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Transcription and Translation Flashcards

1
Q

define transcription?

A

enzymatic synthesis of rna from dna template + forms first step in gene expression (generation of mrna)

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2
Q

define translation?

A

enzymatic synthesis of protein from transcribed gene sequence into functional rna molecule - mRNA

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3
Q

what is transcription catalysed by and what does this complex need?

A

rna polymerase enzyme complex

needs:

  • dsDNA template
  • ribonucleotides (ATP, GTP, CTP + UTP)
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4
Q

which direction does RNA synthesis happen?

A

5’ to 3’ direction

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5
Q

what is a sense strand?

A

carries gene sequence that = copied into RNA mol that = later translated into protein

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6
Q

what is an antisense strand?

A

used as template to generate copy of sense strand via comp base pairing

carries gene seq for rna mol that = non-coding + function as structural/regulatory mol

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7
Q

how many rna polymerase enzymes are there in prokaryotes in eukaryotes?

A

prokaryotes have 1

eukaryotes have 3 rna polymerase enzymes - I, II, III

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8
Q

what is each polymerase enzyme responsible for in eukaryotes?

A

polymerase I - transcribes most rRNA

polymerase II - transcription of all protein coding genes

polymerase III - transcribes tRNAs

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9
Q

what does transcription initiation involve?

A
  • binding of RNA polymerase complex to dsDNA

- polymerase binds to specific regulatory seq on dna that = upstream of gene to = transcribed: promoter seq

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10
Q

what can promoters do?

A

promoters can = switched on weakly/strongly based on seq elements present + if stimulated by bound proteins

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11
Q

what does the dsDNA need to be for the rna polymerase to access the promotor sequence?

A

locally unwound

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12
Q

where will the polymerase start to synthesise the mRNA?

A

at specific nucleotide = start site/initiation site

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13
Q

what does transcription elongation involve?

A
  • rna polymerase covalently adds ribonucleotides to 3’ end of growing mRNA mol in 5’ to 3’ direction
  • polymerase moving in 3’ to 5’ direction along antisense/template dna strand
  • polymerase unwinds dna duplex before rna synthesis activity
  • helix reformed behind polymerase enzyme
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14
Q

how many bases does the e.coli enzyme synthesise?

A

40 bases per second at 37 degrees

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15
Q

what are the 5 subunits the e.coli polymerase consists of?

A

2 alpha subunits - needed for holoenzyme assembly

1 beta subunit - catalytic centre of enzyme + key for initiation + elongation

one beta’ subunit - binds 2 Zn2+ ions to help catalyse joining of ribonucleotides

one w subunit - stabilises assembled holoenzyme

one sigma subunit - responsible for promoter recognition

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16
Q

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

A

sigma 70 factor

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17
Q

the sigma 70 promoter is between how many base pairs long?

A

40-60

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18
Q

in the sigma 70 factor, what is the region from -55 to 20 representing?

A

binds rna polymerase holoenzymes

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19
Q

in the sigma 70 factor, what is the region from -20 to 20 representing?

A

v strongly associated with the holoenzyme

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20
Q

in the sigma 70 factor, what is the region up to -40 representing?

A

needed for efficient transcription

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21
Q

e.coli transcription

step 1 - promoter binding

A
  • core polymerase enzyme( no sigma factor) has non-specific base affinity for dna
  • association of sigma factor makes holoenzyme
  • —> binds to specific promoter sequence with 100 fold inc in affinity
  • holoenzyme always attached to dna + slides along searching for promoter seq at 35 and 10 site
  • closed complex sits awaiting stimulation at certain highly expressed/imp genes
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22
Q

e.coli transcription

step 2 - dna unwinding

A
  • negative supercoiling used at v active gene promoters to help with melting of duplex
  • topology of 35 and 10 sites relative to each other = facilitate changes in dna conformation/topology
  • first unwinding of duplex makes OPEN complex with holoenzyme
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23
Q

e.coli transcription

step 3 - transcription initiation

A
  • rna synthesis happens without primer (unlike dna)
  • all rna has purine at 5’-most end: G more common than A
  • polymerase takes first 2 ribonucleotides + makes phosphodiester bond
  • chain starts with ATP/GTP
  • first 9 bases joined without enzyme moving - transcription can = aborted during formation of any of these bonds
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24
Q

in e.coli what does a growth rate sometimes mean?

