Molecular biology semester 1 Flashcards
(639 cards)
1
Q
Devires, Correns and Tschermak repeated Medels work. Together they confirmed what?
A
- the difference between phenotype and genotype.
- Factors which influence traits are passed on through generations.
- Heredity determinants stay the same between generations.
- Some form of material carries this information.
2
Q
Derives, Correns and Tschermak knew that there was a material that carried genetic information. What did they know that this material must be able to do?
A
- Must be able to be stored.
- Must be also be replicated.
- Must be able to allow variance.
- Must be able to express itself.
3
Q
Miescher discovered what from hospital bandages covered in puss?
A
Nuclein.
4
Q
Why did Miescher knwo that the substance he found on bandages was not protein?
A
It contained no sulphur.
5
Q
What 2 things did Walther Flemming discover via his work on salamander cells?
A
Chromatin and mitosis.
6
Q
What did Boverti discover from looking at ascaris embryos?
A
Meiosis.
7
Q
What did Sutton discover from looking at grasshoppers?
A
That different combinations of chromosomes lead to different stereotypes.
8
Q
Who came up with the theory of inheritance?
A
Sutton and Boverti.
9
Q
What does the theory of inheritance consist off?
A
That chromosomes were needed for embryonic development and ‘factors’ lay among these chromosomes. It coincides with Mendels second law.
10
Q
What did Thomas Hunt Morgan discover regarding phenotypes?
A
He discovered the recessive phenotype in Drosphilla leading to a whit eyed mutant.
11
Q
Apart from discovering the recessive phenotype, what else did Thomas Hunt Morgan discover?
A
That genes lie on chromosomes. He was also the first to use linkage maps.
12
Q
What disorder did Garrod study in his work?
A
Alkaptonuria- a disorder where the patient has black urine and painful joints.
13
Q
Who proposed for the first time that disease acting in a Mendelian way?
A
Archibald Garrod.
14
Q
Archibald Garrod disovered that disease acted in a Mendelian fashion. What else did he discover in regards to genes and metabolism?
A
He discovered that genes were linked to specific defects and that you can have inborn errors in metabolism.
15
Q
Who came up with the ‘One gene one enzyme’ hypothesis?
A
George Beadle and Edward Tatum.
16
Q
What was the main question answered in the ‘One gene one enzyme hypothesis?’
A
‘Is there a link between genes and the enzymes responsible in the metabolic processes of Neurospora casa?
17
Q
What pathway was looked at in the ‘One gene one enzyme’ hypothesis?
A
The production of niacin from tryptophan.
18
Q
What can auxotrophic mutants not grow on?
A
Minimal growth medium.
19
Q
When was the auxotophic mutant able to grow?
A
When Niacin and 3-hydroxyanthranilic acid where added to the medium.
20
Q
What step did the mutant prohibit in the ‘One gene one enzyme’ hypothesis?
A
The synthesis of 3-hydroxyanthranilic acid from kynureine.
21
Q
What was crossed to prove the ‘One gene one enzyme hypothesis?’
A
WT and mutant. There was a one to one segregation proving Mendelian inheritance.
22
Q
What did the transforming principle show?
A
That DNA was the genetic material.
23
Q
Who originally came up with the transforming principle?
A
Fredrick Griffith.
24
Q
Why is the S strain of Strepoccus pneumoniae virulent while the R strain is not?
A
The s strain is smooth as it contains a polysaccharides coat. This protects it from the hosts immune system. the R strain is rough and does not have this coat.
25
When the mouse was treated with heat killed S and live R did the mouse survive and why?
The mouse did not survive. Some form of material had allowed R to transform into the S strain and kill the mouse.
26
It was originally not known what caused the transforming principle. Polysaccharides, lipids, RNA and DNA were also suggested. Why did people think it was not DNA?
They thought that DNA was too simple.
27
Who finally solved the transforming principle by proving that DNA was in fact the genetic material which had passed from the R stain to the S strain?
Oswald Avery, Collin Macleod, Macyln Mccarthy.
28
How was it shown that DNA was what allowed the transforming principle to happen?
Different components of the S strain were destroyed in turn and the mouse was the inoculated with the modified strain. When RNA, Lipids and Polysaccharides were destroyed the mouse died. When DNA was destroyed the mouse lived. This proved that DNA was the heredity material.
29
If repeated would the R strain always become virulent when added to a heat killed S strain?
No. It only becomes virulent if it takes up the genes needed for the coat. It is now known that the virulent DNA taken up would have replaced its non virulent counterparts.
30
What did Hershey and Chase confirm?
That DNA was the genetic material.
31
What bacteriophage did Hershey and Chase work with?
T2 phage. This can attach to the host with tail fibes.
32
What two radioactive elements did Hershey and Chase use to label the protein and the DNA?
S35 and P32.
33
What was present in the supernatent and what was present in the pellet when the T2 phage and the Ecoli were centrifuged?
The phage ghost was present in the suprnatent and the bacterium was present in the pellet.
34
The Tobacco Mosaic Virus causes lessions on leaves. It was used by Fraenkel- Conrat and Singer to show what?
That some viruses contain RNA instead of DNA.
35
The protein and RNA were separated in the labs and combinations of the subunits were painted onto leaves. When did the leaves become infected?
When the RNA was painted onto the leaves and when the reconstructed virus was painted onto leaves. It was also infected when the normal TMV was painted on.
36
A hybrid virus of TMV and _____was used in the second experiment by Conrat and Singer.
HRV - Holmes Ribgrass Virus.
37
Was the coat of TMV or HRV used in the hybrid virus?
TMV.
38
What happened when the leaf was infected by the HRV without the presence of the TMV coat and what did this show?
The leaf was infected and the HRV recovered has a HRV coat. This proved that RNA could direct the production of protein subunits.
39
Are purines or pyramides bigger?
Purines.
40
Who discovered thymine?
Kossel. He also discovered that purines are bigger than pyramides.
41
Levene did not believe that DNA was the genetic material as he thought that it was to simple. What did he discover that turned out to be correct?
He identified the sugars found in nucleotides.
42
Who determined that the ratio of purines and pyamides is the same?
Chargaff.
43
All organisms have the same amount of A=T. true or false ?
False. Some are more AT rich then others.
44
Wilkinson and Franklin discovered that DNA has a regular helix pattern with a turn of 3.5nm. How did they do this?
X ray crystallography.
45
Which base pair is stronger and why?
G=C as it forms three bonds. A=T only forms two.
46
What bond is formed in the major and minor groove?
Glycosidic bond.
47
The center of a DNA molecule is ________.
Hydrophillic.
48
Why does DNA have grooves?
Because the bonds that join the sugar phosphate backbone are not perfectly opposite to each other.
49
How often is there a helical turn in DNA?
Every 10.5bp.
50
Who determined the type of replication used?
Meelson and Stanls.
51
What three models of replication did Meelson and Stanls test?
Conservative, semi conservative and dispersed.
52
In Meelson and Stanls test did they grow the DNA in heavy or light nitrogenfirst?
Heavy.
53
What where the DNA strands centrifuged with in Meelson and Stanls experiment?
Caesium chloride. Heavier strands settled at the bottom and light at the top.
54
How many bands where present in Meelson and Stanls experiment after one generation and what did this mean?
One. This meant it could not be conservative.
55
After two generations how many bands where present in Meelson and Stanls experiment? What did this mean?
Two bands where present (hybrid and light). This meant that replication had to be semi conservative and not dispersed as dispersed only had one band present.
56
What happened to the hybrid band in the dispersed model throughput generations?
It got lighter and lighter.
57
What did John Cairnes do?
Determined the origin of replication in E.coil.
58
John Cairnes used radioactive labels which he visualised with autoradiography. What did this show DNA replication to be in E.coli?
Bidirectional.
59
How did John Cairnes confirm semi conservative replication?
There were two labelled strands in the second generation.
60
How many replication forks are there in E.coli?
2.
61
How many origins of replication are there in Eukaryotes?
Many.
62
What did Arthur Kornberg use to study polymerase activity?
A free cell system.
63
Kornberg separated the proteins in the bacterial cell by electrical charge. By doing this he increased the activity of polymerase by _____ fold.
2000.
64
What did Kornberg assay the DNA polymerase enriched extract with?
Template DNA, Mg2+ and radiolabelled nucleotides.
65
What did Kornberg use to locate long pieces of DNA?
Radioactive markers.
66
In Kornberg's experiment which strand was radioactive?
The new strand.
67
What four things did Kornberg confirm with his experiment?
