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MIMS > Transcription, Translation and Control > Flashcards

Transcription, Translation and Control Flashcards

1
Q

what is a gene? (molecular biology perspective)

A

a sequence of genomic DNA that encodes a single functional RNA

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2
Q

what are exons?

A

expressed segments of DNA

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3
Q

what are introns?

A

intervening non-expressed sequences of DNA

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4
Q

what are coding regions in mRNA flanked by at both ends?

A

UTRs

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5
Q

what are UTRs?

A

untranslated regions

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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

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7
Q

what is transcription?

A

synthesis of ssRNA from a dsDNA template

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8
Q

what enzymes catalyse transcription?

A

RNA polymerases

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9
Q

what direction does transcription occur in?

A

5’ to 3’

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10
Q

what is the name of the DNA strand that’s copied?

A

the coding strand

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11
Q

what is the average half life of mRNA in growing E.coli cells?

A

about 2 minutes

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12
Q

what are the requirements for mRNA formation in prokaryotes?

A

DNA template to copy, riboNTPS (ATP, UTP, GTP, CTP)

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13
Q

what is at the very 5’ end of an RNA chain?

A

a triphosphate group

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14
Q

what is the error frequency for transcription?

A

about 1 error per 10^4 nt

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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

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16
Q

what is the promoter?

A

the nucleotide sequence in the DNA at which the RNA polymerase binds to begin transcription

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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

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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

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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

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20
Q

what are the 2 types of bacterial termination sites?

A

rho independent and rho dependent

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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

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22
Q

what sort of association is there between the Ru stretch and dA template strand?

A

very weak

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23
Q

what is the G-C rich hairpin often preceded by?

A

a 50-90nt region with high C content

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24
Q

what is Rho?

A

an ATP-dependent helicase

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25
Q

what does Rho do?

A

binds the C-rich RNA, may unwind the RNA-DNA duplex while the polymerase is paused at the hairpin

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26
Q

are all of the promoters in E. coli equally efficient?

A

no

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27
Q

what is the common characteristic of the most active E.coli promoters?

A

they most closely match the consensus

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28
Q

what is an operon?

A

a single promoter which controls multiple genes

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29
Q

in what form is mRNA generated from an operon?

A

as a polycistronic transcript (encodes several different polypeptides)

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30
Q

what is the most common sigma factor in E.coli?

A

σ^70

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31
Q

what switches heat shock genes on in heat shock?

A

σ^32

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32
Q

what does σ^32 do?

A

directs RNA polymerase to the heat shock proteins

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33
Q

what do regulatory proteins in transcription do?

A

control frequency of initiation of RNA synthesis in response to the concentrations of particular small molecules

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34
Q

what is a negative regulator/repressor of transcription?

A

one which blocks RNA synthesis when bound to the DNA

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35
Q

how do negative regulators of transcription usually work?

A

binding site on DNA overlaps that of the RNA polymerase so when repressor is bound RNA polymerase can’t access the promoter

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36
Q

what is a positive regulator of transcription?

A

one which when bound to DNA enhances the efficiency of RNA polymerase entry, binding and initiation of transcription

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37
Q

what are the 2 conformations of regulatory proteins?

A

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

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38
Q

how do positive regulators of transcription probably work?

A

provide extra recognition contacts for the RNA polymerase

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39
Q

what does β-galactosidase do?

A

hydrolyses lactose to galactose and glucose

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40
Q

what induces the synthesis of β-galactosidase mRNA?

A

a variety of β-galactosides

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41
Q

what happens to the lac repressor in the absence of an inducer?

A

binds to DNA at an operator site that prevents RNA polymerase binding

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42
Q

what are the inducers for the lac repressor?

A

lactose, non-hydrolysable analogues such as IPTG

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43
Q

what does actinomycin D do?

A

blocks all RNA synthesis immediately as it binds tightly to dsDNA between neighbouring GC base pairs

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44
Q

what does rifamycin do?

A

blocks all bacterial RNA synthesis by binding the β-subunit of RNA polymerase (likely blocks chain elongation)

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45
Q

what does rifamycin not affect?

