Transcription, Translation and Control Flashcards
(311 cards)
1
Q
what is a gene? (molecular biology perspective)
A
a sequence of genomic DNA that encodes a single functional RNA
2
Q
what are exons?
A
expressed segments of DNA
3
Q
what are introns?
A
intervening non-expressed sequences of DNA
4
Q
what are coding regions in mRNA flanked by at both ends?
A
UTRs
5
Q
what are UTRs?
A
untranslated regions
6
Q
what ensures speed and rapid responses to altered environment in prokaryotes?
A
no nucleus, no introns, mRNA is translated while still being transcribed
7
Q
what is transcription?
A
synthesis of ssRNA from a dsDNA template
8
Q
what enzymes catalyse transcription?
A
RNA polymerases
9
Q
what direction does transcription occur in?
A
5’ to 3’
10
Q
what is the name of the DNA strand that’s copied?
A
the coding strand
11
Q
what is the average half life of mRNA in growing E.coli cells?
A
about 2 minutes
12
Q
what are the requirements for mRNA formation in prokaryotes?
A
DNA template to copy, riboNTPS (ATP, UTP, GTP, CTP)
13
Q
what is at the very 5’ end of an RNA chain?
A
a triphosphate group
14
Q
what is the error frequency for transcription?
A
about 1 error per 10^4 nt
15
Q
why is the error rate higher for transcription than DNA replication?
A
the RNA polymerase has no proof-reading 3’-5’ exonuclease activity
16
Q
what is the promoter?
A
the nucleotide sequence in the DNA at which the RNA polymerase binds to begin transcription
17
Q
what does the s factor do?
A
suppresses random non-specific binding of RNA polymerase and induces a high affinity for particular promoters
18
Q
when is the s subunit released?
A
when the RNA chain has grown to about 6-8t. long and can join another core enyzme to initiate synthesis of another RNA chain
19
Q
what are the short motifs conserved upstream of the transcription start site in E.coli?
A
the -10 (Pribnow) box and the -35 box
20
Q
what are the 2 types of bacterial termination sites?
A
rho independent and rho dependent
21
Q
what causes the core enzyme to terminate at rho independent sites?
A
a G-C rich hairpin (variable length) and a run of around 6 Us in the RNA
22
Q
what sort of association is there between the Ru stretch and dA template strand?
A
very weak
23
Q
what is the G-C rich hairpin often preceded by?
A
a 50-90nt region with high C content
24
Q
what is Rho?
A
an ATP-dependent helicase
25
what does Rho do?
binds the C-rich RNA, may unwind the RNA-DNA duplex while the polymerase is paused at the hairpin
26
are all of the promoters in E. coli equally efficient?
no
27
what is the common characteristic of the most active E.coli promoters?
they most closely match the consensus
28
what is an operon?
a single promoter which controls multiple genes
29
in what form is mRNA generated from an operon?
as a polycistronic transcript (encodes several different polypeptides)
30
what is the most common sigma factor in E.coli?
σ^70
31
what switches heat shock genes on in heat shock?
σ^32
32
what does σ^32 do?
directs RNA polymerase to the heat shock proteins
33
what do regulatory proteins in transcription do?
control frequency of initiation of RNA synthesis in response to the concentrations of particular small molecules
34
what is a negative regulator/repressor of transcription?
one which blocks RNA synthesis when bound to the DNA
35
how do negative regulators of transcription usually work?
binding site on DNA overlaps that of the RNA polymerase so when repressor is bound RNA polymerase can't access the promoter
36
what is a positive regulator of transcription?
one which when bound to DNA enhances the efficiency of RNA polymerase entry, binding and initiation of transcription
37
what are the 2 conformations of regulatory proteins?