1. A free 3' end is needed for replication
2. The DNA needs to be double stranded.
3. DNA is made 5' to 3'
4. All four nucleotides are needed along with cofactor Mg2+.
68
What are the three distinct regions in DNAP1?
1. DNA synthesis domain.
2. 3'--> 5' proofreading exonuclease domain.
3. 5'-->3' primer removal exonuclease domain.
69
What enzyme makes primers?
Primase.
70
What is the role of tropisomerase in DNA replication?
Binds ahead of the replication fork causing and relives the strain placed on it as it unravels.
71
What is the role of helicase in DNA replication?
It breaks the hydrogen bonds between the 2 strands allowing the DNA to unwind and from a replication fork.
72
What is the role of single stranded binding proteins (SSB'S) in DNA replication?
They prevent re annealing of the separate strands by binding to the individual strands and stabilising them.
73
What is the main polymerase used in DNA replication?
DNAP3.
74
What is the main polymerase used in DNA repair?
DNAP1.
75
In addition to DNAP1 and DNAP3 how many other polymerases are there?
3.
76
There is _______ replication of the leading DNA strand and _______ replication of the lagging strand. ______ _______ are found in the lagging strand. The lagging opposes the fork movement.
Continuous, discontinuous, Okazaki fragments.
77
What is RNP?
A ribonuceloprotien (RNA bound to protein.)
78
What two things can stabilise the RNA structure?
1. Base pairing.
| 2. Base stacking interactions.
79
RNA is folded through intramolecular base paining into what?
Short double stranded stem loops.
80
What do short RNA helices often form?
Stem loops and hairpins.
81
________ interactions in RNA define its 3D shape,
Tertiary.
82
What are the 3 possible tertiary interactions found in RNA?
1. Long range base pairing
2. Coxial stacking of helices ( resulting in a longer helix.)
3. A minor motif.
83
Is the major groove of RNA deeper and narrower than that of B form DNA or shallower and broader?
Deeper and narrower?
84
Is the minor groove of RNA deeper and narrower than that of B form DNA or shallower and broader?
Shallower and broader.
85
What groove do RNA binding proteins usually bind to?
The minor groove.
86
RNA binding proteins include divalent metal cations. Why do they bind in the minor groove?
They interact with the phosphodiester backbone to equal out the charge of the phosphate.
87
Some nucleotides are post transcriptionally modified, especially __RNA.
t.
88
What noncanocial base pair is known as the 'wobble' base pair?
G-U.
89
Is the GU or GA base pair more common?
GA.
90
Is '1,6' relevant to the purines or the pyramides?
Purines.
2,3,4 is relevant to the pyramides.
91
Can DNA or RNA contain base triplets?
RNA.
92
Why is it hard to predict the 3D structure of RNA?
As noncanocial base pairs can form.
93
What are tetraloops?
Four RNA nucleotides stacked on top of each other.
94
Loops in RNA are always four nucleotides in size. True or false?
False. They can be larger.
95
Why are noncanocial base pair interactions important in RNA?
They can alter the dimension if the RNA helix which is important in specific binding interactions. This can including the binding of the second elongation factor.
96
What percentage of cellular RNA is mRNA?
5%.
97
What percentage of cellular RNA is rRNA?
75%.
98
What percentage of cellular RNA is tRNA?
10%.
99
Apart from mRNA, tRNA and rRNA there are many other small rnas within the cell. What are four examples of these?
1. Small nuclear RNA
2. Small nucleolar RNA
3. micro RNA
4. regulatory RNA.
100
What are all RNAs transcribed as?
Larger precussor molecules with are subsequently processed into mature functional RNAs.
101
What cleaves specific structures from within the RNA?
endoribonuclease.
102
Where do exoribonucleases degrade the RNA from?
The free end of the molecule.
103
What are the proteins called that target polymerase to the promoter regions of specific genes?
Sigma factors.
104
Does RNAP require additional primers or helicases?
No.
105
Where are the two promoter regions found in E.coli?
35 and 10 nucleotides upstream.
106
What do highly conserved/ highly transcribed genes have in regards to their promoter?
A promoter that highly matches the consensus sequence of the 35 and 10(pribnow) boxes.
107
Is the promoter 35 nucleotides upstream or 10 nucleotides upstream in E.coli called the pribnow box?
10 nucleotides.
108
Why is the transcription of RNA an essentially irreversible reaction?
As the phosphate produced when a nucleotide is added to the chain is irreversibly hydrolysed.
109
How many core subunits are there in RNAP found in E.coli and what are they?
alpha, alpha, gamma, beta, beta'.
110
Where is the DNA binding groove found in RNAP found in E.coli?
Between the beta and beta' subunits.
111
What subunits are catalytic in RNAP found in E.coli?
Beta/ beta'.
112
What subunits bind transcription factors in the RNAP found in E.coli?
Alpha.
113
What is the purpose of the gamma subunit in the RNAP from E.coli?
It allows assembly and stability.
114
Apart from increasing the affinity to promotors, what can sigma factors do?
Decrease non specific DNA binding.
115
What is a holenzyme?
An RNA polymerase bound to a transcription factor.
116
What sigma factor is used most in E.coli?
70.
117
What sigma factor is involved in heatshock?
32.
118
What sigma factor is involved in nitrogen metabolism?
54.
119
What did this experiment show?
A filter binding assay of protein and radiolabelled DNA was set up. the filter trapped protein which bound to DNA at a varying rate. You can visualise where the DNA is due to its radioactivity. When unlabelled DNA was added it formed a DNA/protein complex on the rate dependant of the breakdown the radioactive DNA/protein complex. The rate of breakdown was much slower when a ______ was used instead of ______.
It showed that sigma factors allow tighter binding of RNAP to DNA.
Haloenzyme
RNAP.
120
What three things are involved in transcription initiation in prokaryotes?
1. Promoter binding
2. DNA unwinding
3. Primer synthesis.
121
In prokaryote transcription initiation what happens when the primer reaches 18 nucleotides long?
The alpha subunit of RNAP is released (which binds transcription factors) and the NUSA protein binds.
122
Why does the NUSA protein bind during transcription initiation in E.coli?
It imparts processivity to the RNAP.
123
What two things can the NUSA protein prevent in prokaryote transcription?
1. Polymerase stalling.
| 2. Premature transcription termination.
124
What two methods of transcription termination are present in prokaryotes?
1. Intristic termination.
| 2. rho dependant termination.
125
There are two methods of transcription termination in prokaryotes. What do they both involve?
Destabilisation of the RNA/DNA heteroduplex with the active site of RNAP. This allows the RNA to dissociate from the RNA/DNA complex.
126
What structural characteristic of RNA allows for intrinsic termination in prokaryotes?
The stem loop structure with a GC rich stable base region.
127
What base pairs destabilise the RNA/DNA structure?
ru-da.
128
Where does the polymerase pause in intrinsic termination in prokaryotes?
The hairpin structure.
129
What method of transcription termination in prokaryotes is ATP dependant as it involves an ATP dependant helicase?
rho-dependant termination.
130
What terminator sequence allows rho to bind in termination in prokaryotes?
C rich terminator sequences.
131
What happens to the polymerase when rho initially binds to the c rich terminator sequence?
It pauses.
132
Why can rho unwind base pairing between the RNA and DNA?
It has helicase activity.
133
What has a hexameric ATPase ring structure which can open to allow threading?
rho.
134
At what step in rho dependant termination is ATP hydrolysed?
When rho meets RNAP.
135
In what type of organism does compartmentalisation occur?
Eukaryotes.
Compartmentalisation is when transcription and translation occur separately.
136
How many distinct RNA polymerases are there in eukaryotes?
3.
137
Which of the eukaryote RNA polymerases contains a CDA tail?
pol2.
138
What does RNAP1 code for in eukaryotes?
rRNA.
139
What does RNAP2 code for in eukaryotes?
mRNA.
140
What does RNAP3 code for in eukaryotes?
5s, tRNA.
141
How many specific regions does RNAP1 encode for in eukaryotes?
5.
142
How many specific regions does RNAP2encode for in eukaryotes?
3.
143
How many specific regions does RNAP3 encode for in eukaryotes?
7.
144
Mitrochondrial and chloroplast genes are encoded for by RNA polymerase 1. True of False?
False. They have their own polymerases.
145
What do eukaryotes lack that prokaryotes do not?
Sigma factors.
146
Eukaryotes do not have sigma factors. What do they use instead?
Transcription factors.
147
What facilities the assembly of the preinitation complex on the promoter of polymerase 2?
General transcription factors.