A

eukaryotic cells

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46
Q

what is snRNA involved in?

A

splicing

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47
Q

what does snRNA stand for?

A

small nuclear RNA

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48
Q

how many types of RNA polymerase are there in eukaryotes?

A

3

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49
Q

what is the most complex RNA polymerase in eukaryotic cells?

A

RNA Pol II

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50
Q

what is the location of RNA Pol I in eukaryotic cells?

A

nucleolus

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51
Q

what is the location of RNA Pol II in eukaryotic cells?

A

nucleoplasm

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52
Q

what is the location of RNA Pol III in eukaryotic cells?

A

nucleoplasm

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53
Q

what is the product of RNA Pol I?

A

45S pre-rRNA

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54
Q

what is the product of RNA Pol II?

A

pre-mRNA, snRNAs, miRNAs

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55
Q

what is the product of RNA pol III?

A

tRNA, 5S rRNA, other small RNAs

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56
Q

how many subunits do the eukaryotic RNA polymerases have?

A

around 12

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57
Q

what are DNA promoters in eukaryotic cells?

A

sequences in the vicinity of the transcription start site required for accurate and efficient initiation of mRNA synthesis, core promoter and upstream elements

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58
Q

what are the 4 types of DNA elements to which transcription factors bind?

A

the core promoter element, upstream promoter elements, enhancer sequences and response (regulatory) elements

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59
Q

what is the promoter ‘core’ element in eukaryotes?

A

an A/T rich TATA box centered around 25bp from the transcription start site

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60
Q

what is the key difference between the eukaryotic promoter core element and the prokaryotic Pribnow box?

A

eukaryotic promoter core element is further upstream from the start site

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61
Q

what aids RNA pol II to achieve accurate initiation of transcription?

A

the basal transcription factors: YFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH

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62
Q

is initiation of transcription in eukaryotes specific?

A

no

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63
Q

what is the pre-initiation complex (PIC)?

A

the general transcription factor and RNA pol II assembled on the core promoter elements

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64
Q

what does the PIC need for transcription initiation?

A

the addition of NTPs

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65
Q

what is TFIID?

A

a complex of TATA binding protein (TBP) and a number of other TAFs (TBP-associated factors)

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66
Q

what initiates PIC assembly?

A

TBP recognising the TATA box

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67
Q

what does TFIIF do?

A

binds RNA pol II and brings it into the PIC?

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68
Q

what is the PIC?

A

the pre-initiation complex

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69
Q

what happens to the structure of DNA bound to TBP?

A

it is sharply bent

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70
Q
A
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71
Q

what other elements than the TATA box are essential for efficient DNA transcription in eukaryotes?

A

DNA elements within the -50 to -150 region that regulate transcription by interaction with transcription factors

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72
Q

what does transcription work against in eukaryotes?

A

repression by histones

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73
Q

what are transcription factors?

A

sequence-specific DNA-binding proteins with separate DNA-binding domains and activation domains

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74
Q

what techniques can be used to assay the presence of DNA binding proteins and their recognition sequences in DNA?

A

DNA footprinting, chromatin immunoprecipitation (ChIP)

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75
Q

what post-translational modifications are histones subject to?

A

acetylation, methylation, phosphorylation, ubiquitination

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76
Q

what is ChIP?

A

chromatin immunoprecipitation (a technique that allows you to determine the binding sites of DNA binding proteins in cells)

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77
Q

what does acetylation of a free amino group in Lys residues do?

A

reduces the net positive charge of the Lys

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78
Q

which genes are acetylated histones preferentially found in?

A

active genes where the chromatin is less tightly packed

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79
Q

what do histone acetylase do to gene expression?

A

activate

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80
Q

what do histone deacetylases do to gene expression?

A

inhibit

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81
Q

what are HATs found as components of?

A

repressor transcription factors

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82
Q

what type of protein is CBP?

A

a histone acetylase

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83
Q

what does CBP stand for?

