polIII transcripts?
PolIII.
587
Why are RNA ATPases essential in splicing?
They allow for kinetic proofreading ensuring that the correct substrate is bound.
588
What two RNA ATPases allow for the assembly of the splicesome?
Prp5 and Prp28.
589
What RNA ATPase allows the first splicing transesterfication reaction to take place?
Prp2.
590
What is the role of the RNA ATPase Prp22?
Allows the splicesome to be disassembled.
591
Prp16 drives a structural rearrangement of the splicesome before the second splicing reaction occurs. What does this allow for?
The degradation of the exons if they are not correctly aligned after ATP hydrolysis.
592
Yeast Prp16 mutants have a decreased ATPase activity. What does this mean?
There is a decreased fidelity of splicing due to an increase in the splicing of weak/ noncanonical splice sites.
593
Prp16 is involved in kinetic proofreading- what can it be known as?
A molecular clock.
594
What does Prp16 allow to happen in wild type cells, other than the structural rearrangement of the splicesome?
Selected weak splicesites to be rapidly degraded before the second splicing step.
595
What do in vitro splicing reactions contain?
P32 radiolaballed substrates containing a single intron or an appropriate size.
596
What can be followed as a function of time with in vitro splicing reactions involve gels?
Changing level of substrates, intermediates and products.
597
How can pre-MRNA splicing be tested in vitro (2 things)?
Depleting nuclear extracts of specific proteins allow you to see if the proteins are related to splicing.
RNA substrates can be synthesised with an altered structure do identity key nucleotides and their key interactions.
598
Describe the basic concept of kinetic proofreading?
If ATP hydrolysis is efficient the time frame will only be long enough for a certain molecular event to occur. If it is incorrect there is not enough time for incorporation and the reaction is taken out of equilibrium. If there is a mutant, e.g. Prp16 then hydrolysis takes longer and incorrect incorporation has time to occur.
599
What is pre-mRNA processing coupled to?
Transcription.
600
What does the C terminal domain of RNAPOLII contain?
Heptapeptide serine rich repeat YSPTSPS.
601
What end of the gene is Ser 5 phosphorylated in the C terminal domain of RNAP2?
5'.
602
What end of the gene is Ser 2 phosphorylated in the C terminal domain of RNAP2?
3'.
603
What does different phosphorylation patterns of the heptapeptide serine rich repeat YSPTSPS allow for?
Recruitment of different processing complexes. Capping at the start, splicing in the middle an polyadneylation at the end.
604
In what two ways can splicing occur?
Intron definition or exon definiton.
605
In what systems does splicing occur through intron definition?
In systems such as yeast where there are few introns and the introns are small.
606
In what systems does splicing occur through exon definition?
In most mammalian pre-mRNAs where there are multiple large introns and the exon sequences are relatively small.
607
What is the role of splicing factors?
Promote intron or exon recognition.
608
Name one example where splicing factors aid with intron recognition?
Srm160 and Srm3000 form a molecular bridge between U1 and U2.
609
What has to assemble to allow exon recognition?
Cross-exon recognition complexs. T
610
What do cross-exon recognition complexs do?
Provide bridging interactions between snurps and proteins associated to sequences within the exon through the use of SR proteins which can mediate protein /RNA and protein/protein interactions.
611
What do the RNA binding SR proteins contain?
Serine and arginine.
612
What common RNA binding domain do SR proteins contain?
RRM- RNA Recognition motif.
613
SR proteins can recognise sequence elements within exons that can promoter exon recognition. What are the these elements?
ESEs (exonic splicing enhancers).
614
U2AF is a splicing factor. What does it do?
Binds the polypryimidne tract that aids U2 binding to the branchpoint.
615
What is the structure of the splicing factor U2AF?
Heterodimer.
616
What defines exon boundaries?
Protein interactions that bridge U1 and U2 snurps.
617
What is reclusive splicing?