1 which binds to a specific sequence on the DNA at the promoter to be controlled, one which doesn't bind to the DNA sequence
38
how do positive regulators of transcription probably work?
provide extra recognition contacts for the RNA polymerase
39
what does β-galactosidase do?
hydrolyses lactose to galactose and glucose
40
what induces the synthesis of β-galactosidase mRNA?
a variety of β-galactosides
41
what happens to the lac repressor in the absence of an inducer?
binds to DNA at an operator site that prevents RNA polymerase binding
42
what are the inducers for the lac repressor?
lactose, non-hydrolysable analogues such as IPTG
43
what does actinomycin D do?
blocks all RNA synthesis immediately as it binds tightly to dsDNA between neighbouring GC base pairs
44
what does rifamycin do?
blocks all bacterial RNA synthesis by binding the β-subunit of RNA polymerase (likely blocks chain elongation)
45
what does rifamycin not affect?
eukaryotic cells
46
what is snRNA involved in?
splicing
47
what does snRNA stand for?
small nuclear RNA
48
how many types of RNA polymerase are there in eukaryotes?
3
49
what is the most complex RNA polymerase in eukaryotic cells?
RNA Pol II
50
what is the location of RNA Pol I in eukaryotic cells?
nucleolus
51
what is the location of RNA Pol II in eukaryotic cells?
nucleoplasm
52
what is the location of RNA Pol III in eukaryotic cells?
nucleoplasm
53
what is the product of RNA Pol I?
45S pre-rRNA
54
what is the product of RNA Pol II?
pre-mRNA, snRNAs, miRNAs
55
what is the product of RNA pol III?
tRNA, 5S rRNA, other small RNAs
56
how many subunits do the eukaryotic RNA polymerases have?
around 12
57
what are DNA promoters in eukaryotic cells?
sequences in the vicinity of the transcription start site required for accurate and efficient initiation of mRNA synthesis, core promoter and upstream elements
58
what are the 4 types of DNA elements to which transcription factors bind?
the core promoter element, upstream promoter elements, enhancer sequences and response (regulatory) elements
59
what is the promoter 'core' element in eukaryotes?
an A/T rich TATA box centered around 25bp from the transcription start site
60
what is the key difference between the eukaryotic promoter core element and the prokaryotic Pribnow box?
eukaryotic promoter core element is further upstream from the start site
61
what aids RNA pol II to achieve accurate initiation of transcription?
the basal transcription factors: YFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH
62
is initiation of transcription in eukaryotes specific?
no
63
what is the pre-initiation complex (PIC)?
the general transcription factor and RNA pol II assembled on the core promoter elements
64
what does the PIC need for transcription initiation?
the addition of NTPs
65
what is TFIID?
a complex of TATA binding protein (TBP) and a number of other TAFs (TBP-associated factors)
66
what initiates PIC assembly?
TBP recognising the TATA box
67
what does TFIIF do?
binds RNA pol II and brings it into the PIC?
68
what is the PIC?
the pre-initiation complex
69
what happens to the structure of DNA bound to TBP?
it is sharply bent
70
71
what other elements than the TATA box are essential for efficient DNA transcription in eukaryotes?
DNA elements within the -50 to -150 region that regulate transcription by interaction with transcription factors
72
what does transcription work against in eukaryotes?
repression by histones
73
what are transcription factors?
sequence-specific DNA-binding proteins with separate DNA-binding domains and activation domains
74
what techniques can be used to assay the presence of DNA binding proteins and their recognition sequences in DNA?
DNA footprinting, chromatin immunoprecipitation (ChIP)
75
what post-translational modifications are histones subject to?
acetylation, methylation, phosphorylation, ubiquitination
76
what is ChIP?
chromatin immunoprecipitation (a technique that allows you to determine the binding sites of DNA binding proteins in cells)
77
what does acetylation of a free amino group in Lys residues do?
reduces the net positive charge of the Lys
78
which genes are acetylated histones preferentially found in?
active genes where the chromatin is less tightly packed
79
what do histone acetylase do to gene expression?
activate
80
what do histone deacetylases do to gene expression?
inhibit
81
what are HATs found as components of?
repressor transcription factors
82
what type of protein is CBP?
a histone acetylase
83
what does CBP stand for?