A

CREB binding protein

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84
Q

what are AP1 and AP2?

A

DNA elements recognised by their dimeric sequence-specific binding of transcription factors c-Fos and c-Jun

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85
Q

what is GRE?

A

a glucorticoid response element

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86
Q

what is CRE?

A

a cAMP response element

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87
Q

how long do enhancer sequences tend to be?

A

around 100bp

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88
Q

how does the enhancement of transcription occur when enhancers are far away from promoters?

A

via DNA bending

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89
Q

where can response elements be found?

A

both within far-away enhancers and close to promoters

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90
Q

how do hormones and 2nd messengers couple external stimuli to gene expression?

A

by modifying the structure or location of transcription factors that bind them

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91
Q

what does CREB stand for?

A

cAMP response element-binding transcription factor

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92
Q

what does phosphorylation of CREB enable?

A

binding of the CBP co-activator which can bind the basal transcription machinery

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93
Q

what do the sex hormones + adrenal cortical hormones derive from?

A

cholesterol and retinoic acid, thyroid hormone and vitamin D3

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94
Q

what gene family are the hormone response elements in genes members of?

A

the steroid-thyroid hormone receptor or nuclear receptor gene family

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95
Q

what does activation of the GR by cortisol binding involve?

A

dissociation of hsp90 allowing receptor dimerisation and GRs movement into the nucleus

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96
Q

what is the GR?

A

the glucorticoid receptor

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97
Q

what is hsp90?

A

90kDs heat shock protein

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98
Q

why are many transcription factors dimers?

A

because of the symmetry of their response elements on both of the strands

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99
Q

what is MyoD?

A

a transcription factor present in myoblasts which controls expression of muscle-specific genes

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100
Q

what is Oct-2?

A

a tissue specific TF which regulates expression of light and heavy-IgG genes in B cell lymphocytes

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101
Q

what does Hox stand for?

A

homeotic/homeodomain-containing

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102
Q

what do Hox genes encode?

A

transcription factors

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103
Q

what do all Hox genes share?

A

the homeodomain which binds specific promoter DNA sequences in genes that encode segment identity proteins

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104
Q

what do mouse Hox genes specify?

A

the differences between cells along the head-to-tail axis

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105
Q

what do mutations in the Ant gene do?

A

transform the antennae into legs

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106
Q

do transcription co-activators/repressors bind to DNA?

A

no

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107
Q

how do transcription co-activator/repressors work?

A

interact with activator or repressor TFs through protein:protein interactions

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108
Q

what does c-Fos form with c-Jun?

A

the TF AP1 (a dimer)

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109
Q

what are the normal levels of c-Fos in the cell?

A

very low

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110
Q

what do patients with fibrous dysplasia show high levels of in the bone lesions?

A

c-Fos expression

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111
Q

what is P53?

A

a transcription activator when phosphorylated by a cyclin-dependent kinase

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112
Q

what end does pre-mRNA receive post-transcriptional modifications at?

A

both the 5’ and 3’

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113
Q

what does P53 act as in healthy cells?

A

a tumour suppressor

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114
Q

what is the modification pre-mRNA receives at the 5’ end?

A

the 5’ cap

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115
Q

what is the modification pre-mRNA receives at the 3’ end?

A

a 3’ poly(A) tail

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116
Q

what are the roles of the 5’ cap?

A

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

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117
Q

how is the eukaryotic 5’ cap formed?

A

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

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118
Q

how many nucleotides long is the poly(A) tail of eukaryotic mRNAs initially?

A

around 240nt

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119
Q

what is addition of the poly(A) tail coupled to?

A

transcriptional termination

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120
Q

when does the polyA tail shorten?

A

in transport to the cytoplasm + as mRNA ‘ages’ in cytoplasm

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121
Q

what is the role of the polyA tail?

A

protects mRNA from 3’ exonucleases and controls degradation rate of mRNAs, enhances rate of transcription

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122
Q

what is the evidence that the polyA tail controls the degradation rate of mRNAs?