A splicing method where exceptionally large introns (>50kb) preMRNA gets spliced while it is made meaning the factors are assembling at the 5' end before the 3' end is made. The Intron is removed gradually through the regeneration of the 5' splice site at the spliced junction.
618
What percentage of human genetic diseases caused by point mutations are not caused by a change in the amino acid sequence but through changes in exon definition?
15%.
619
What is an example of a disease caused by incorrect splicing/ changes in exon definition?
Spinal musclular autotrophy. (SMA)
620
Explain how the SMN protein relates to Spinal musclular autotrophy.
The SMN protein is needed for the function assembly of SNURP particles. It is encoded for by two genes, SMN1 and SMN2 which only differ through a single point mutation in exon 7 of the SMN2 gene, this mutation causes the gene to be poorly expressed as it blocks the exotic splice site . As the SN2 gene is poorly expressed the SR protein SF2 needs to bind to its weak 3' splice site upstream of exon 7. SF2 recognises the exonic splicing enhancer which is mutated in people with SMA, causing incorrect splicing and a unstable protein. This mutation is actually found in the SMN1 gene and is homozygous.
621
Alternative splicing patterns are relatively rare in mammalian pre-MRNAs. True or false?
False, they occur in the majority.
622
What is an advantage of alternative splicing patterns?
Increased diversity of coding potential.
623
In what two ways can alternative splicing occur?
In a tissue specific manner or within the same cell as a result of programmed change.
624
Name an example where preMRNA splicing happens in a tissue specific manner?
Striated and smooth muscle forms of tropomyosin.
625
What determines whether alternative splicing patterns are used?
Alternative splicing factors.
626
Do alternative splicing factors activate or repress splicing?
Both.
627
What are alternative splicing factors classed as?
RNA binding proteins.
628
Where do alternative splicing factors bind to?
Enhancers or silencers close to the splice sites.
629
What is sex lethal (sxl)
A splice site repressor.
630
Where is sex lethal transcribed in early embryogenesis?
PE promotor in female flies.
631
Where is sex lethal transcribed in later embryogenesis?
PL promoter in both male and female flies.
632
How does sex lethal act as a splice site repressor?
Binds to intronic silencing elements blocking U2AF biding.
633
What pre-mRNAs does sex lethal bind to?
late sxl and tra (transformer) pre-mRNAs.
634
Sxl lethal allows exotic exclusion events in female flies. Why is this important?
Avoids incorporation of premature termination signals which cause the generation of non functional proteins in male flies.
635
Sxl lethal can bind to transformer (tra) pre-mRNAS blocking URF2 binding. What is the role of tra?
It is a splice site activator which activates alternative splicing in the 3' splicesite of the doublesex transcript when the SR protein tra2 is also present.
636
What does the alternative splicing of doublesex transcript in the presence/ absence of tra generate?
Sex specific transcripts with distinct C termini through exon exclusion and the use of polyadneylation sites.
637
What is the role of the different isoforms of doublesex?
It is a transcriptional repressor on sets of genes that control sexual differentiation.
638
Where is it thought pre-mRNA splicing has evolved from allowing intronic diversity?
Self-splicing introns.
639
How do group 1 introns splice?
Though the use of a guanine nucleotide cofactor that releases a linear intron.
640
How do group 2 introns splice?
Through a analogous mechanism involving a branchpoint adenosine and the generation a lariat intermediate.
641
What do self splicing introns have which allows them to fold and allowing splicing?
A defined tertiary structure.
642
What is an advantage of pre-mRNA splicing?
Allows for intronic diversity.
643
What travels through the nuclear pores?
RNA and protein.
644
There is a large amount of traffic through nuclear pores. What travels through of the pore for ribosome synthesis alone every minute?
Thousands of ribosomes are exported.
| Tens of thousands of ribosomes are imported.
645
What are nucleopores made of?
Approximately 30 different proteins called nuclear porins.
646
Nuclear pore complexes are large structures. How many MDa are they in size?
60.
647
What is the channel of nuclear pore aligned with?
FG-repeat nuceloporins.