CREB binding protein
84
what are AP1 and AP2?
DNA elements recognised by their dimeric sequence-specific binding of transcription factors c-Fos and c-Jun
85
what is GRE?
a glucorticoid response element
86
what is CRE?
a cAMP response element
87
how long do enhancer sequences tend to be?
around 100bp
88
how does the enhancement of transcription occur when enhancers are far away from promoters?
via DNA bending
89
where can response elements be found?
both within far-away enhancers and close to promoters
90
how do hormones and 2nd messengers couple external stimuli to gene expression?
by modifying the structure or location of transcription factors that bind them
91
what does CREB stand for?
cAMP response element-binding transcription factor
92
what does phosphorylation of CREB enable?
binding of the CBP co-activator which can bind the basal transcription machinery
93
what do the sex hormones + adrenal cortical hormones derive from?
cholesterol and retinoic acid, thyroid hormone and vitamin D3
94
what gene family are the hormone response elements in genes members of?
the steroid-thyroid hormone receptor or nuclear receptor gene family
95
what does activation of the GR by cortisol binding involve?
dissociation of hsp90 allowing receptor dimerisation and GRs movement into the nucleus
96
what is the GR?
the glucorticoid receptor
97
what is hsp90?
90kDs heat shock protein
98
why are many transcription factors dimers?
because of the symmetry of their response elements on both of the strands
99
what is MyoD?
a transcription factor present in myoblasts which controls expression of muscle-specific genes
100
what is Oct-2?
a tissue specific TF which regulates expression of light and heavy-IgG genes in B cell lymphocytes
101
what does Hox stand for?
homeotic/homeodomain-containing
102
what do Hox genes encode?
transcription factors
103
what do all Hox genes share?
the homeodomain which binds specific promoter DNA sequences in genes that encode segment identity proteins
104
what do mouse Hox genes specify?
the differences between cells along the head-to-tail axis
105
what do mutations in the Ant gene do?
transform the antennae into legs
106
do transcription co-activators/repressors bind to DNA?
no
107
how do transcription co-activator/repressors work?
interact with activator or repressor TFs through protein:protein interactions
108
what does c-Fos form with c-Jun?
the TF AP1 (a dimer)
109
what are the normal levels of c-Fos in the cell?
very low
110
what do patients with fibrous dysplasia show high levels of in the bone lesions?
c-Fos expression
111
what is P53?
a transcription activator when phosphorylated by a cyclin-dependent kinase
112
what end does pre-mRNA receive post-transcriptional modifications at?
both the 5' and 3'
113
what does P53 act as in healthy cells?
a tumour suppressor
114
what is the modification pre-mRNA receives at the 5' end?
the 5' cap
115
what is the modification pre-mRNA receives at the 3' end?
a 3' poly(A) tail
116
what are the roles of the 5' cap?
protects RNA from degradation by 5' exonucleases; increases efficiency of splicing of 5' proximal introns; required for export to cytoplasm; distinguishes complete transcripts from other RNA fragments; necessary for efficient translation initiation
117
how is the eukaryotic 5' cap formed?
1 phosphate is removed from the 5'pppN end of a newly made RNA, an enzymes adds GMP from GTP to give G5'ppp5'N- makes a 5'-5' bond; the added G is immediately methylated at the 7 position by S-adenosylmethionine (forming 7-Methyl G); a second methyl group is usually added to 2'OH of the first and second ribose
118
how many nucleotides long is the poly(A) tail of eukaryotic mRNAs initially?
around 240nt
119
what is addition of the poly(A) tail coupled to?
transcriptional termination
120
when does the polyA tail shorten?
in transport to the cytoplasm + as mRNA 'ages' in cytoplasm
121
what is the role of the polyA tail?
protects mRNA from 3' exonucleases and controls degradation rate of mRNAs, enhances rate of transcription
122
what is the evidence that the polyA tail controls the degradation rate of mRNAs?