A

poly(A) tails shorten more rapidly in short-lived mRNAs like the c-Fos oncogene

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3
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123
Q

where is pre-mRNA cleaved to generate 3’ ends with a poly(A) tail?

A

between a highly conserved AAUAAA sequence and a less conserved GU/U-rich sequence

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124
Q

what adds the poly(A) tail to pre-mRNA?

A

poly(A) polymerase

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125
Q

how many adenosines does poly(A) polymerase add at a time?

A

around 250

126
Q

what is reverse transcriptase?

A

an RNA-dependent DNA polymerase

127
Q

what are microarrays?

A

a hybridisation based technology that allows one to measure amounts of mRNA

128
Q

what is the disadvantage of microarrays over sequencing?

A

can only identify known sequences for which the microarray contains a ‘probe’

129
Q

what are cDNA libraries used for?

A

to determine mRNA sequence, express eukaryotic proteins, characterise regulatory sequences in untranslated region

130
Q

what are genomic DNA libraries used for?

A

to determine gene structure, to characterise sequences that regulate transcription, to express bacterial proteins

131
Q

what process is used to remove introns from pre-mRNA?

A

RNA splicing

132
Q

how long is the average human exon?

A

200 bases

133
Q

how long is the average human intron?

A

thousands of bases

134
Q

what happens to capped pre-mRNA before further processing?

A

it is bound by RNPs that ensure RNA stability and prevent ‘knots’

135
Q

what does splicing require?

A

snRNPs

136
Q

what does snRNP mean?

A

small nuclear ribonucleoprotein particles

137
Q

what are snRNPs (definition)?

A

a series of small nuclear RNAs present in ribonucleoprotein particles

138
Q

what are the snRNAs known as and why?

A

U1, U2 etc. because they are uridine rich

139
Q

what are snRNPs transcribed from + by (except U6)?

A

multicopy genes by RNA pol II

140
Q

what are many of the antibodies produced in lupus against?

A

proteins of the snRNPs

141
Q

what do the majority of introns begin and end with?

A

begin with a 5’ GU dinucleotide and end with an AG

142
Q

what does the short conserved consensus sequence the splice site at the end of introns base pair with?

A

U1 snRNA

143
Q

what base pairs with U2 snRNA at the 3’ end of the intron?

A

the branch point adenosine

144
Q

what binds protein U2AF at the 3’ end of the intron?

A

polypyrimidine tract around 10nt, pyrimidine rich

145
Q

what is an RNA with catalytic activity called?

A

a ribozyme

146
Q

what does spliceosome assembly require?

A

ATP

147
Q

what does the spliceosome contain?

A

pre-mRNA, snRNPs, additional proteins

148
Q

what is the spliceosome?

A

a large ribonucleoprotein complex

149
Q

what does the spliceosome do?

A

mediates splicing

150
Q

what forms the catalytic core of the spliceosome?

A

U2 and U6 snRNAs

151
Q

what transcribes U6?

A

RNA pol III

152
Q

what do mutations in the more highly conserved positions of consensus splice site sequences do?

A

either completely inactivate splicing or sometimes result in the use of nearby ‘cryptic splice site’ with similar sequences

153
Q

what percentage of human genetic diseases are estimated to be caused by mutations that destroy functional splice sites/create new ones?

A

15%

154
Q

what are the modes of alternative splicing?

A

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
Q

what may changing the splice sites do?

A

introduce termination codons or change reading frames

156
Q

how many isoforms of the WT1 protein are generated by alternative splicing/use of alternative translation start sites/RNA editing?

A

over 20

157
Q

what can mutations in WT1 lead to?

A

Wilms’ tumour (childhood kidney cancer), severe kidney disease, gonadal dysgenesis (male-female sex reversal)

158
Q

what is the WT1 protein?

A

a transcription factor that recognises GC- or TC-rich promoter sequences

159
Q

what is the the structure of the WT1 protein?

A

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
Q

what is Frasier syndrome?

A

a developmental defect that affect kidney and gonads due to intronic mutation disrupting the alternative splice site in WT1

161
Q

what is RNA editing?