148
What does the preinitiation complex allow?
RNAP2 to bind to the right place.
149
What is TBP also known as?
The TATA box binding protein?
150
Where is TBP found?
In TFIID.
151
The TBP is found in TFIID. What does this bind to in transcription initiation in eukaryotes?
The DNA.
152
Multiple Transcription factors bind once the TBP in TFIID has bound to the DNA. What are these transcription factors and in what order do they bind in?
TFIIB, TFIIF, TFIIE, TFIIH
153
The binding of what transcription factors allows RNAP2 to bind to the DNA in transcription initiation in eukaryotes?
TF11F.
154
Do eukaryotes use intrinsic termination, rho dependant termination or the torpedo model of termination to stop transcription?
The torpedo model.
155
What is RNAP2 termination coupled to in eukaryotes?
3' mRNA processing.
156
In eukaryotes transcription is coupled to 3' mRNA processing. What carries out this processing?
Cleavage/polyadenylation complex.
157
What does cleavage by the cleavage/polyadneylation complex allow in transcription termination in eukaryotes?
The downstream fragment to be degraded 5' to 3' by the exonuclease Xrn2.
158
What exonuclease allows very quick degradation the downstream fragment in the torpeado model of termination?
Xrn2.
159
Allow compartmentalisation is slower what does it allow?
Nuclear processing event. This means that there can be greater diversity in the mRNA's produced.
160
What does CTD on RNAP2 contain?
Tandem repeats of serine rich heptapeptide.
161
How does the CTD tail on RNAP2 change during transcription?
Serine phosphorylation is varied.
162
How is the CTD tail phosphorylated when capping occurs?
It is phosphorylated at position 5.
163
How is the CTD tail phosphorylated when polyadneylation occurs?
It is phosphorylated at position 2.
164
What does the varying pattern of phosphorylation in the CTD tail allow transcription to be coordinated with?
RNA processing.
165
How does RNAP3 terminate transcription in eukaryotes
With stretches of T's. This is similar to intristic termination in prokaryotes.
166
Can slowing down cause RNAP's to dissociate?
Yes.
167
Translation can not happen at varying rates. True or False?
False.
168
Why does capping occur to the 5' end of mRNA transcripts in eukaryotes?
So the ends are not susceptible to exonucleases.
169
What process must occur before polyadneylation to expose the 3' end of the mRNA transcript?
Splicing.
170
What post transcriptional modification of mRNA happens first?
5' capping.
171
What is a ribonucleoprotein complex also known as?
A splicesome.
172
In eukaryotes do mRNA's encode a single peptide?
Yes.
173
Is the poly(A) tail transcribed?
No.
174
What often ends up degrading the poly(A) tail in eukaryotes?
Xrn2.
175
The cleavage/polyadenylation complex carries out polyadenylation. What two types of factors does it contain to allow it to do this?
1. Cleavage factors.
| 2. Specificity factors.
176
RNA processing reactions mainly take place in the cytoplasm. True or false?
False. They mainly take place in the nucleus.
177
Where is it thought that the splicesome could have evolved from?
Autocatalytic activity of self splicing ribozymes.
| A ribozyme is an RNA that can splice itself.
178
Does the cap structure, poly(A) tail or both prevent degradation?
Both.
179
Apart from preventing degradation, what is the purpose of the cap and poly(A) tail added to the mRNA after transcription?
They can help to promote translation.
180
What does the UTR stand for?
Untranslated region. These are found either side of the ORF.
181
What linkage is found in the mRNA cap structure that is not found anywhere else in nature?
5'-5' triphosphate linkage.
182
What is added to all mRNA and pol2 transcripts that is methylated in the n7 position?
A guanosine residue.
183
The poly(A) tail is the same length in all organisms. True or false?
False, the length varies. In humans it is around 200 and in yeast it is around 700.
184
When will a mRNA no longer be used?
When the poly(A) tail has become to short. The tail degrades throughout its lifetime.
185
What cleavage/polyadneylation complex processes the 3' end of the mRNA. What enzyme actually adds on template adenylates to the transcript?
Poly(A) polymerase.
186
What is the consensus sequence which allows polyadenylation at the 3' end?
AAUAAA.
187
Is Poly(A) polymerase DNA dependant or independent?
Independent. RNAP is an example of a DNA dependant polymerase.
188
ATP is not required for post translation modification of eukaryotic mRNA. True or False?
False, it is required for polyadneylation.
189
What did hybridisation of DNA and mature mRNA show?
That some parts of the DNA were removed (exons). This was shown when DNA and mature mRNA were annealed to each other which resulted in there being large loops of DNA not annealed to mRNA.
190
There is 5 times more exon than intron. True or false?
False. There is 5x more intron.
191
What are the three highly conserved splicesites in mRNA called?
5' splicesite, branchpoint and the 3' splicesite.
192
Which splicesite is the flowing?
| GU
5' splicesite.
193
Which splicesite is the flowing?
| AC
Branchpoint.
194
Which splicesite is the flowing?
| AG
3' splicesite.
195
What assembles the splicesome?
SNURP's.
196
What are SNURP's?
Small nuclear RNPs.
197
How many distinct ribonucleic particles, involving small nuclear RNA, does a SNURP contain?
5.
198
Introns are released in the form of a ______.
Lariat.
199
Dos the splicesome assemble on the pre mRNA intron or exon?
Intron
200
How many transesterfication reactions does splicing involve?
2.
201
Does splicing require ATP?
No.
202
What is the first step of splicing?
2' hydroxyl group on branchpoint adenosine attacks the 3' phosphate on the 5' exon. This forms a 5'-2' phosphodiester bond which gives rise to a looped lariat. This allows the release of the 5' exon.
203
What is the second step of splicing?
The generated 3' hydroxyl group attacks the 5' phosphate on the 3' exon. This releases the lariat and joins the two exons.
204
What is the significance of Tetrahymena thermophila in transcription?
it contains self splicing RNA.
This was shown in vitro in the absence of protein.
205
In what way are the introns in Tetrahymena thermophila similar to nuclear pre-RNA?
The intron undergoes two transesterifciation reactions.
206
Where is it thought that nuclear pre-RNA evolved from?
Self splicing introns, such as those found in Tetrahymena thermophila.
207
What is the definition of gene expression?
A process in which information from a gene is synthesised into a functional gene product.
208
Why is gene expression very important? (3 reasons)?
1. Avoids chaos
2. Less energy wasted/ better use of resources
3. Allows response to environmental change.
209
What type of control is an instant response?
Fine control.
210
What can fine control do to proteins?
Make them functional/ non functional.
211
What can fine control do to enzymes?
Turn them on and off.
212
Fine control can make proteins functional/non functional and can turn enzymes on/off. What can bring about these changes?
Covalently bounded ligands.
213
What does irreversible fine control involve?
Proteolytic processing.
214
In reversible fine control ligands can covalently bind to molecules. What else can happen which also brings about a change in gene expression?
Specific amino acids can have functional groups attached or removed.
215
Phosphorylation, acetylation and ubiquitination, allosterism and feedback inhibition are all examples of what?
Reversible modifications involved in instant responses/ course control.
216
Acetylation is an example of a reversible instant response involved in fine control. Where does this acyl group come from?
Acetyl coA.
217
Ubiquitination is an example of a reversible instant response involved in fine control. The donor molecule is ubiquitin. What is the main purpose of ubiquitination?
It tags proteins to be degraded. It is involved in cyclin and cell cycle determination.
218
What enzyme turns the pyruvate dehydrogenase complex on?
Enzyme PDH phosphatase.
219
What enzymes turns the pyruvate dehyrodgenase complex off?
Enzyme PDH kinase.
220
What type of reversible modification involves the pyruvate dehydrogenase complex?
Phosphorylation.
221
When the pyruvate dehydrogenase complex is bound to a phosphate group is it active or inactive?
Inactive.
222
What is allosterism?
Reversible ligand binding.
223
In allosterism an inhibitor can bind to the active site. What other type of molecule is involved in allosterism and can influence enzyme activity?
An enhancer.
224
What happens in feedback inhibition ( an example of reversible fine control)?
The products of a reaction interact with the enzymes active site to ensure that too much of something isn't made. It also slows down the overall reaction.
225
DHAP catalysing the first step of aromatic amino acid biosynthesis is an example of what type of reversible fine control?
Feedback inhibition. The aromatic amino acids can inhibit the pathways.
226
What type of control are delayed responses also known as?
Course control.
227
What type of gene expression control results in slower changes that are long term?
Course control.