poly(A) tails shorten more rapidly in short-lived mRNAs like the c-Fos oncogene
123
where is pre-mRNA cleaved to generate 3' ends with a poly(A) tail?
between a highly conserved AAUAAA sequence and a less conserved GU/U-rich sequence
124
what adds the poly(A) tail to pre-mRNA?
poly(A) polymerase
125
how many adenosines does poly(A) polymerase add at a time?
around 250
126
what is reverse transcriptase?
an RNA-dependent DNA polymerase
127
what are microarrays?
a hybridisation based technology that allows one to measure amounts of mRNA
128
what is the disadvantage of microarrays over sequencing?
can only identify known sequences for which the microarray contains a 'probe'
129
what are cDNA libraries used for?
to determine mRNA sequence, express eukaryotic proteins, characterise regulatory sequences in untranslated region
130
what are genomic DNA libraries used for?
to determine gene structure, to characterise sequences that regulate transcription, to express bacterial proteins
131
what process is used to remove introns from pre-mRNA?
RNA splicing
132
how long is the average human exon?
200 bases
133
how long is the average human intron?
thousands of bases
134
what happens to capped pre-mRNA before further processing?
it is bound by RNPs that ensure RNA stability and prevent 'knots'
135
what does splicing require?
snRNPs
136
what does snRNP mean?
small nuclear ribonucleoprotein particles
137
what are snRNPs (definition)?
a series of small nuclear RNAs present in ribonucleoprotein particles
138
what are the snRNAs known as and why?
U1, U2 etc. because they are uridine rich
139
what are snRNPs transcribed from + by (except U6)?
multicopy genes by RNA pol II
140
what are many of the antibodies produced in lupus against?
proteins of the snRNPs
141
what do the majority of introns begin and end with?
begin with a 5' GU dinucleotide and end with an AG
142
what does the short conserved consensus sequence the splice site at the end of introns base pair with?
U1 snRNA
143
what base pairs with U2 snRNA at the 3' end of the intron?
the branch point adenosine
144
what binds protein U2AF at the 3' end of the intron?
polypyrimidine tract around 10nt, pyrimidine rich
145
what is an RNA with catalytic activity called?
a ribozyme
146
what does spliceosome assembly require?
ATP
147
what does the spliceosome contain?
pre-mRNA, snRNPs, additional proteins
148
what is the spliceosome?
a large ribonucleoprotein complex
149
what does the spliceosome do?
mediates splicing
150
what forms the catalytic core of the spliceosome?
U2 and U6 snRNAs
151
what transcribes U6?
RNA pol III
152
what do mutations in the more highly conserved positions of consensus splice site sequences do?
either completely inactivate splicing or sometimes result in the use of nearby 'cryptic splice site' with similar sequences
153
what percentage of human genetic diseases are estimated to be caused by mutations that destroy functional splice sites/create new ones?
15%
154
what are the modes of alternative splicing?
use of alternative 5' splice sites and a common 3' splice site; use of a common 5' splice site with alternative 3' splice sites
155
what may changing the splice sites do?
introduce termination codons or change reading frames
156
how many isoforms of the WT1 protein are generated by alternative splicing/use of alternative translation start sites/RNA editing?
over 20
157
what can mutations in WT1 lead to?
Wilms' tumour (childhood kidney cancer), severe kidney disease, gonadal dysgenesis (male-female sex reversal)
158
what is the WT1 protein?
a transcription factor that recognises GC- or TC-rich promoter sequences
159
what is the the structure of the WT1 protein?