A

an RNA processing reaction other than capping, splicing or 3’-end formation, that occurs after transcription and changes the nucleotide sequence of RNA

162
Q

what is the major type of RNA editing in the nucleus of higher eukaryotes?

A

base (nucleotide) conversion

163
Q

what are the best characterised RNA editing reactions?

A

hydrolytic deaminations

164
Q

what happens in hydrolytic deaminations?

A

a genomically encoded cytidine (C) or adenosine (A) is converted to uridine (U) and inosine (I) respectively

165
Q

what is NF1?

A

a common hereditary disease which predisposes individuals to tumours of the CNS and PNS

166
Q

what does NF1 stand for?

A

neurofibromatosis 1

167
Q

what type of protein is neurofibromin?

A

a tumour suppressor

168
Q

what protein does the NF1 gene encode?

A

neurofibromin

169
Q

what does neurofibromin contain?

A

a GAP domain

170
Q

what does GAP domain stand for?

A

GTPase activating protein domain

171
Q

what does the GAP domain do in neurofibromin?

A

interacts with Ras to regulate signal transduction

172
Q

what does poly(A) tail shortening do?

A

destabilises mRNA

173
Q

what is poly(A) tail shortening dictated by?

A

sequence in the 3’UTR of mRNAs

174
Q

what do the 3’UTR regions of c-Fos mRNAs have binding sites for?

A

regulatory proteins that specifically target the mRNA for degradation

175
Q

what does a rise in c-Fos RNA stimulate?

A

re-entry of G0 cells into the cell cycle

176
Q

what is the major pathway of RNA degradation in eukaryotes initiated by?

A

deadenylation followed by decapping and 5’-3’ exonuclease action

177
Q

what does NMD stand for?

A

nonsense mediated decay

178
Q

what leads to the NMD pathway?

A

mutations introduce premature stop codons into mRNA. detected and the mRNA is degraded

179
Q

what happens in NMD?

A

histone mRNA is polyuridylated, some mRNAs are degraded by endonucleases, miRNAs and RNAi destabilise mRNAs

180
Q

what is polyuridylation?

A

addition of a poly(U) tail

181
Q

how many triplet codons are there?

A

64

182
Q

how many stop codons are there (denote termination)?

A

3

183
Q

what are the codons that denote termination?

A

UGA, UAG, UAA

184
Q

what are the sense codons?

A

the 61 codons that specify amino acids

185
Q

what are the nonsense codons?

A

the 3 codons specifying termination/stop

186
Q

what are synonym codons?

A

codons which specify the same amino acid

187
Q

what is the function of tRNAs?

A

adaptors between an appropriate amino acid and its codon on mRNA

188
Q

what are the largest gene family?

A

tRNA genes

189
Q

how many tRNA genes does the human genome contain?

A

around 1300

190
Q

what are the common features of the different species of tRNA?

A

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
Q

what are the 2 principal active sites on tRNA?

A

the acceptor arm and the anticodon loop

192
Q

what is tRNA charging with an amino acid catalysed by?

A

aminoacyl tRNA synthetases

193
Q

what are the activated intermediates in protein synthesis?

A

amino acid esters in which the carboxyl group of an amino acid is linked to tRNA

194
Q

how may different types of activating enzyme for tRNA do cells contain?

A

20

195
Q

what is RS?

A

an aminoacyl-tRNA synthetase

196
Q

what is the mRNA code interpreted by?

A

base pairing between the trinucleotide codon in the mRNA and the trinucleotide anti-codon sequence in tRNA

197
Q

why is the last base in the codon recognised with less stringency than the 1st two?

A

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
Q

what does the wobble hypothesis account for?

A

codon 34 degeneracy

199
Q

what are ribosomes composed of?

A

several rRNAs and proteins

200
Q

what proportion of the mass of ribosomes is RNA?

A

2/3

201
Q

what proportion of the mass of ribosomes is protein?

A

1/3

202
Q

what size particles do ribosomes engaged in elongation exist as?