228
Is the regulation in the amount of proteins an example of course control or fine control?
Course control.
229
Course control involves long term negative responses. True or False?
False. it can also involve long term positive responses.
230
Course control can occur at different levels. What level of control is more important in prokaryotes?
Transcriptional.
231
What happens at the 5' end of mRNA in prokaryotes as soon as it becomes available?
A ribosome will attach.
232
In prokaryotes can multiple ribosomes attach to as single mRNA transcript?
Yes.
233
If the sequence is exactly the same in multiple sequences/places is it said to be conserved sequence or a consensus sequence?
A conserved sequence. a consensus sequence has a high frequency of similarity.
234
What do DNA binding proteins do?
Regulate the rate in which RNAP binds to the promoter in prokaryotes.
235
How do DNA binding proteins bind to the DNA.
Through DNA recognition motifs- the most common of these being the helix turn helix.
236
What part of the DNA do DNA binding proteins bind to?
Major groove.
237
What does each helix turn helix motif contain?
A recognition helix.
238
The amino acid side chains of the recognition helix interact with what?
Exposed base pairs on the DNA.
239
DNA binding protiens can bind to DNA via common motifs, such as the helix turn helix. These can bind to DNA via the side chains on the recognition helix and exposed base pairs. What happens next?
A second helix lies on top to help position the recognition helix. It can also increase the binding affinity.
240
Positive control with DNA binding proteins increases the rate of transcription. True or false?
True.
Negative control will decrease the rate of transcription.
241
How many genes are there approximately in the E.coli genome?
400-600 genes.
242
Around 4000 proteins can be encoded for by E.coli. How many will be encoded for at a given time?
600-800.
243
When are constitutively expressed genes expressed?
Always. Their genes and enzymes are always needed.
244
What is the role of housekeeping genes?
They encode proteins that help with maintenance.
245
Housekeeping genes are important but are not constitutively expressed. True or false?
False. They tend to be constitutively expressed, at least in a tissue specific manner.
246
it is assumed that the expression of housekeeping genes is unaffected by what?
Experimental conditions.
247
The expression of facultative/responsive/adaptive genes depends on what?
Environmental stimuli.
248
Can inducible genes be switched on or off?
On.
249
Can repressible genes be switched on or off?
Off. (They are also off when transcription is prevented.)
250
Gene expression can be looked at from the mRNA level or from the protein level. Which is more important?
Protein level, however looking at the at the mRNA level is easier.
251
What 6 things can influence the level of protein production?
1. The number of genes for that protein in the genome.
2. Time spent in transcription/translation.
3. Promotor efficiency.
4. Promoter strength.
5. Variable strength of the ribosomal binding site.
6. The half life of the mRNA made.
252
Why is EFTU expressed often expressed at a different level to other proteins in the E.coil genome?
There are 2 copies of the EFTU gene in the genome. Most genes only have one copy.
253
What causes variation in promoter strength?
Variation in the promoter sequence.
254
What sequence varies to distinguish the strength of the ribosomal binding site?
The shine delgaro sequence (this helps recruit rRNA). Ribosomal binding sites that greatly match the shine delgaro sequence will bind with more strength and efficiency to the rRNA that weaker sequences would.
255
What is this sequence also known as ?
UAAGGAG.
The shine delgaro sequence.
256
Prokaryotic mRNA is very stable. True or false?F
False. They are metabolically unstable and have a very short half life- normally less than 10 minutes.
257
What did Barbara McClintock discover through her work in maize seeds?
Transposons.
258
What are transposons?
Small pieces of DNA that can insert itself into another place in the genome where it reversibly changes the expression of other genes.
259
Transposons reversibly change the expression of other genes, however is this there sole purpose?
Yes. They are only involved in expression and regulation.
260
What did James Monard, Francois Jacob and Andre Lwoff discover?
Operons.
261
Are regulatory proteins encoded separately or together in the genome?
Separately.
262
What do regulatory proteins influence the behaviour off?
RNA polymerase.
263
Regulatory proteins bind to one specific promoter. True or false?
False. They can bind to multiple promoters.
264
What determines the concentration of regulatory proteins?
Binding of specific ligands.
265
Regulatory proteins have many purposes, including informing the cell of any new carbon sources and they can influence the transcription rates of many genes. Do they use positive regulation, negative regulation or both?
Both. They can both increase or decrease the rate of transcription.
266
What is a regulon?
Multiple genes (or operons) at different locations in the genome under coordinate control. The protein products of these genes/operons are normally all related to a specific pathway or function.
267
Do operons making up a regulon all have the same promoter?
They can do, but they can also all have a different promoters.
268
What was the first regulon to be described?
The arginine regulon.
269
What is converted into arginine by the arginine regulon?
Glutamate.
270
How many steps are involved in the synthesis of arginine from glutamate?
8.
271
If arginine is added to a system expressing the arginine regulon there is ___ fold reduction in the amount of arginine produced.
10. If arginine is removed there the production goes back to normal within a minute.
272
What repressor is found at the start of all arg genes?
argR.
273
What is the arginine regulatory gene called?
argR (underlined).
274
If there are mutations in the argR repressor what happens?
The gene is constitutively expressed and arginine is always produced.
275
When arginine is present the argR repressor is ______ expressed. This binds to the argR gene and expression is repressed.
Constitutively.
276
In arginine biosynthesis via the arginine regulon what does arginine act as when it is present?
A co repressor.
277
What type of repressor can the argR repressor be described as and why?
A aporepressor as it is only activated when bound to arginine.
278
What is the promoter region on genes involved in arginine synthesis called?
The ARG box. This is where argR can bind, if it is bound to arginine.
279
What is the ARG box made up of?
a 218bp palindromic sequence.
280
What properties of the ARG box can vary to cause differential expression in the ARG genes?
Its location and any variation in its sequence.
281
The location and sequence of the ARG box can alter arg gene expression. Why?
They help control how strongly argR (bound to arginine) binds/ how much activity the repressor impairs.
282
What two arg genes are identical?
E and F.
283
Why is the synthesis of arginine never fully repressed?
As the genes carA and carB are involved in the conversion of ornithine to cysteine, which is involved in the conversion of glutamate into arginine. As these are not arg genes they are not controlled by argR.
284
All arg genes are single genes apart from 4 which are on the same operon. What are these?
E-C-B-H.
285
All arg genes are co-ordinately expressed, but are they transcribed in equal amounts?
No.
286
What technique was used to show differential expression in arg genes with and without arginine present?
Microarray. This involves looking at mRNAs produced when arginine is/isn't present.
287
Are all genes in the arg regulon directly involved in the production of arginine?
No. Some are involved in transport and other related events. There is also a possibility that some genes involved in the arginine production are yet to be discovered.
288
What sigma factor has the highest affinity to gram negative bacteria?
sigma 70.
289
What does a sigma factor have to be in to displace sigma 70 from RNAP in gram negative bacteria?
Higher concentrations than sigma 70.
290
What sigma factor is involved in flagella formation?
Sigma 28.
291
What temperature change will induce the production of 20 proteins which will go on to protect host celluar proteins?
37 degrees- 42 degrees.
292
Chaperon proteins can be found all over the genome. What four things can they do related to the heatshock response?
1. Support protein folding
2. Prevent cellular degradation
3. Change membrane stability
4. Control some enzymes.
293
Is sigma 70 regulated at transcriptional level or at a translational level?
Translational level.
294
The secondary structure of sigma 32 is found in the mRNA that encodes for sigma 32. What is this mRNA called?
rpoh mRNA.
295
The secondary structure of sigma 32 is found in the gene that encodes it: the rpoh gene. What effect does this have on the production of sigma 32?
The production of sigma 32 is dramatically decreased as its secondary structure in the rpoh transcript blocks ribsome access.
296
What change occurs to the rpoh gene when the temperature rises?
The secondary structure of sigma32 found in the rpoh gene melts. This allows ribosomal access and the production of more sigma 32. This process is very rapid.
297
What is involved in the primary mechanism of sigma 32 regulation?
The presence of the secondary structure of sigma 32 in the mRNA that encodes it (rpoh).
298
What is involved in the secondary mechanism of sigma 32 production, in place when the temperature is 37 degrees ?
1. Chaperons binding to and inhibiting sigma 32 from activating heatschock proteins.
2. Proteases degrading heat shock proteins.
299
What happens to the secondary mechanisms of sigma 32 production when the temperature rises to 42 degrees?
Chaperons dissociate from the sigma 32 as they are needed to help prevent protein degradation. The level of active sigma 32 rises dramatically due to the primary mechanism.