2 domain protein with an N-terminal Pro and Glu rich activation domain and a C-terminal DNA-binding protein with 4 Zn fingers
160
what is Frasier syndrome?
a developmental defect that affect kidney and gonads due to intronic mutation disrupting the alternative splice site in WT1
161
what is RNA editing?
an RNA processing reaction other than capping, splicing or 3'-end formation, that occurs after transcription and changes the nucleotide sequence of RNA
162
what is the major type of RNA editing in the nucleus of higher eukaryotes?
base (nucleotide) conversion
163
what are the best characterised RNA editing reactions?
hydrolytic deaminations
164
what happens in hydrolytic deaminations?
a genomically encoded cytidine (C) or adenosine (A) is converted to uridine (U) and inosine (I) respectively
165
what is NF1?
a common hereditary disease which predisposes individuals to tumours of the CNS and PNS
166
what does NF1 stand for?
neurofibromatosis 1
167
what type of protein is neurofibromin?
a tumour suppressor
168
what protein does the NF1 gene encode?
neurofibromin
169
what does neurofibromin contain?
a GAP domain
170
what does GAP domain stand for?
GTPase activating protein domain
171
what does the GAP domain do in neurofibromin?
interacts with Ras to regulate signal transduction
172
what does poly(A) tail shortening do?
destabilises mRNA
173
what is poly(A) tail shortening dictated by?
sequence in the 3'UTR of mRNAs
174
what do the 3'UTR regions of c-Fos mRNAs have binding sites for?
regulatory proteins that specifically target the mRNA for degradation
175
what does a rise in c-Fos RNA stimulate?
re-entry of G0 cells into the cell cycle
176
what is the major pathway of RNA degradation in eukaryotes initiated by?
deadenylation followed by decapping and 5'-3' exonuclease action
177
what does NMD stand for?
nonsense mediated decay
178
what leads to the NMD pathway?
mutations introduce premature stop codons into mRNA. detected and the mRNA is degraded
179
what happens in NMD?
histone mRNA is polyuridylated, some mRNAs are degraded by endonucleases, miRNAs and RNAi destabilise mRNAs
180
what is polyuridylation?
addition of a poly(U) tail
181
how many triplet codons are there?
64
182
how many stop codons are there (denote termination)?
3
183
what are the codons that denote termination?
UGA, UAG, UAA
184
what are the sense codons?
the 61 codons that specify amino acids
185
what are the nonsense codons?
the 3 codons specifying termination/stop
186
what are synonym codons?
codons which specify the same amino acid
187
what is the function of tRNAs?
adaptors between an appropriate amino acid and its codon on mRNA
188
what are the largest gene family?
tRNA genes
189
how many tRNA genes does the human genome contain?
around 1300
190
what are the common features of the different species of tRNA?
75-90nts; high proportion of unusual modified bases; unpaired sequence CCA at 3' end with aminoacylated terminal 3' or 2'OH; base-paired clover leaf structure in 2D folded into L-shape; acceptor arm and the anticodon loop at distal ends of molecule
191
what are the 2 principal active sites on tRNA?
the acceptor arm and the anticodon loop
192
what is tRNA charging with an amino acid catalysed by?
aminoacyl tRNA synthetases
193
what are the activated intermediates in protein synthesis?
amino acid esters in which the carboxyl group of an amino acid is linked to tRNA
194
how may different types of activating enzyme for tRNA do cells contain?
20
195
what is RS?
an aminoacyl-tRNA synthetase
196
what is the mRNA code interpreted by?
base pairing between the trinucleotide codon in the mRNA and the trinucleotide anti-codon sequence in tRNA
197
why is the last base in the codon recognised with less stringency than the 1st two?
the sugar phosphate backbone around the 'wobble position' in the tRNA is quite flexible so first base of anticodon can adopt different position and form non-standard base pairs with the 3rd base of the codon
198
what does the wobble hypothesis account for?
codon 34 degeneracy
199
what are ribosomes composed of?
several rRNAs and proteins
200
what proportion of the mass of ribosomes is RNA?
2/3
201
what proportion of the mass of ribosomes is protein?
1/3
202
what size particles do ribosomes engaged in elongation exist as?
70S/80S
203
which ribosome subunit binds mRNA first?
the small subunit
204
where does the large subunit join the small subunit on mRNA?
at the initiating AUG codon
205
what is a polyribosome/polysome?
several ribosomes simultaneously translating the same mRNA molecule
206
what is the directionality of mRNA translation?