A

70S/80S

203
Q

which ribosome subunit binds mRNA first?

A

the small subunit

204
Q

where does the large subunit join the small subunit on mRNA?

A

at the initiating AUG codon

205
Q

what is a polyribosome/polysome?

A

several ribosomes simultaneously translating the same mRNA molecule

206
Q

what is the directionality of mRNA translation?

A

5’ end to 3’ end

207
Q

what is the directionality of protein synthesis?

A

from the N to C direction

208
Q

why can RNAs be simultaneously translated as they are being made in bacteria?

A

because they are synthesised in the 5’ to 3’ direction

209
Q

what group do all newly synthesised bacterial proteins start off as joined to?

A

formyl-methionine

210
Q

what happens to the formyl-methionine attached to all newly synthesised bacterial proteins?

A

the formyl group is rapidly removed, methionine may also be remove but more slowly depending on nature of the next a.a. residue

211
Q

what does all protein synthesis start with in eukaryotes?

A

methionine

212
Q

what is the initiator codon?

A

AUG (represents Met)

213
Q

what starts the polypeptide chain at the start of the reading frame?

A

a special initiator tRNA

214
Q

what is the difference between Met-tRNAi and Met-tRNA?

A

Met-tRNAi is used for initiation and Met-tRNA recognises AUG codons during elongation

215
Q

what other initiator codes are sometimes used in bacteria?

A

GUG and UUG

216
Q

what characterises the correct initiation site on mRNA?

A

presence of a polypurine tract located a short distance nearer to the 5’ end of the RNA than the AUG initiation codon

217
Q

what is a polypurine tract?

A

the Shine-Dalgamo sequence: 5’…GGAGG…3’

218
Q

how does the 30S ribosomal subunit recognise the polypurine tract? (prokaryotes)

A

base-pairing between the …GGAGG.. in the mRNA and a ..CCUCC.. sequence at the end of the 16S rRNA in the 30S subunit

219
Q

what is a polycistronic mRNA species?

A

mRNA with information coding for more than 1 protein

220
Q

what does each cistron of a polycistronic mRNA have?

A

its own AUG initiation codon and Shine-Dalgarno sequence

221
Q

are the cistrons in polycistronic mRNA translated together or independently of each other?

A

independently

222
Q

what is an example of polycistronic mRNA?

A

the lac operon

223
Q

can eukaryotic mRNA be polycistronic?

A

no

224
Q

what does the process of selection of the initiation codon involve in eukaryotes?

A

the 40S ribosomal subunit binds to the 5’ end of the mRNA then scans (migrates) toward the 3’ end

225
Q

what does the 5’ cap of the mRNA bind to recruit the 40S subunit of the ribosome?

A

the cap binding protein complex eIF4F

226
Q

what does eIF4F stand for?

A

eukaryotic initiation factor

227
Q

how many subunits does eIF4F have?

A

3

228
Q

what is scanning of the 40S ribosomal subunit coupled to?

A

hydrolysis of ATP

229
Q

translation in eukaryotes is usually cap-dependent- what does this mean?

A

it requires a 5’m^7GpppG cap on the mRNA

230
Q

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?

A

by binding the small ribosomal subunit

231
Q

what is internal initiation of translation used by?

A

some viral mRNAs, some cellular mRNAs

232
Q

what is the viral mRNA that uses internal initiation of translation like?

A

uncapped, 5’ UTRs are unusually long (400-1300nt), multiple AUG codons

233
Q

what directs the binding of the ribosome to mRNA to the correct initiator AUG in internal initiation of translation?

A

the internal ribosome entry site (IRES)

234
Q

how is an IRES sequence defined experimentally? (name of method)

A

using dicistronic assays

234
Q

what does tetracycline do?

A

inhibits binding of aminoacyl-tRNA to the 30S subunit

235
Q

what is a dicistronic assay?

A

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
Q

what do oxazolidines do?

A

inhibit formation of initiation Met tRNAi-30S ribosomal subunit-mRNA complex in prokaryotes

236
Q

what IRES is a major drug target in several pharmaceutical companies?