648
What is the role of the FG-repeat nucleoporins that align the nuclear pore?
They form a hydrophobic gel that restricts passive diffusion of molecules less than 40Kd. Some molecules and proteins will shuffle back and forth.
649
The FG-repeat nucleotide porins restrict the passive diffusion of smaller molecules through the nuclear pores. How are larger proteins and complexs transported?
Through facilitated diffusion in their folded state.
650
What is required to facilitate transport through the nuclear pore?
Carrier proteins that interact with the cargo molecule and the FG-repeat nucleoporins.
651
How many times larger than a ribosome is a nuclear pore?
14.
652
What does a molecule need to be to be transported across the nuclear pore?
Soluble in the microenvironment- e.g interact with the FG nuclearporins.
653
What has been used to study nuclear transport?
Digitonin-permeabilised Xenopus oocytes.
Digitionin is a steroid that can solubilise lipids.
654
What is import of a receptor protein into the nucleus dependant on?
Cytosolic extracts and a nuclear localisation signal.
655
What is the nuclear localisation signal of the large T antigen of the Sv40 virus rich in?
Lysine.
656
What is an example of a cytoplasmic protein required for nuclear import?
Ran.
657
How was the cytoplasmic protein Ran identified?
Through assaying fractionated cytosolic extracts.
658
What is ran classed as and what can it function as because of this?
It is a small GTPase which can function as a molecular switch.
659
Is the Ran guanine nucleotide exchange factor (Ran GEF) a nuclear protein or a cytoplasmic protein?
Nuclear.
660
Is the RNA GTPase activating protein (Ran GAP) a nuclear protein or a cytoplasmic protein?
Cytoplasmic.
661
What proteins directly interact with cargo molecules to mediate nuclear transport?
Karyopherins.
662
What two classes of Karyopherins are there?
Importins and exportins.
663
Why can karyopherins interact with nuclear porins?
As they interact with hydrophobic FG-repeat nucleoporins.
664
When will karyopherins interact tightly with RAN?
When it is bound to GTP, so in the nucleus.
665
When will karyopherins dissociate from RAN?
When GTP is hydrolysed, so in the cytoplasm.
666
How do importins bind to their cargo molecules?
Through the NLS sequences.
667
How is a cargo protein released from the nucleus?
Binding of the importin/ cargo complex to Ran/GTP in the nucleus, which causes the release of the cargo protein.
668
When will exporting unbind their cargo?
Upon lose of Ran binding, so when ran is bound to GDP.
669
How do exporting bind to their cargo molecules?
NES sequences (nuclear export sequences).
670
In what three ways is the directionality of transport across the pores ensured?
1. Different localisation of Ran GEF and Ran GAP.
2. Affinity of karyophorins for their cargos and for GTP-bound Ran.
3. Differential affinity of karyopherins for their cargo molecules upon interaction/ loss of Ran binding.
671
Some RNAS are transported through the nuclear pore by karyopherins. What is an example of a specific exportin used for the export of tRNA?
exportin-t.
672
How are ribosome exported through the nuclear pore?
Through interaction of their protein subunits with the karyophorins.
673
What type of RNA is exported in a Ran independent manner?
mRNA, it is exported through the TAP transporter.
674
What does the TAP transport protein bind to to export mRNA?
To the mRNA and the FG nucleoporins.
675
What is mRNA binding to TAP dependant on?
The exon junction complex (EJC).
676
When is the EJC placed onto the mRNA?
After splicing at the splice site.
677
What RNA binding protein can be found in the Exon Junction Complex?
Ref - RNA export factor.
678
mRNA, Ref and Tap can be involved in what can be described as a 'molecular hand over event' What is this?
Ref is originally bound to the mRNA, but it can also bind to TAP. When Ref binds to TAP it weakens the Ref-mRNA interaction and stimulates the mRNA-TAP interaction.
679
What happens to the TAP/ mRNA complex once the mRNA has been exported?
It is dissembled through the chaperone activity of RNA helices Dbp5.