300
What can sigma 32 activate when the temperature increases from 37 degrees to 42 degrees?
Heat shock proteins involved in the heat shock response.
301
SOS regulation in bacteria involves the expression of how many independent genes?
40+.
302
SOS regulation involves the expression of 40+ independent genes. What are the 5 things these genes are involved in?
1. Protection
2. Repairs
3. Replication
4. Mutagenesis
5. Metabolism of DNA.
303
What are the two key proteins involved in SOS regulation?
LexA and RecA.
304
What type of protein is LexA?
A repressor protein.
305
Where does RexA bind?
To SOS boxes.
306
In normal conditions there is no expression of the SOS box due to the presence of LexA. True or False?
False. There is often still Basel levels of transcription.
307
What does RecA have a role in?
DNA repair.
308
Can LexA or RecA function as a protease?
RecA.
309
What activates RecA?
ssDNA. RecA forms filaments with ssDNA which activates it.
310
LexA is activated by RecA. What does this allow it to do?
Cleave itself.
311
What determines how tightly LexA binds to the SOS box?
The sequence in the SOS box.
312
Where is LexA removed from first?
SOS boxes with a weak consensus.
313
LexA is first removed from SOS boxes with a weak consensus. What are the genes normally transcribed by these boxes involved in?
Nucleotide extension repair.
314
Does Nucleotide extension repair allow for high fidelity repair?
Yes.
315
Is urvA encoded for by weak or strong SOS boxes?
Weak.
316
Is umc encoded for by weak or strong SOS boxes?
Strong.
317
After weak SOS boxes have been expressed stronger SOS boxes will always been expressed. True or false?
False. Strong SOS boxes will only be expressed if the response from the weak boxes was not sufficient to repair the damage.
318
What are the genes encoded for by strong SOS boxes involved in?
Mutagenic repair.
319
SuIA is encoded for by strong SOS boxes. What does it prevent?
Cell division. It does this by fillimentation and and induction of umudc dependant mutagenic repair.
320
Sigma factors levels can be regulated through altering levels of transcription and translation. What else can regulate levels of sigma factors?
Proteolysis of levels of sigma factors.
321
The activity of sigma factors can only be regulated by the binding of anti sigma factors and not through ligand binding. True or false?
False. Ligand binding can also change the activity of sigma factors.
322
Different sigma factors are active throughout the sporulation process. This allows the cell to enter the sporulation and then stay in its vegetative state. Through controlling gene expression sort of cell division will sigma factors ultimately allow in a sporylating cell?
Asymmetric cell division.
323
In asymmetric cell division a septum forms at one end of the cell. What does this trap?
Some of the forespores chromosome in the mothers cell.
324
What allows a cell to sporylate?
The presence of alternative sigma factors.
325
What does the forespore have to develop in order to become independent from the mother?
A protective cortex and coat,
326
What happens to the mother in asymmetric cell division?
She sacrifices herself for the daughter cell. The mother cell will lysis and die.
327
When will a bacterial cell enter a vegetative cycle?
When the conditions are unfavourable for binary fission.
328
Where is sigma F expressed?
In the forspore compartment.
329
Where is sigma E expressed?
In the mothers compartment.
330
Where is sigma G expressed?
In the forspore compartment.
331
Where is sigma K expressed?
In the mothers compartment.
332
Which is the correct order for sigma factor activation is sporulation?
1. FEKG
2. FKEG
3. FEGK
4. FGEK
FEGK.
333
What is SPOIIAB?
An anti-sigma factor.
334
What can SPOIIAB bind to?
sigma F
335
What is the purpose of SPOIIAB?
It ia an antisigma factor that holds sigma F inactive in the mothers compartment.
336
How is sigma F activated in the forspore compartment?
By the use of anti-anti-sigma factor SPOIIAA. This binds to anti-sigma factor SPOIIAB allowing sigma F to be activated.
337
What does a two component regulatory allow both +ve and -ve bacteria to do?
Respond to the external environment.
338
A two component regulatory systems involves the ______ protein ______ ______.
Sensory, histidine kinase.
339
Where is the sensory protein histidine kinase, involved in the two component regulatory system found?
Spanning the membrane. There is also a response regulator protein within the cell.
340
In the two component regulatory system when the sensory protein ______ ______detects a signal molecule it ________ a _______ residue on its _______ ________.
Histidine kinase, phosphorylates, histdine, intracellular domain.
341
In the two component regulatory system when the sensory protein histidine kinase detects a signal it phosphorylates a histidine residue on its intraceullar domain. A ______ ______ is then sent to a _______ molecule on the _______ _______ ______ which is then activated by ________ and will go to alter _____ ______.
Phospho relay, aspartate, response regulator protein, phosphorylation, gene expression.
342
What is the first component of the two component regulatory system?
Histidine kinase.
343
What is the second component of the two component regulatory system?
Aspartate residue on the response regulator protein.
344
What is Quorum sensing used for?
To alter gene expression.
345
What change is responded to in quorum sensing?
Population density.
346
What molecule, produced by bacteria, diffuses out of the cell and has little effect at low concentration. However at higher concentrations it can alter gene expression in other members of the population?
Autoinducer.
347
How does the density of an autoinducer molecule increase?
Through an increase in population.
348
What has to be reached in order for an autoinducer molecule to regulate gene expression in a population?
The threshold level.
349
What method of altering gene expression is used by Vibrio fischeri ( a type of bacteria).
Quorum sensing.
350
What organism has a symbiotic relationship with Vibrio fischheri bacteria?
Eupryma scolopes, The Hawaiian bobtailed squid.
351
What can Vibrio fischeri bacteria produce?
Light.
352
What are the 5 structural genes that encode LuxL in Vibrio fischeri?
Lux C, D, B, A ,E.
353
What does the gene Lux L encode for in Vibrio fischeri?
Enzyme Luciferase. This allows for the production of light.
354
What are the two regulatory egnes involved in the production of the enzyme luciferase?
LUXL and LUXR.
355
What autoinducer molecule is used in the Viberio fischei's light production?
HSL, Homoserine lactone.
356
What happens when the autoinducer molecule HSL binds to the regulatory gene LUXR?
Transcription is activated of the luciferase operon.
357
When there is a very high concentration of LUXR what type of feedback is used?
Negative.
358
How is quorum sensing used in Pseudomonas aeruginosa?
In the production of biofilms.
359
Why is quorum sensing useful in the production of virulence factors?
It allows the bacteria to multiply without the host knowing.
360
What initiation codon is used 99% of the time?
Methionine- AUG.
361
What does the 'wobble' phenomenon in the genetic code allow?
One tRNA molecule to recognise multiple codon sequences.
362
What are the 21st and 22nd amino acids?
Selenocysteine and pyrrolysine.
363
Are selenocysteine and pyrrolysine used as initiation or termination codons?
Termination.
364
There is one ORF per transcript. True or false?
False. There are multiple.
365
What does 16/mRNA base pairing select?
The right AUG initiation start codon.
366
What sequence is this?
AGGA.
The shine delgaro sequence.
367
What is found 12 nuceltoides upstream of the initiation AUG codon?
The shine delgaro sequence- AGGA.
368
Where do shine deglaro/ anti shine delgaro interactions position the initiation codon?
In the ribosomal P site.
369
Brenner and Crick showed that poly(u) RNA directed the synthesis of what?
Phenylalanine.
370
What did the following experiment show?
An E.coli extract that was active in translation was isolated. The mRNA was degraded and made inactive by RNAse. RNA was then added with amino acids, one of which was radiolabelled. The protein made was then precipitated with TCA and collected by filter binding.
It showed the process of translation. The rdiolaballed amino acid would only be present if the transcript contained the appropriate codon.
371
Francis and Crick proved that the genetic coding is read a series of overlapping triplets. They also showed that the code was non punctuated and degenerate. What did they analyse to do this?
Bacteriophages.
372
There are very few stop codons. True or false?
True.
373
When will aminoacyl-tRNA bind to the ribosome?
When its cognate codon is present.
374
How many sense codons are there?
61.
375
How many nonsense codons are there?
3.
376
What are meth and tryp examples of?
Amino acids that are no degenerate.
377
Are similar amino acids encoded for by similar codons?
Yes.
378
Do all organisms have 61 sense tRNA's?
No.
379
Many tRNA's have a modified nucleotide in the first position of the anticodon. If G is present in the first position what can it identify codons ending in?
C or U.
380
What can inosine base pair with?
A, C or U.
381
tRNA leu (IAG) can identify what 4 codons?