A

next polymerase holoenzyme is queued up waiting for next initiation

decision to transcribe is fast —> 1-2sec

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25
e.coli transcription step 4 - mRNA elongation
sigma factor makes ternary(3 comp) complex with: - polymerase - dna - nacent dna - polymerase moves forward + clears the promoter - allows re-initiation by new holoenzyme if need be - as polymerase moves along dna, constant area of 17bp unwound at an given time at 40 bp per sec - 12bp hybrid between temp strand + mRNA kept constant —> one turn of rna-dna helix - sigma factor released once polymerase = moving + now free to make new holoenzyme
26
e.coli transcription step 5 - termination
- once polymerase reaches stop signal — > dissociates from dna - effect of stalling polymerase + now weak association between rna + dna template causes dissociation of core enzyme
27
what is the most common stop signal?
formation of rna hairpin generated by self-comp sequence areas in mRNA
28
what is the structure of the stop signal often described as?
- GC rich —> v stable causing polymerase to stall | - 4/more U residues after GC-loop —> weakly associated with A residues on template strand
29
for most genes transcriptional controls are what?
paramount
30
what is transcription of a gene controlled by?
regulatory region of dna near site of transcription - common in prokaryotes
31
where do gene regulatory proteins bind?
on outside of dna by recognising specific sequences bases mostly on inside however, edges of base pairs on surface
32
what do the exposed edges provide opportunities for?
interaction via HB + hydrophobic interactions in minor and major grooves
33
where is the pattern for each of 4 base pairs unique?
major groove most regulatory proteins bind at major groove
34
how many nucleotide bases do the gene regulatory regions of dna contain?
less than 20
35
the lac operon
- e.coli lactose used as source of carbon - enzymes for lactose metabolism only generated when lactose available - controllably expressed from central control expression cassette = operon
36
what 3 enzymes are expressed when lactose is available?
- lacY (galactoside permease) : allows lactose uptake from media - lacZ (beta galactosidase): converts lactose to glucose and galactose and allolactose - lacA (thiogalactoside transketolase): physiological function in lactose metabolism unknown expression = polycistronic mRNA
37
lac operon function
- binding of lacI to operater = cooperative - binding of lacI to Olac promotes binding of rna polymerase - low level expression of lacZYA in absence of lactose - lactose converted to allolactose —> binds to lacI protein —> dissociates from operator site dna - once operator sequence free —> strong level transcription induced
38
primary structure of proteins
sequence of AA in polypeptide chain
39
secondary structure of proteins
folded structures that form within polypeptide due to interactions between atoms of backbones
40
tertiary structure of proteins
overal 3D shape | interactions between R groups of AA that make up protein
41
quaternary structure
multiple polypeptide chains
42
in prokaryotes, what is translation?
co-transcriptional happen at same time
43
where does translation happen?
in cytoplasm
44
what are ribosomes responsible for?
reading mRNA sequence + assembling protein based upon message
45
ribosome properties
- diameter of 10nm - made of ribosomal rna (65%) - made of ribosomal proteins (35%)
46
what are the different units of ribosomes characterised based on?
rate at which sediment measured in svedburg units (S)
47
what does the small ribosomal subunit contain?
decoding centre needed for reading mRNA
48
what is the large ribosomal subunit contain?
peptidyl transferase centre (catalytic RNA)
49
what are polyribosomes?
multiple ribosomes loaded onto mRNA strand
50
what sites do tRNAs occupy?
A, P and E
51
A site
acceptor site, where amino-acyl tRNA lands
52
P site
peptidyl-tRNA site, occupied by last AA added
53
E site
exit site, where tRNA sits once AA transferred tRNA leaves ribosome from E site
54