680
Where is the RNA helices Dbp5 localised?
Cytoplasmic fibrils of the nuclear pore complex. Here art can dissemble the TAP/mRNA complex.
681
How can proteins be expressed in a particular area of the cell?
Through mRNA localisation.
682
In what three ways can mRNA localisation occur?
1. Random diffusion and anchoring of mRNA through interaction with tethered proteins.
2. Active directional transport of the mRNA along microtubules or actin filaments.
3. Selected degradation or protection from degradation.
683
What type of RNA binding protein allows the specific localisation of the mRNA?
Zip code binding proteins.
684
What specific sequence elements within the mRMA allow it to be localised?
Zip code sequence.
685
Where within the mRNA is the zip code sequence found?
The 3' end of the UTR.
686
What does the ASH1 transcript in yeast encode?
A transcriptional repressor of the HO gene (ASH stands for asymmetric silencing of HO.)
687
What is the role of the HO endonuclease gene in yeast.
It induces mating type switching.
688
Does the HO endonuclease induce mating type switch in the yeast mother or daughter cell?
Mother.
689
In whats yeast cells is the HO repressor expressed?
The daughter cells, meaning only the mother cells encode the HO endonuclease and switch mating type.
690
Ash1 binds to the upstream repression sequence of the HO gene and recruits what?
Rpd3 histone deacetylase complex (HDAC).
691
What is the role of the Rp3d histone deacetylase complex (HDAC) in yeast?
It is recruited by ASH1 to repress HO gene.
692
Where in the cell is the ASH1 mRNA localised?
The distal tip of anaphase cells (daughter cells).
693
How is the ASH1 mRNA localised in yeast cells?
Through activated transport along actin.
694
What three things are required for the active transport of the ASH1 mRNA transcript?
She2, She3, Myo4.
695
She2 is required for the active transport of the ASH1 mRNA transcript. What is it?
A zip code binding protein.
696
She 3 required for the active transport of the ASH1 mRNA transcript. What is it?
A adaptor protein.
697
Myo4 is required for the active transport of the ASH1 mRNA transcript. What is it?
A motor protein.
698
The HO endonuclease in yeast cells allows the mother cell to switch mating types, this is repressed in the daughter cell through localisation of the ASH1 transcript. What mating type does the daughter cell hence have?
The mothers original mating type.
699
Apart from the ASH1 transcript in yeast, name another occasion when localisation of the maternally encoded mRNAs plays a key role in development?
In flies where it establishes axial patterning.
700
What is a transcriptional regulator in flies whose major function is to block genes that promote the expression of posterior tails?
Hunchback.
701
What 'hunchback' be classified as?
A transcriptional regulator.
702
Where must hunchback be expressed in an early embryo.
Anterior end.
703
Hunchback in flies represses the expression of posterior tails meaning it needs to be active in the anterior region but inactive in the posterior region. What makes it inactive in the posterior region by blocking its expression?
Nanos.
704
What does nanos bind to in the hunchback transcript ?
The nano response element (NRE) found at the 3' UTR.
705
Explain how nanos works.
The nanos gene is active in the posterior end of fly embryos to prevent the transcription of the hunchback transcript. It does this by binding to the NRE (nanos response element) at the 3' end of the UTR in the nanos transcript. This blocks its translation and promotes deadenylation.
706
What does deadenylation involve?
Shorting of the poly(A) tail.
707
Nanos regulates hunchback in flies, but what regulates nanos?
Oskar protein.
708
Where is Oskar protein found in a fly embryo?
The Oskar protein is found at the posterior end of the fly embryo where it prevents the degradation of nanos.
709
How does the Oskar protein prevent the degradation of nanos?
It blocks its deadenylation.
710
How is Oskar protein localised to the posterior pole?
Through the motor protein kinesin along the microtubules. via active transport.
711
What localises nanos?
Oskar.
712
What gene does hunchback block?
Posterior gap gene.
713
In what two ways can translational control be regulated?