5' end to 3' end
207
what is the directionality of protein synthesis?
from the N to C direction
208
why can RNAs be simultaneously translated as they are being made in bacteria?
because they are synthesised in the 5' to 3' direction
209
what group do all newly synthesised bacterial proteins start off as joined to?
formyl-methionine
210
what happens to the formyl-methionine attached to all newly synthesised bacterial proteins?
the formyl group is rapidly removed, methionine may also be remove but more slowly depending on nature of the next a.a. residue
211
what does all protein synthesis start with in eukaryotes?
methionine
212
what is the initiator codon?
AUG (represents Met)
213
what starts the polypeptide chain at the start of the reading frame?
a special initiator tRNA
214
what is the difference between Met-tRNAi and Met-tRNA?
Met-tRNAi is used for initiation and Met-tRNA recognises AUG codons during elongation
215
what other initiator codes are sometimes used in bacteria?
GUG and UUG
216
what characterises the correct initiation site on mRNA?
presence of a polypurine tract located a short distance nearer to the 5' end of the RNA than the AUG initiation codon
217
what is a polypurine tract?
the Shine-Dalgamo sequence: 5'...GGAGG...3'
218
how does the 30S ribosomal subunit recognise the polypurine tract? (prokaryotes)
base-pairing between the ...GGAGG.. in the mRNA and a ..CCUCC.. sequence at the end of the 16S rRNA in the 30S subunit
219
what is a polycistronic mRNA species?
mRNA with information coding for more than 1 protein
220
what does each cistron of a polycistronic mRNA have?
its own AUG initiation codon and Shine-Dalgarno sequence
221
are the cistrons in polycistronic mRNA translated together or independently of each other?
independently
222
what is an example of polycistronic mRNA?
the lac operon
223
can eukaryotic mRNA be polycistronic?
no
224
what does the process of selection of the initiation codon involve in eukaryotes?
the 40S ribosomal subunit binds to the 5' end of the mRNA then scans (migrates) toward the 3' end
225
what does the 5' cap of the mRNA bind to recruit the 40S subunit of the ribosome?
the cap binding protein complex eIF4F
226
what does eIF4F stand for?
eukaryotic initiation factor
227
how many subunits does eIF4F have?
3
228
what is scanning of the 40S ribosomal subunit coupled to?
hydrolysis of ATP
229
translation in eukaryotes is usually cap-dependent- what does this mean?
it requires a 5'm^7GpppG cap on the mRNA
230
how do translational factors that bind the cap structure on mRNA direct the recruitment of the 40S ribosomal subunit to the 5' end of mRNA?
by binding the small ribosomal subunit
231
what is internal initiation of translation used by?
some viral mRNAs, some cellular mRNAs
232
what is the viral mRNA that uses internal initiation of translation like?
uncapped, 5' UTRs are unusually long (400-1300nt), multiple AUG codons
233
what directs the binding of the ribosome to mRNA to the correct initiator AUG in internal initiation of translation?
the internal ribosome entry site (IRES)
234
how is an IRES sequence defined experimentally? (name of method)
using dicistronic assays
234
what does tetracycline do?
inhibits binding of aminoacyl-tRNA to the 30S subunit
235
what is a dicistronic assay?
when a potential sequence is inserted between the protein coding frames of 2 reporter genes in a plasmid vector- if the inserted sequence is an IRES then translation of the 2nd gene will occur
235
what do oxazolidines do?
inhibit formation of initiation Met tRNAi-30S ribosomal subunit-mRNA complex in prokaryotes
236
what IRES is a major drug target in several pharmaceutical companies?
the Hepatitis C IRES
236
what does the anticodon of Met-tRNAi base pair with to define the reading frame?
the initiating AUG codon
236
how many sites do ribosomes have for binding charged tRNA?