A

the Hepatitis C IRES

236
Q

what does the anticodon of Met-tRNAi base pair with to define the reading frame?

A

the initiating AUG codon

236
Q

how many sites do ribosomes have for binding charged tRNA?

A

2

236
Q

when does the EFTu-GDP form of elongation factor dissociate from the ribosome?

A

following GTP hydrolysis

236
Q

what is needed for aminoacyl-tRNAs to bind the A-site?

A

an elongation factor protein and GTP

237
Q

why do viruses use IRES?

A

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
Q

what is the only way peptide bonds can be formed in the elongation phase of translation?

A

transfer of a peptide from the P-site to an amino group of aminoacyl-tRNA in the A-site

237
Q

what is peptide bond formation catalysed by in elongation in translation?

A

the peptidyl transferase activity of the large ribosomal subunit

237
Q

what does recent evidence suggest there is an additional third of near the P site?

A

a tRNA binding site, the exit (E) site through which discharged tRNA leaves the ribosome

237
Q

what is the elongation factor protein in prokaryotes known as?

A

EFTu

237
Q

what is the elongation factor protein known as in eukaryotes?

A

eEF-1

237
Q

what is translocation (in translation)?

A

the ribosome moving along the mRNA by 3 nucleotides

237
Q

what is peptidyl transferase an example of?

A

a ribozyme

237
Q

what does translocation (in translation) require in eukaryotes?

A

the elongation factor eEF-2 and GTP

237
Q

what does translocation do as well as moving the ribosome along the mRNA?

A

shifts the peptidyl-tRNA from the A-site to the P-site whilst ejecting the deacylated tRNA from the P-site

238
Q

why are cellular IRESs useful?

A

they direct translation in situations where cap-dependent translation is reduced- during mitosis, apoptosis, in stress conditions

238
Q

what does translocation (in translation) require in prokaryotes?

A

the elongation factor EFG and GTP

239
Q

what signifies that the ribosome is ready for the elongation phase of protein synthesis?

A

formation of the 80S or 70S initiation complex

240
Q

what is the ribosome binding site for peptidyl tRNA?

A

the P-site

241
Q

what is the ribosome binding site for aminoacyl-tRNA?

A

the A-site

242
Q

how is translation terminated?

A

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
Q

what does chloramphenicol do?

A

inhibits peptidyl transferase activity of the 50S subunit in prokaryotes

243
Q

what does diptheria toxin catalyse?

A

covalent modification of translation elongation factor eEF2- inhibits its function in translocation in eukaryotes leading to cell death

244
Q

why is chloramphenicol relatively toxic to humans?

A

it affects mitochondrial protein synthesis

244
Q

what does ricin do?

A

its glycosidase activity removes a single A from the eukaryotic 28S rRNA and thus inactivates the ribosome

245
Q

what does erythromycin do?

A

binds to 50S subunit at entrance to peptide exit tunnel, block progression of the nascent peptide, in prokaryotes

246
Q

how many ribosomes can 1 risin molecule inactivate?

A

50000

247
Q

what are the 2 types of modifications to control translation?

A

global and specific

248
Q

what do global modifications to initiation factors by phosphorylation do?

A

affect translation of all cellular RNAs

249
Q

what stage of translation is control almost always exerted at?

A

initiation

250
Q

what is eIF2?

A

an initiation factor

251
Q

what does eIF2 do?

A

forms a complex with initiator tRNA and GTP and binds to the 40S subunit along with mRNA

252
Q

what does phosphorylation do to eIF2 activity?

A

reduces it leading to global inhibition of protein synthesis

253
Q

what increases the activity of one eIF2 kinase?

A

the presence of dsRNA

254
Q

what does eIF2 kinase do?

A

inhibits eIF2 by phosphorylating it

255
Q

how does eIF2 kinase prevent viral replication?

A

dsRNA, a byproduct of viral infection, increases eIF2 kinase activity, eIF2 kinase inhibits eIF2, protein synthesis is inhibited preventing viral replication

256
Q

what happens in specific modification?