1. Globally
| 2. Transcript specific level
714
When will translation be widely down regulated?
In response to stress (e.g. nutrient deprivation, UV, heat shock, infection.)
715
What can stress activated protein kinases phosphorylate?
The alpha subunit of eIF2 (initiation factor).
716
When the sell is stressed specific protein kinases can phosphorylate the initiation factor eIF2. What does this do?
eIF2-P is a competitive inhibitor to eIF2 and binds to the GEF eIF2B preventing the eIF2 dissociating.
717
What mRNAs can be more efficiently translated when the eIF2 levels are lower?
Stress induced- this relates to the phosphorylation of eIF2 under stress.
718
When something is globally regulated what does this mean?
Regulation occurs through the whole cell or for a particular type of mRNA.
719
Why does preventing the eIF2-P from leaving the GEF stop it functioning as an initiator?
For eIF2 to work as an initiaton factor it needs to be recharged by GTP, this can not happen if it can not disassociate from GDP when the GEF is blocked.
720
What GEF binds to eIF2?
eIf2B.
721
Is there more eIF2B or eIF2 in the cell?
eIF2.
722
In what state can some mRNAs be stored in in the cytoplasm?
Translationally dormant.
723
When will translationally dormant mRNAs be activated?
When the cell is exposed to the appropriate signal.
724
What is different about translationally dormant mRNAS?
They are polyadenylated in the cytoplasm and not in the nucleus.
725
Translationally dormant mRNAS have very short poly(A) tails. How are they trapped in the cytoplasm?
They are stored by a complex which traps the cap structure.
726
Name two scenarios where regulation of masked mRNA complexs is important?
1. Early metazoan oocyte development.
| 2. Synaptic plasticity.
727
Regulation of masked mRNA is important in synaptic plasticity. What is this?
Synaptic plasticity is the 'ability of synapse connections between neuronal axons and dendrites to change in strength in different flux of neurotransmitters- this is the basis of learning and memory'.
728
When dormant in the cytoplasm, what does the mRNP particles bind to?
Maskin.
729
In the dormant state in the cytoplasm the mRNP particles bind to masking, What is the role of Maskin.
Maskin blocks the cap binding protein eIF4E binding to eIF4G and Gld2.
730
Maskin blocks the cap binding protein eIF4E binding to eIF4G and Gld2. What is eIF4G?
A deadenylase. - stops the mRNP being degraded.
731
Maskin blocks the cap binding protein eIF4E binding to eIF4G and Gld2. What is Gld2?
A cytoplasmic poly(A) polymerase- stops the mRNP being activated.
732
What two things does translational activation of the mRNP complex involve?
Restructuring the MRNP complex and cytoplasmic polyadneylation.
733
Synaptic plasticity involves translationally dormant mRNP in the cytoplasm. How is this activated?
Through the NMDA glutamate receptor.
734
Metazoan embryogenesis involves translationally dormant mRNP in the cytoplasm. How is this activated?
Through the interaction of progesterone with its receptor.
735
Maskin is invloed in the storage of dormant mRNPS by blocking eIF4E binding to the cap of the mRNA. How does masking bind to the mRNP?
Through the cytoplasmic polyadenyaltion element CPE.
736
Apart from maskin what can the CPE element bind?
CREB, also helps store mRNPs. in the cytoplasm.
737
Maskin and CREB work to store dormant mRNP in the cytoplasm. How?
They can lock the structure into a compact shape.
738
What is the role of CREB in the storage of dormant mRNP?
When it is phosphorylated it opens up the cap structure which releases Gld2 allowing polyadenylation.
739
What does CPEB stand for?
Cytoplasmic polyadneylation binding protein.
740
When CREB is phosphorylated it stops maskin binding to eIF4E allowing it to bind with Gld2. What does this cause?
Extension of the poly(A) tail.
741
When CREB is phosphorylated it releases ma and what else from the complex?
Deadenylase PARN.
742
Once the dormant mRNP is activated and has a poly(A) added by Gld2 what can happen to it?