2
236
when does the EFTu-GDP form of elongation factor dissociate from the ribosome?
following GTP hydrolysis
236
what is needed for aminoacyl-tRNAs to bind the A-site?
an elongation factor protein and GTP
237
why do viruses use IRES?
they encode a protease which cleaves one of the cap-binding factors so host cell protein synthesis is compromised and viral protein synthesis can then use cellular translational machinery
237
what is the only way peptide bonds can be formed in the elongation phase of translation?
transfer of a peptide from the P-site to an amino group of aminoacyl-tRNA in the A-site
237
what is peptide bond formation catalysed by in elongation in translation?
the peptidyl transferase activity of the large ribosomal subunit
237
what does recent evidence suggest there is an additional third of near the P site?
a tRNA binding site, the exit (E) site through which discharged tRNA leaves the ribosome
237
what is the elongation factor protein in prokaryotes known as?
EFTu
237
what is the elongation factor protein known as in eukaryotes?
eEF-1
237
what is translocation (in translation)?
the ribosome moving along the mRNA by 3 nucleotides
237
what is peptidyl transferase an example of?
a ribozyme
237
what does translocation (in translation) require in eukaryotes?
the elongation factor eEF-2 and GTP
237
what does translocation do as well as moving the ribosome along the mRNA?
shifts the peptidyl-tRNA from the A-site to the P-site whilst ejecting the deacylated tRNA from the P-site
238
why are cellular IRESs useful?
they direct translation in situations where cap-dependent translation is reduced- during mitosis, apoptosis, in stress conditions
238
what does translocation (in translation) require in prokaryotes?
the elongation factor EFG and GTP
239
what signifies that the ribosome is ready for the elongation phase of protein synthesis?
formation of the 80S or 70S initiation complex
240
what is the ribosome binding site for peptidyl tRNA?
the P-site
241
what is the ribosome binding site for aminoacyl-tRNA?
the A-site
242
how is translation terminated?
protein called release factor (RF) binds to the ribosome A-site in response to a termination codon; binding modulates ribosome peptidyl transferase activity so it transfers the polypeptide to H2O, peptidyl-tRNA hydrolysed leading to release of the completed polypeptide
243
what does chloramphenicol do?
inhibits peptidyl transferase activity of the 50S subunit in prokaryotes
243
what does diptheria toxin catalyse?
covalent modification of translation elongation factor eEF2- inhibits its function in translocation in eukaryotes leading to cell death
244
why is chloramphenicol relatively toxic to humans?
it affects mitochondrial protein synthesis
244
what does ricin do?
its glycosidase activity removes a single A from the eukaryotic 28S rRNA and thus inactivates the ribosome
245
what does erythromycin do?
binds to 50S subunit at entrance to peptide exit tunnel, block progression of the nascent peptide, in prokaryotes
246
how many ribosomes can 1 risin molecule inactivate?
50000
247
what are the 2 types of modifications to control translation?
global and specific
248
what do global modifications to initiation factors by phosphorylation do?
affect translation of all cellular RNAs
249
what stage of translation is control almost always exerted at?
initiation
250
what is eIF2?
an initiation factor
251
what does eIF2 do?
forms a complex with initiator tRNA and GTP and binds to the 40S subunit along with mRNA
252
what does phosphorylation do to eIF2 activity?
reduces it leading to global inhibition of protein synthesis
253
what increases the activity of one eIF2 kinase?
the presence of dsRNA
254
what does eIF2 kinase do?
inhibits eIF2 by phosphorylating it
255
how does eIF2 kinase prevent viral replication?
dsRNA, a byproduct of viral infection, increases eIF2 kinase activity, eIF2 kinase inhibits eIF2, protein synthesis is inhibited preventing viral replication
256
what happens in specific modification?
binding of an RNA-binding protein to a specific motif in the 5' or 3' UTR of an mRNA represses/rarely activates translation of the individual mRNA
257
what is ferritin and what does it do?
intracellular protein which protects the cell from iron accumulation by sequestering ion
258
what is a conserved feature of mRNAs encoding ferritin?
presence of unique hairpin structure called the IRE in the 5' UTR
259
what does IRE stand for?
iron response element
260
what is the specific binding for the IRE?
iron regulatory protein (IRP)
261
what happens when the IRE is bound by the IRP when iron levels are low?