A

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
Q

what is ferritin and what does it do?

A

intracellular protein which protects the cell from iron accumulation by sequestering ion

258
Q

what is a conserved feature of mRNAs encoding ferritin?

A

presence of unique hairpin structure called the IRE in the 5’ UTR

259
Q

what does IRE stand for?

A

iron response element

260
Q

what is the specific binding for the IRE?

A

iron regulatory protein (IRP)

261
Q

what happens when the IRE is bound by the IRP when iron levels are low?

A

IRP binds to the hairpin with high affinity

262
Q

what does an increase in iron availability do to IRP + the IRE?

A

reduces the affinity of IRP to the IRE, allowing ferritin synthesis to proceed

263
Q

up to what percentage of human genes is regulated by miRNAs?

A

30%

264
Q

what are the initial pre-miRNA transcripts transcribed by?

A

RNA pol II

265
Q

how do ssmiRNAs inhibits target mRNA expression?

A

base pair imperfectly with the target mRNAs and inhibit their expression by inhibiting translation or by degrading the mRNA/degrading the protein

266
Q

what is siRNA?

A

small interfering RNA, synthetic

267
Q

what is the activity of siRNAs known as?

A

RNA interference (RNAi)

268
Q

what is RNAi?

A

RNA interference

269
Q

why is translation regulation by amino acids restricted to prokaryotes?

A

it requires co-transcription and translation

270
Q

what are riboswitches?

A

RNA sequences in the 5’UTR or in introns that bind directly to small molecules such as thiamine to regulate translation

271
Q

how do thiamine levels directly control the rate of thiamine synthesis?

A

by regulating the enzymes required for its synthesis

272
Q

what 2 major pathways control protein degradation?

A

the lysosomal route and proteasomal route

273
Q

what are the 2 main processes of protein delivery for degradation?

A

chaperone-mediated autophagy and macroautophagy

274
Q

what is required for chaperone-mediated autophagy?

A

proteins must contain a specific signal sequence (KFERQ in single letter amino acid code)

275
Q

which organ is especially geared for macroautophagy in amino acid starvation?

A

the liver

276
Q

what does the proteasome mediate?

A

destruction of short-lived proteins and primarily proteins that are ubiquitinated

277
Q

how many amino acids does ubiquitin have?

A

76

278
Q

what is E1 and what does it do?

A

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
Q

what is E2 and what does it do?

A

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
Q

what is E3 and what does it do?

A

Ub ligase, takes the Ub from E2 and ligates it to the e-NH2 groups of lysines in the protein destined for destruction

281
Q

how are more Ubs added to the first Ub added to a protein to form poly-ubiquitins?

A

by linking the C-terminal glycine to an internal lysine in the next Ub

282
Q

what enzyme rapidly removes ubiquitins?

A

Ub peptidases

283
Q

what is the proteasome?

A

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
Q

what is the simplified sequence of degradation of cyclin B?(4)

A

recognition, dissociation, translocation, destruction

285
Q

what happens in the recognition stage of degradation of cyclin B?

A

ubiquitinated protein binds to proteins in the cap

286
Q

what happens in the dissociation stage of degradation of cyclin B?

A

an ATP-dependent process unravels the protein and spits out the Ubs

287
Q

what happens in the translocation stage of degradation of cyclin B?

A

the protein is fed into the shaft where it reaches the inner 2 rings responsible for proteolysis

288
Q

what happens in the destruction stage of degradation of cyclin B?

A

protein is entirely degraded into 8 amino acid peptides which are released

289
Q

what is the N-end rule?

A

there are 5 specific amino acids which, when present at the N-terminus of a protein, cause it to be degraded faster

290
Q

what are PEST sequences?

A

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
Q

what does D stand for in the D-box?

A

destruction

292
Q

what is the D box?

A

RxxLxxxxN/D/E which was first discovered in the N-terminus of cyclins- seems to be required for cyclin ubiquitination

293
Q
A

MIMS (14 decks)

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