1. CUC
2. UUC
3. CUA
4. UUA
382
How are selonoproteins made?
By incorporating the amino acid selenocysteine.
383
Selenium is essential for many organisms but not humans. True or false?
False.
384
why is tRNA sec unusual?
It is unusually long and has its own elongation factor.
385
What can tRNA sec sometimes encode?
UGA (selenocysteine).
386
Pyrrolysin was first discovered in 2002. UAG can encode for pyrrolysine in some ______.
Archaebacteria.
387
How can you differentiate between different tRNA molecules?
By the enzyme responsible for amino aceytlation.
388
How many aminoacyl-tRNA synthases are there and how many are required for each incorporation?
There are 20. 1 is needed each time.
389
What is the second genetic code?
The ability of the enzyme aminoacyltransferases to charge the tRNA with the correct amino acid.
390
tRNA molecules are generated by longer precursor molecules. True or false?
True.
391
tRNA is generated by .longer precursor molecules. What enzyme generates the 5' end of the tRNA molecule via cleavage to create a phosphate group?
RNAaseP.
392
Is RNAseP and endoribonuclease or exoribonuclease?
Endoribonuclease.
393
Can endonucleases and exonucelases generate the 3' end of tRNA?
Yes.
394
What is the finally modification event in the production of a tRNA molecule?
The terminal sequence CCA is added to the 3' end by the enzyme tRNA nucleotidyltransferase.
395
What enzyme adds the terminal CCA sequence to a tRNA molecule at its 3' end?
tRNA nucleotidyltransferase.
396
How much of a tRNA molecule is spliced by tRNA ligases?
10%.
397
What type of RNA has a universally conserved cloverleaf secondary structure?
tRNA.
398
How many nucletoides is a tRNA molecule roughly made off?
75. Some have a variable arm with an additional helix making them slightly longer.
399
What are the four parts of a tRNA molecule?
1. Acceptor stem
2. D arm
3. Anticodon arm
4. TΨC arm.
400
What type of RNA have the 5' and 3' end drawn together?
tRNA.
401
tRNAs are extensively modified, and some modifcaitons such as D, TΨC. are found in most tRNAs. True or false?
True.
402
Where does the amino acid attach to on a tRNA molecule?
3' hydroxyl of the 3'A.
403
When do modifications of tRNA occur?
Post transcriptionally, they are carried out by enzymes.
404
What shape is tRNA folded into?
An L shape.
405
What makes the tRNA a flat L shape?
Coaxical stacking of helices and base pairing between ends of the TΨC and D loops. There can then be tertiary interactions between these two groups.
406
What is this process describing?
The amino acid group attaches to the 3' hydroxyl group of the terminal nucleotide within the tRNA via a covalent ester linkage.
Charging of tRNA's.
407
What does aminoacetylation generate?
AMP and pyrophosphate.
408
A single enzyme charges all isoacceptor tRNas. True or false?
True.
409
Aminoacyl-tRNA synthetases must recognise all isoacceptor tRNAS. To do this do they use positive identity elements, negative identity elements or both?
Both. The structural characteristics of the acceptor stem, and anticodon loops are examples of positive identity elements while modified nucleotides are eamples of negative elements.
410
Why is it difficult for enzymes to recognise the correct tRNA?
As the structure of all tRNA molecules is very similar.
411
During amino acid selection by the aminoacyl-tRNA synthetase, the difference in binding energy is not sufficient to allow adequate discrimination of cognate (correct) and non-cognate (incorrect) amino acids. True or false?
True.
412
What are the two functional regions of a tRNA molecule?
The anticodon loop and the aminoacyl loop.
413
How is the amino acid chemically trapped as an aminoacyladenylate group?
Through ATP hydrolysis via aminoacyl tRNA synthetase.
414
Once the amino acid is trapped as an aminoacyladenylate group what one of two things can happen to it?
1. It can be joined to the tRNA.
| 2. It can be dissociated from the enzyme.
415
For a cognate amino acid, is the reaction for tRNA aminoacylation faster or slower than the reaction allowing it to leave the enzyme?
Faster, which is why it will be able to bind.
416
Why does the aminoacyl tRNA synthetase have to hydrolyse ATP?
It brings the system out of equilibrium. This allows the amino acid group to be trapped.
417
Why is it not possible for a non-cognate amino acid to undergo tRNA aminoacylation?
Because it is not bound to the enzyme aminoacyl tRNA synthetase for long enough.
418
Where else are proofreading mechanisms found that are similar to tRNA charging?
1. tRNA selection by ribosomes.
| 2. Formation of large protein complexes.
419
How many subunits are always found in ribosomes?
2.
420
What is found in each of the ribosomal subunits?
A large RNA molecule (60%), and 20-25% unique protein.
421
Where does codon and anticodon subunit bind to on the RNA?
The decoding centre on the small subunit.
422
What subunit in the ribosome allows the formation of the peptide bond?
Large subunit.
423
What subunit is the polypeptide exit tunnel found?
Large subunit.
424
What can be clustered at the sites of functional importance on a ribosome?
Post transcriptionally modified nucleotides.
425
What is peptidyltranseferase activity is catalyses by
rRNA.
426
What does rRNA synthesis require?
Hundreds of proteins and snoRNAs.
427
Is rRNA transcription and processing tightly coupled?
Yes.
428
How many S units (sedimentation units) are found in prokaryotic ribosomes?
70s.
429
How many RNAs are found in the prokaryotic ribosomes small unit?
One 16s.
430
How many RNAs are found in the prokaryotic large subunit?
One large RNA (23s) and one small (5s).
431
How is the large subunit is split in a eukaryotic ribosome?
Into two molecules based paired to each other.
432
How many sedimentation units are found in a eukaryotic ribosome?
80s
433
Where are ribosomes synthesised?
In a nucleolus.
434
Is pre-rRNA extensively modified?
Yes.
435
Ribosomal proteins produced in the cytoplasm are transported to where?
Into the nucleus to be assembled into pre RNA.
436
Do all ribosomal proteins bind to the rRNA during transcription?
No. Some bind afterwards.
437
What is ribose-2' hydroxyl methylation an example of?
A pre-rRNA modification.
438
The modification of uradine- pseudouradine in pre-rRNA is also known as what?
Pseudouridylation.
439
What two modifications occur to pre-rRNA?
1. Ribose-2'hydroxyl methylation
| 2. Modification of uradine to pseudouradine (pseudoridylation).
440
Are proteins added to the rRNA once it has left the nucleus?
Yes.
441
Anticodons and the aminoacyl group lie close together on the tRNA. True it false?
True.
442
What type of reaction is involved in peptide bond formation?
Nucleophilic attack.
443
In eukaryotes how is the start codon found to allow translation to occur?
The 40s subunit binds to the mRNA and scans the transcript.
444
What is the size of the two subunits found in the prokaryotic ribosomes?
30S and 50L.
445
What is the size of then two subunits found in eukaryotic ribosomes?
40S and 60L.
446
What do translation factors control?
Assembly and disassembly of the ribosome at the start and end of translation.
447
How can translation be blocked at a molecular level?
By toxins and antibiotics.
448
Where is the initiator codon at the start of translation?
The ribosomal P site.
449
How many distinct met-tRNAs are there and why?
2. to distinguish between a normal codon and the start codon.
The elongator reads aug in the orf and the initiator reads the initiation codon.
450
What differences are there between the elongation met-tRNA and the initiator met-tRNA?
They initiator as it's 5' nucleotide not base paired and has fewer modified nucleotides in general.
451
All tRNAs but initiator-met are associated with EFTU in prokaryotes. What transcription factor is initiator met associated too?
IF2.
452
What forms the ternary complex in translation initiation in prokaryotes?
IF2, GTP and f-mettTNA.
453
The ternary complex in translation initiation in prokaryotes consists of f-met-tRNA, IF2 and GTP. What does this bind to once it has formed?
The 30s small ribosomal subunit complexed to mRNA.
454
What positions the initiator tRNA at he start codon in translation in prokaryotes?
The binding of the ternary complex to 30s subunit bound to mRNA.
455
Once the 30s subunit binds to the ternary complex what can then bind?
50s subunit.
456
Once the 50s subunit has bound to the 30s subunit bound to the ternary complex the ribosome is ready for translation. What needs to happen before it can be ready for translation (in prokaryotes)?
IF2 has to be released via GTP hydrolysis.
457
What is the only tRNA that can bind to the P site without binding to the A site first?
Initiator met tRNA.
458
All eukaryotic mRNA has a methylated g7 cap. True or false?