It can be circulated through interactions with eIF4G, eIF4E or PABP.
743
Fe2+ are cofactors for important cellular proteins. Name two examples of these proteins?
1. Cytochrome oxidases.
| 2. Ribonucleotide reductases.
744
Fe2+ is an important cofactor with cells and its level is controlled through translational control of mRNA encoding what?
Transferrin receptor.
| Ferretin.
745
To increase Fe2+ in the cell expression of what is increased/ decreased?
Transferrin receptor increased, ferritin decreased.
746
What type of receptor is transferrin?
Membrane associated.
747
What is ferritin?
Cytosolic iron biding protein.
748
To increase cellular Fe2+ levels the transferrin receptor expression is increased and ferritin expression is decreased. How does this happen?
Through Iron Regulatory Proteins (IRPs) binding to Iron Response Elements (IREs) in the ferritin and transferrin.
749
What do iron regulatory proteins act as?
Iron sensors.
750
When Fe2+ levels are high what do IRPs interact with?
Fe2+.
751
When Fe2+ levels are low what do IRPs interact with?
IRE.
752
When Fe2+ levels are low IRPs interact with IREs within the transcripts. What does this do to ferritin?
IRPS binds to the 5' leader within the sequence blocking the ribosome scanning and translation.
753
When Fe2+ levels are low IRPs interact with IREs within the transcripts. What does this do to transferrin?
IRPS bind to the 3' UTR stabilising the mRNA by preventing binding of AUBPS increasing translation efficiency.
754
What sequences are masked in transferrin when iron is low to prevent the degradation of the transcript?
ARE sequences.
755
How many residues are added to the poyl(A) tail in nuclear polyadneylation?
Roughly 70.
756
Where is the poly(A) tail gradually shortened?
In the cytoplasm.
757
The poly(A) tail is gradually shortened in the cytoplasm. What does this?
Deadenylases (adenylate specific endonuclease.)
758
How short does the poly(A) tail have to be for the mRNA to be rapidly degraded?
10 residues.
759
Why will the mRNA be rapidly degraded at 10 residues?
As the poly(A) binding protein PABP can not bind to the olio nucleotide as it is too short.
760
PABP can not bind to the mRNA when the poly(A) tail is below 10 residues. What is the consequence of this?
Requires a loss of interaction between the 5' end and the 3' of the MRNA decreasing translation efficiency causing it to be degraded.
761
Deadenylation is a slow process within the cell, true or false?
False, the time for deadnyation is transcript specific.
762
Cytoplasmic mRNA biding proteins specifically interact with a sequence within the mRNA imparting on its gene expression. What four processes can be affected by this?
1. Translation initiation.
2. Polyadenylation.
3. RNA localisation.
4. Increase or decrease in deadenylation.
763
What form of mRNA is less susceptible to degradation?
The circular form of mRNA, caused by interactions by proteins at the 3' and 5' end.
764
What removes the M7G cap structure of the deadenylated mRNA?
The heterodimeric decapping enzyme.
765
What does the heterodimeric recapping enzyme consist of?
Dcp1 and Dcp2.
766
How does the decapping enzyme work?
Hydrolyses the 5'-5' triphosphate linkage, releasing m7GDP and generating a 5' RNA with a monophosphate group.
767
The decapping enzyme generates a 5' RNA with a monophosphate group. What is this a good substrate for?
The 5'-3' exonuclease Xrn1.
768
What can happen to some deadenylated transcripts?
Exonucleolytic degradation.
769
Some deadenylated transcripts can be further degraded by exonucleolytic degradation. What direction does this occur in?
3' to 5'.
770
What does exonucleolytic degradation involve?
The exosome ribonuclease complex contain a 3' to 5' exoribonuclease.
771
Exonucleolytic degradation by Xrn1 is rapid whereas exonucelytic degradation by the exosome ribonuclease complex is slow. True or false?
False, they are both rapid.
772
What steps are the rate limiting steps of mRNA turnover?
Deadenylation and decapping.