although the bulk of each A,P and E sites lie in large subunit, where are the sites completed?
in subunit
55
what are transfer RNAs?
adaptor molecules that deliver AA to ribosome
56
what is the primary structure of tRNAs between?
60 and 95 most commonly 75
57
tRNA secondary structure?
- clover leaf | - significant levels of intra-tRNA HB sites
58
what is the genetic code a continuous run of?
3 nucleotide triplets —> codons
59
describe translation in words of codons?
codons read and info used to insert AA into growing polypeptide
60
describe the Nirenberg experiment?
- synthesised mRNA with repeating nucleotides - added them to test tube containing e.coli cell lysate - isolated resulting proteins + looked for AA incorporated - some triplet codes identified
61
what were the conclusions made from the Nirenberg experiment?
- UUU = mRNA codon for phenylalanine - AAA = mRNA codon for lysine - CCC = mRNA codon for proline
62
what are the components needed for translation?
- mRNA - tRNA - ribosome - GTP - initiation factors - elongation factors
63
define translocation in translation?
movement of ribosome along mRNA
64
what are the 3 stages in translation?
- initiation - elongation - termination
65
translation - initiation
- initiation complex made from ribosome, mRNA, initiator tRNA - initiator tRNA enters P site - all subsequent tRNAs enter A site only - 3 initiation factors + GTP needed
66
what is the initiator tRNA?
formulated methionine tRNA specifically recognises AUG codon
67
what does the elongation step involve?
- aminoacyl-tRNA delivery - peptide bond formation - translocation
68
translation - elongation
- P site occupied whilst A site = empty - ribosome complex maintains 6bp contact with mRNA —> stops frameshifting - elongation proceeds until termination codon appears in A site
69
why are the 3 elongation factors recruited to initiation complex?
all can bind to GDP/GTP - help deliver aminoacyl-tRNAs (GTP hydrolysis causes release of EF-Tu) - helps regenerate release of EF-Tu-GDP complex - translocase: uses energy from GTP hydrolysis to eject tRNA from P site + moves peptidyl-tRNA into P site from A site
70
define termination in terms of translation?
process whereby ribosome = dissociated from mRNA
71
translation - termination
- no tRNAs that recognise stop codons - protein release factors interact with codons + bring about release of pp chain - release factor causes peptidyl transferase to transfer pp to water than next tRNA - protein released - EF-G and RF needed for dissociation of ribosome complex from mRNA + removal of uncharged tRNA from p site
72
what are the release factors involved in termination?
- RF1 recognises UAA and UAG - RF2 recognises UAA and UGA - RF3 helps RF1 and RF2
73
what are the trp operons found in e.coli?
group of genes that encode biosynthetic enzymes for AA tryptophan - rare AA
74
what is the trp operon regulated by?
trp repressor when bound to tryptophan, trp repressor blocks expression from operon
75
what is tryptophan biosynthesis also regulated by?
attenuation
76
what is attenuation?
mechanism based on coupling of transcription + translation
77
when is attenuation used?
when tryptophan levels = high + effectively stops progression of ribosome along mRNA mol
78
what is the leader sequence in attenuation?
control sequence between operator and first gene of operon
79
what does the leader sequence do?
encodes short polypeptide + also contains attenuator sequence
80
describe attenuation in the tryptophan operon?
- attenuator doesn't encode polypeptide - when transcribed into mRNA—> has self comp sections + forms hairpin structures - attenuator pp has 2 tryptophan residues - if no tryptophan, ribosome stalls and anti-terminator hairpin structure genreated - this allows transcription + translation to process
81
how are anti-terminators generated?
using sequences 2 and 3 terminator generated using 3 and 4 on emerging mRNA
82
what are the properties of proteins?
- has C,O,H,N - has shape - has defined mass - absorbs light in UV range - has both charged + uncharged AA - has hydrophilic + hydrophobic AA

PHA111 (30 decks)

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