True.
459
What does the ternay complex involve in translation consist of in eukaryotes?
Initiation factor eIF2, initiator tRNAmet and GTP.
460
Is eIF4F involved in translation initiation in prokaryotes or eukaryotes?
Eukaryotes.
461
Once the 40s subunit binds to the ternary complex in eukaryotes what then happens before the 60s subunit can bind?
The compels is directed to the 5' end through interactions with the initiation complex eIF4F.
462
What sort of activity does eIF4F have?
RNA helicase activity. This allows it to scan the membrane 5'-3'.
463
What sequence does eIF4F recognise?
The kozak sequence.
464
When is eIF4F hydrolysed?
Once the kozak sequence has been found.
465
When does the 60s subunit bind to the rest of the complex in eukaryotic translation?
Once the kozak sequence has been located by eIF4F and IF2 has been released through the hydrolysis of GTP. The GTPase eIF2 is also needed.
466
What is the GTPase eIF5 needed for?
It is needed to allow the 60s subunit to bind in eukaryotes.
467
What recognises stop codons?
Termination factors.
468
Virus and a small number of endogenous mRNAs encode more than one ORF. In these cases ribosomes can be assembled at a specialised structure in the RNA. What is this structure called?
Internal ribosome entry site.
469
What are these examples of?
RF1, RF2 and eRF1.
Release factors.
470
What release factor is found in eukaryotes?
eRF1.
471
Release factors are molecular mimics of what?
tRNAS.
472
When a realise factors binds to the A site what does it trigger?
Peptide hydrolysis.
473
When a release factors binds to the ribosomal A site the peptide bound is hydrolysed. What is released from the P site?
An uncharged tRNA.
474
What releases RF1 from the A site?
The binding of RF3. This causes GTP hydrolysis.
475
what three things allow the dissociation of the ribosome after translation has been terminated?
Initiation factors, a ribosome recycling factor and EF-G.
476
How many tRNA binding sites are found on a ribosome?
3.
477
Once the protein has been generated on the large subunit of the ribosome where does it go?
Passes through the ribosome and exits through the exit channel.
478
How many ribosomal proteins are needed to assembly rRNA?
Around 50-80.
479
The structure of ______ defines the structure of the ribosome.
rRNA.
480
Why do rRNA transcription and translation need to be tightly coupled?
To allow correct assembly of the ribosome.
481
What does the first transcript produced in rRNA contain?
Many rRNA's: the transcript gets split up.
482
What does the peptidyl transferase reaction form?
A peptide bond.
483
Peptide bond formation/ peptidyl transferase reaction involves nucleophilic attack of what groups?
The alpha amino group on the amino-acyl tRNA and the carboxyl group on the peptidyl tRNA.
484
Where do anticodons react with adjacent codons?
At the decoding centre.
485
The amino acyl group and the peptidyl transferase groups juxtapose where?
The peptidyltranseferase centre.
486
What are the 3 tRNA binding sites called?
A- aminoacyl
P- peptidyl
E- Exit.
487
What ribosome binding sites are occupied before translation?
A and P.
488
What ribosome binding sites are occupied after translation?
P and E.
489
At the start of translation is the P sore filled before A or is the A site filled before P?
A peptidyl tRNA is present at the P site, then an aminoacyl tRNA binds to the A site. The nucleophilic reaction of peptide bond formation can then occur.
490
After the peptide bond formation where is the peptidyl group transferred to?
To the tRNA in the A site.
491
What type of tRNA leaves the ribosome at the P site?
Deacyl tRNA.
492
What are EFTU and EF1A?
Elongation factors. It is their job to bring the the tRNA bound to an amino acid to the ribosome.
493
What elongation factor is found in prokaryotes?
EFTU.
494
What elongation factor is found in eukaryotes?
EF1A.
495
What drives translocation in eukaryotes?
EFG.
496
What drives translocation in prokaryotes?
EF2.
497
What two elongation factors are GTPases?
EFTU and EGG.
498
How many GTP molecules are hydrolysed for each amino acid incorporated?
2.
499
How many phosphodiester linkages are spent per amino acid incorporated in translation?
4.
2 of these are from the GTP hydrolysis of 2 GTP molecules and the other two from the hydrolysis of one ATP molecule to form AMP. It is an energetically expensive process.
500
What do many antibiotics target?
Prokaryotic ribosomes.
501
What do many toxins target?
Eukaryotic ribosomes.
502
What does diptheria do?
Adds an adenosine residue to a modified histidine residue in EF2.
503
What cleaves a purine ring in the sarcin/ricin loop bound by EF2? (This is the part of the ribosome that is identified for elongation by EF2)
Ricin.
504
How many ribosomes can ricin inactivate in a minute?
1500.
505
What are ricin and plant lectins classed as?
N-glycosidases. These can depurinate A4324 blocking EFT2 binding.
506
What do some viruses block?
Scanning mechanisms.
507
What is an operon?
A cluster of genes under the control of a single regulatory signal or promoter.
508
Are polycistronic genes only found in prokaryotes?
Generally yes.
509
What has to happen for lactose to enter glycolysis?
2 6 carbon rings are broken in half.
510
What does LacY encode?
Lactose permease.
511
What is LacZ encode ?
Beta-Galactasidase.
512
What is lactose broken down to?
Galactose and glucose.
513
What is the structure of B-galactosidase?
A homotetromer. Each molecule has its own active site.
514
How many helices are found in the N and C terminal domains in LacY permase?
6.
515
What does LacY permase cotransport?
Lactose and hydrogen.
516
When a regulatory protein prevents transcription of the Lac operon is this negative or postive control?
Negative.
517
What is LacI.
Repressor for the lac operon.
518
What does LacI bind to?
LacO.
519
What structure does LacI have?
A tetromer.
520
How many operating sequences does LacI have?
2. One binds to the promoter and one upstream.
521
What structure does LacI make prevent RNAP from transcribing genes?
A loop.
522
What converts a small proportion of lactose into allolactose?
Beta-Galactosidase.
523
What can allolactose bind to?
LacI.
524
What happens to LacI when allolactose binds to it?
It changes shape preventing it from binding to the repressor.
525
What is IPTG?
A molecular mimic to allolactose.
526
An experiment was performed with a radiolaballed lac repressor, phage DNA with a lac operator sequence an phage DNA without a lac operon. This was then centrifuged in a glycerol gradient. What did this experiment show?
That LacI binds specifically to the LacO operator sequence.
527
What is LacA involved in?
Detox. It produces thiogalactoside transacetylase. It is not essential.
528
What is involved in the positive regulation of the lac operon?
Regulatory proteins activate transcription when glucoseis not present.
529
What type of control is allolactose involved in?
Negative.
530
What organisms use diauxic growth?
Bactria. At first the cell used glucose even if others sugars are available. They will only metabolise other sugars if glucose is not present.
531
What type of control of the Lac operon is cAMP involved in?
Positive.
532
What enzymes converts ATP to cAMP?
Adenylate cyclase.
533
What inhibits adenylate cyclase?
Glucose.
534
When there are high amounts of glucose in the cell how much cAMp is there?
A small amount as adenylate cyclase is inhibited.
535
When theere are low levels of glucose in the cell how much cAMP is present?
High amounts as adnylate cyclase is able to produce cAMP from ATP.
536
what is CRP?
The cAMP receptor protein (also known as CAP).
537
What can CRP form enabling it to bind to the DNA sequence?
A homodimer.
538
What needs to be bound to CRP allowing it to undergo a conformational change and recruit pol2, allowing transcription?
cAMP.
539
Where is the CRP binding sequence found?
Upstream to the genes involved in metabolism of lactose.
540
If lactose is being used in the cell instead of glucose what other sugar needs to be present?
Allolactose.
541
What happens to the lac operon when low amounts of lactose are present?
The lacI repressor binds.
542
What happens to the lac operon when there are high levels of lactose?
Allolactose binds to lacI meaning it can not bind.
543
What happens to the lac operon when glucose is present?
No cAMP is produced meaning it can not bind to the CRP activator.
544
What happens to the lac operon when no glucose is present?
Adenylate cyclase can produce cAMP which can bind to CRP allowing it to act as an activator.
545
Why is there incomplete expression of the lac operon?
As BG is needed to suppress its own expression.
546
When there is glucose and lactose are present is the lac operon expressed?
Yes, as basel levels.
547
What can arabinose be turned into/
Xylulose 5' phosphate.
548
Is the reaction converting glucose or arabinose to xyulose 5' phosphate prefered?
Glucose.