773
What two turnover pathways can occur to mRNA?
1. 5'-3' slow deadneylation then decapping follow by rapid degradation.
2. 3'-5' decay without recapping (deadneylation happens first.)
774
What enzymes are involved in mRNA degradation also involves recapping?
Deadenylase, decapping enzyme containing Dcp 1 and 2 subunits and Xrn1.
775
What enzymes are involved in mRNA turnover when recapping does not occur?
Deadenylase and the exosome.
776
mRNAs have inherent deadenylation and decamping rates however some transcripts can be altered in response to specific signals. Name an example of this.
Iron repose proteins (IRPs) regulate the stability of transferrin (try) mRNA.
777
What are AUBPs and what do they do?
They are A/U rich element binding proteins which can regulate mRNA turnover rates.
778
What do AUBPs bind to?
AREs (A/U rich sequence elements).
779
Where are AREs found?
3' UTR.
780
Do AUBPS increase or decrease the stability of mRNA?
They can do both- in regards to Trf mRNA they decrease mRNA stability hence why IRPs are needed to prevent them binding.
781
Are AU rich sequences typically found in stable or unstable mRNAS?
Unstable.
782
ARE mediated decay can be repressed or stimulated by specific factors. Name an example that increases stability.
HUR.
783
ARE mediated decay can be repressed or stimulated by specific factors. Name an example that decreases stability/
AUF1.
784
What is an ARE?
A/U rich sequence element with 3' UTR.
785
Where can AREs be found?
Early response genes.
786
Early response genes contain AREs. What are early response genes?
A set of genes that are transiently induced in quiescent cells when they are treated with a serum- they help induce cell growth.
787
Early response genes are rapidly introduced which can provide a short burst of gene expression. What there fact or early response genes results in a high burst of gene expression?
The high instability of their transcripts.
788
Apart from early response genes, what other genes contain instability elements?
Cytokines that drive the progression through the cell cycle.
789
What does the AUF1 protein do?
Stimulates ARE-mediated degradation of the mRNA.
790
what is HuR?
A AUBP which stabilises ARE contain mRNAs.
791
Can AUF1 or HuR be phosphorylated at different sites allowing the control of the mRNA stability?
Both.
792
Can AUF1 or HuR be alternatively spliced allowing the control of mRNA stability?
AUF1.
793
AUF1 and HuR can come in different isoforms allowing mRNA stability to be controlled. What stimulates the production of different isoforms?
A change in the signalling pathway.
794
What can nonsense mutations within an ORF generate?
Early stop codons which causes proteins to be truncated.
795
Truncated proteins can cause genetic defects meaning they are not normally expressed. how does the cell do this (2 ways)?
1. Through degradation by the proteasome complex as truncated protein do not fold correctly.
2. Through the use of quality control mechanisms that detect the early stop codons. This often involves the NMD pathway.
796
Are proteins with nonsense mutations slowly or rapidly degraded?
Rapidly.
797
What does the NMD pathway stand for?
The nonsense mediated decay pathway.
798
For the NMD Pathway to be active what has to happen?
The mRNA has to be actively translated.
799
The NMD pathway has to be activated by active tribulation. how does this work?
The early stop codon will be detected by the interactions between the ribosome and the termination codon aswell as the structure of the mRNP which will be placed over the 3' UTR. If the stop codon is not in the right place the ribosome may not interact with the mRNP domain triggering the transcript to be targeted to NMD.
800
Apart from when transcripts contain premature stop codons what else is NMD important from?
The elimination of nonproductively arranged immunoglobulin genes.
801
What can NMD do to normal genes?
Keep expression at low levels.
802
What triggers NMD?
Absence of interaction between the ribosome and the mRNP 3' domain.
803
What steps does regulation of expression pathways occur at?
Rate limiting steps.
804
At what level are most goes primarily regulated at?
Level of transcription.
805
What are four ways in which transcriptional control occur at?
Through alternative splicing, mRNA localisation, translational control and mRNA degradation.