IRP binds to the hairpin with high affinity
262
what does an increase in iron availability do to IRP + the IRE?
reduces the affinity of IRP to the IRE, allowing ferritin synthesis to proceed
263
up to what percentage of human genes is regulated by miRNAs?
30%
264
what are the initial pre-miRNA transcripts transcribed by?
RNA pol II
265
how do ssmiRNAs inhibits target mRNA expression?
base pair imperfectly with the target mRNAs and inhibit their expression by inhibiting translation or by degrading the mRNA/degrading the protein
266
what is siRNA?
small interfering RNA, synthetic
267
what is the activity of siRNAs known as?
RNA interference (RNAi)
268
what is RNAi?
RNA interference
269
why is translation regulation by amino acids restricted to prokaryotes?
it requires co-transcription and translation
270
what are riboswitches?
RNA sequences in the 5'UTR or in introns that bind directly to small molecules such as thiamine to regulate translation
271
how do thiamine levels directly control the rate of thiamine synthesis?
by regulating the enzymes required for its synthesis
272
what 2 major pathways control protein degradation?
the lysosomal route and proteasomal route
273
what are the 2 main processes of protein delivery for degradation?
chaperone-mediated autophagy and macroautophagy
274
what is required for chaperone-mediated autophagy?
proteins must contain a specific signal sequence (KFERQ in single letter amino acid code)
275
which organ is especially geared for macroautophagy in amino acid starvation?
the liver
276
what does the proteasome mediate?
destruction of short-lived proteins and primarily proteins that are ubiquitinated
277
how many amino acids does ubiquitin have?
76
278
what is E1 and what does it do?
the ubiquitin activating enzyme, it uses ATP to covalently link the C-terminal glycine in Ub to a specific SH-group in E1 forming a thioester bond, in step 1 of Ub linking to Lys
279
what is E2 and what does it do?
the ubiquitin-conjugating enzyme, takes Ub from E1 and ligates it to its own SH group, in step 2 of Ub joining to Lys
280
what is E3 and what does it do?
Ub ligase, takes the Ub from E2 and ligates it to the e-NH2 groups of lysines in the protein destined for destruction
281
how are more Ubs added to the first Ub added to a protein to form poly-ubiquitins?
by linking the C-terminal glycine to an internal lysine in the next Ub
282
what enzyme rapidly removes ubiquitins?
Ub peptidases
283
what is the proteasome?
a giant protein assembly made up of a multi protein 'cap' at each end and a hollow shaft composed of 4 rings, 2 outer rings and 2 inner rings
284
what is the simplified sequence of degradation of cyclin B?(4)
recognition, dissociation, translocation, destruction
285
what happens in the recognition stage of degradation of cyclin B?
ubiquitinated protein binds to proteins in the cap
286
what happens in the dissociation stage of degradation of cyclin B?
an ATP-dependent process unravels the protein and spits out the Ubs
287
what happens in the translocation stage of degradation of cyclin B?
the protein is fed into the shaft where it reaches the inner 2 rings responsible for proteolysis
288
what happens in the destruction stage of degradation of cyclin B?
protein is entirely degraded into 8 amino acid peptides which are released
289
what is the N-end rule?
there are 5 specific amino acids which, when present at the N-terminus of a protein, cause it to be degraded faster
290
what are PEST sequences?
motifs containing P, E, S and T a.a.s are often phosphorylated on S or T and and aid destruction which can be Ub-dependent or independent
291
what does D stand for in the D-box?
destruction
292
what is the D box?
RxxLxxxxN/D/E which was first discovered in the N-terminus of cyclins- seems to be required for cyclin ubiquitination
293