549
What can xyulose 5' phosphate be made into?
Glucose intermediates. It is also used in purine synthesis.
550
CRP bound to cAMP can positively regulate the lac operon. Can they positvely regulate any other operons?
Yes. The ara operon.
551
Where are the genes ARA B-A-D found?
On the ara operon.
552
Where are the genes ARA F-G-H-E found?
Not on the ara operon as they are involved in the transport of arabinose.
553
Why does IPTG last longer in the cell than arabinose?
As it is not broken down by BG.
554
What model is AraC involvced in?
The light switch model.
555
AraC is a repressor and activator. True or false?
True.
556
What does the binding of arabinose to AraC do?
It changes the position of AraC on the DNA binding domain.
557
When arabinose is bound to AraC how are the DNA binding domains positioned?
Side by side.
558
When arabinose is not bound to AraC where are the DNA binding domains found?
Apart from each other.
559
When the DNA is looped transcription can not occur. Where must the DNA binding domains be positioned in order for the DNA to be looped?
Apart.
560
What DNA binding domains are used when arabinose is present and the DNA binding domains lay next to each other?
O2 and I2.
561
What DNA binding domains are used when arabinose is not present and the DNA binding domains are apart?
I1 and I2.
562
What is the strongest binding site on the ara operon?
I1.
563
When no arabinose is present a loop forms in the DNA. Where isdoes CRP bind in relation to this loop?
It binds to the middle of the loop.
564
What operon uses transcriptional attenuation?
TRP operon.
565
In the trp operon why does the ribosome stall when tryptophan is not present?
The leader peptide contains tryptophan residues.
566
When tr is not present the ribosome stalls on the trp operon covering what region and forming a hairpin in what regions?
Region 1 is covered meaning regions 2 and 3 form hairpin.
567
When tryptophan is not present a hairpin is formed on regions 2 and 3. Does this mean transcription can proceed?
Yes.
568
When tryptophan is present the leader peptide can be synthesised. Where does the ribosome stall?
The termination codon.
569
When tryptophan is present what regions does the hairpin cover?
3 and 4.
570
When tryptoiphan is present a hairpin is formed in regions 3 and 4. What follows this hairpin?
a stretch of u's.
571
rho independent termination is used in prokaryotes. Where else is a similar system used?
In the trp operon when trptophan is predsent. It causes the RNAP to fall off and transcription to not proceed.
572
Can plants or animals make tryptophan?
Plants. Animals can not.
573
Is tryptophan energetically expensive to make?
Yes.
574
How many genes are involved in the trp operon?
5, EDCBA.
575
what is trpR?
The trp repressor involved in negative control of the trp operon.
576
What needs to bind to trpR in order for it to bind to the operoator region and block transcription?
Tryptophan.
577
What is Trpl?
Leader protein which forms mutually exclusive hairpins in the trp operon.
578
When a hairpin forms in regions 2 and 3 what is it called?
A anti terminator.
579
Transcriptional attentuation results in a __ fold difference in the amount of trp produced.
10.
580
trpR transcriptional control results in a ___ fold difference in the amount of trp produced.
70.
581
When trp is not present there is a ___ fold difference in trp production.
700.
582
What two things do you need for efficent transcription?
1. A good match to the shine delgaro sequence.
| 2. The shine delgaro sequence to be readily available.
583
What 2 things do antisense RNAs control?
1. Translation initiation.
| 2. mRNA stability.
584
What can antisense RNAs bind to to distrupt translation initiation?
The shine delgaro sequence.
585
In what organism do Omp porins use beta barallels to form holes in the membrane?
Gram negative bacteria.
586
Do OmpC porins form large or small holes in the membrane?
Small.
587
Do OmpF porins form large or small holes in the membrane?
Large.
588
What type of porins are Omp porins?
General diffusion porins- they will allow diffusion of toxins or nutrients as long as they are soluble.
589
When there are high nutrients and low toxins what porins will be used?
C.
590
When there are high nutrients and high toxins what porins will be used?
C.
591
When there are low nutrients and high toxins what porins will be used?
C.
592
When there are low nutirents and low toxins what porins will be used?
F.
593
What two mechanisms regulate the Omp porin system?
1. Two component system.
| 2. Antisense RNAs.
594
What kinase is used in the expression of omp porins?
Envz.
595
What regulator is used in the expression of omp porins?
OmpR.
596
What genes are involved in the expresison of Omp porins?
OmpC and OmpF.
597
What does MicA asRNA bind?
OmpA mRNA.
598
What does MicF asRNA bind?
OmpF mRNA.
599
What does MicC asRNA bind?
OmpC mRNA.
600
OmpR, MarA, SoxS and RoB are all examples of what?
Transcriptional regulators.
601
When is OmpF expression limited (4 things)?
1. Increased osmolarity (OmpR)
2. Toxins (MarA)
3. Oxidative stress (SoxS)
4. Peptide antibiotics (Rob).
602
What is a riboswitch?
A regulatory sequence of mRNA molecule that binds a small molecule resulting in a change to the structure of mRNA and a change in mRNA expression,
603
Where are riboswitches often found?
In the 5'UTR.
604
What are the three parts of a riboswitch?
1. Aptamer
2. Switching sequence
3. EXpression platform.
605
What part of the riboswitch binds a metabolite to change to change the structure of the riboswitch?
Aptamer. Normally the binding of a particular molecule prevents the transcription of that molecule.
606
What part of the riboswitch changes the secondary structure?
The switching sequence.
607
What part of the riboswitch modulates gene expression?
The expression platform.
608
Where is Sam1 found?
Upstream of the genes for methionine biosynthesis?
609
What typeof bacetria contain the riboswitch Sam1?
Gram positive.
610
Whatstructure is formed by the Sam1 riboswitch when methinone is present?
A hairpin. This leads to termination.
611
That does the Thi box control synthesis off?
Thiamine pyrophosphate.
612
What is thiamine pyrophosphate?
Cofactor for vitamin B1.
613
What operon does the thi box control?
ThiCEFCH.
614
When no TTP is bound to the thi box riboswitch what is available?
Shine delgaro sequence.
615
What happens to the shine delgaro sequence when TTP is bound to the thibox?
The shine delgaro sequence is based paired.
616
How many tRNA binding sites are found on a ribosome?
3.
617
Once the protein has been generated on the large subunit of the ribosome where does it go?
Passes through the ribosome and exits through the exit channel.
618
How many ribosomal proteins are needed to assembly rRNA?
Around 50-80.
619
The structure of ______ defines the structure of the ribosome.
rRNA.
620
Why do rRNA transcription and translation need to be tightly coupled?
To allow correct assembly of the ribosome.
621
What does the first transcript produced in rRNA contain?
Many rRNA's: the transcript gets split up.
622
What does the peptidyl transferase reaction form?
A peptide bond.
623
Peptide bond formation/ peptidyl transferase reaction involves nucleophilic attack of what groups?
The alpha amino group on the amino-acyl tRNA and the carboxyl group on the peptidyl tRNA.
624
Where do anticodons react with adjacent codons?Where do anticodons react with adjacent codons?
At the decoding centre.
625
The amino acyl group and the peptidyl transferase groups juxtapose where?
The peptidyltranseferase centre.
626
What are the 3 tRNA binding sites called?
A- aminoacyl
P- peptidyl
E- Exit.
627
What ribosome binding sites are occupied before translation?
A and P.
628
What ribosome binding sites are occupied after translation?
P and E.
629
At the start of translation is the P sore filled before A or is the A site filled before P?
A peptidyl tRNA is present at the P site, then an aminoacyl tRNA binds to the A site. The nucleophilic reaction of peptide bond formation can then occur.
630
After the peptide bond formation where is the peptidyl group transferred to?
To the tRNA in the A site.
631
What type of tRNA leaves the ribosome at the P site?
Deacyl tRNA.
632
What are EFTU and EF1A?
Elongation factors. It is their job to bring the the tRNA bound to an amino acid to the ribosome.
633
What elongation factor is found in prokaryotes?
EFTU.
634
What elongation factor is found in eukaryotes?
EF1A.
635
What drives translocation in eukaryotes?
EFG.
636
What drives translocation in prokaryotes?
EF2.
637
What two elongation factors are GTPases?
EGG.
638
How many GTP molecules are hydrolysed for each amino acid incorporated?
2.
639
How many phosphodiester linkages are spent per amino acid incorporated in translation?
4.
2 of these are from the GTP hydrolysis of 2 GTP molecules and the other two from the hydrolysis of one ATP molecule to form AMP. It is an energetically
