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PHA111 > Transcription and Translation > Flashcards

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
Q

e.coli transcription

step 4 - mRNA elongation

A

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
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26
Q

e.coli transcription

step 5 - termination

A
  • once polymerase reaches stop signal — > dissociates from dna
  • effect of stalling polymerase + now weak association between rna + dna template causes dissociation of core enzyme
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27
Q

what is the most common stop signal?

A

formation of rna hairpin generated by self-comp sequence areas in mRNA

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28
Q

what is the structure of the stop signal often described as?

A
  • GC rich —> v stable causing polymerase to stall

- 4/more U residues after GC-loop —> weakly associated with A residues on template strand

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29
Q

for most genes transcriptional controls are what?

A

paramount

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30
Q

what is transcription of a gene controlled by?

A

regulatory region of dna near site of transcription - common in prokaryotes

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31
Q

where do gene regulatory proteins bind?

A

on outside of dna by recognising specific sequences

bases mostly on inside however, edges of base pairs on surface

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32
Q

what do the exposed edges provide opportunities for?

A

interaction via HB + hydrophobic interactions in minor and major grooves

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33
Q

where is the pattern for each of 4 base pairs unique?

A

major groove

most regulatory proteins bind at major groove

34
Q

how many nucleotide bases do the gene regulatory regions of dna contain?

A

less than 20

35
Q

the lac operon

A
  • 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
Q

what 3 enzymes are expressed when lactose is available?

A
  • 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
Q

lac operon function

A
  • 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
Q

primary structure of proteins

A

sequence of AA in polypeptide chain

39
Q

secondary structure of proteins

A

folded structures that form within polypeptide due to interactions between atoms of backbones

40
Q

tertiary structure of proteins

A

overal 3D shape

interactions between R groups of AA that make up protein

41
Q

quaternary structure

A

multiple polypeptide chains

42
Q

in prokaryotes, what is translation?

A

co-transcriptional

happen at same time

43
Q

where does translation happen?

A

in cytoplasm

44
Q

what are ribosomes responsible for?

A

reading mRNA sequence + assembling protein based upon message

45
Q

ribosome properties

A
  • diameter of 10nm
  • made of ribosomal rna (65%)
  • made of ribosomal proteins (35%)
46
Q

what are the different units of ribosomes characterised based on?

A

rate at which sediment

measured in svedburg units (S)

47
Q

what does the small ribosomal subunit contain?

A

decoding centre

needed for reading mRNA

48
Q

what is the large ribosomal subunit contain?

A

peptidyl transferase centre (catalytic RNA)

49
Q

what are polyribosomes?

A

multiple ribosomes loaded onto mRNA strand

50
Q

what sites do tRNAs occupy?

A

A, P and E

51
Q

A site

A

acceptor site, where amino-acyl tRNA lands

52
Q

P site

A

peptidyl-tRNA site, occupied by last AA added

53
Q

E site

A

exit site, where tRNA sits once AA transferred

tRNA leaves ribosome from E site

54
Q

although the bulk of each A,P and E sites lie in large subunit, where are the sites completed?

A

in subunit

55
Q

what are transfer RNAs?

A

adaptor molecules that deliver AA to ribosome

56
Q

what is the primary structure of tRNAs between?

A

60 and 95

most commonly 75

57
Q

tRNA secondary structure?

A
  • clover leaf

- significant levels of intra-tRNA HB sites

58
Q

what is the genetic code a continuous run of?

A

3 nucleotide triplets —> codons

59
Q

describe translation in words of codons?

A

codons read and info used to insert AA into growing polypeptide

60
Q

describe the Nirenberg experiment?

A
  • 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
Q

what were the conclusions made from the Nirenberg experiment?

A
  • UUU = mRNA codon for phenylalanine
  • AAA = mRNA codon for lysine
  • CCC = mRNA codon for proline
62
Q

what are the components needed for translation?

A
  • mRNA
  • tRNA
  • ribosome
  • GTP
  • initiation factors
  • elongation factors
63
Q

define translocation in translation?

A

movement of ribosome along mRNA

64
Q

what are the 3 stages in translation?

A
  • initiation
  • elongation
  • termination
65
Q

translation - initiation

A
  • 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
Q

what is the initiator tRNA?

A

formulated methionine tRNA

specifically recognises AUG codon

67
Q

what does the elongation step involve?

A
  • aminoacyl-tRNA delivery
  • peptide bond formation
  • translocation
68
Q

translation - elongation

A
  • 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
Q

why are the 3 elongation factors recruited to initiation complex?

A

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
Q

define termination in terms of translation?

A

process whereby ribosome = dissociated from mRNA

71
Q

translation - termination

A
  • 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
Q

what are the release factors involved in termination?

A
  • RF1 recognises UAA and UAG
  • RF2 recognises UAA and UGA
  • RF3 helps RF1 and RF2
73
Q

what are the trp operons found in e.coli?

A

group of genes that encode biosynthetic enzymes for AA tryptophan - rare AA

74
Q

what is the trp operon regulated by?

A

trp repressor

when bound to tryptophan, trp repressor blocks expression from operon

75
Q

what is tryptophan biosynthesis also regulated by?

A

attenuation

76
Q

what is attenuation?

A

mechanism based on coupling of transcription + translation

77
Q

when is attenuation used?

A

when tryptophan levels = high + effectively stops progression of ribosome along mRNA mol

78
Q

what is the leader sequence in attenuation?

A

control sequence between operator and first gene of operon

79
Q

what does the leader sequence do?

A

encodes short polypeptide + also contains attenuator sequence

80
Q

describe attenuation in the tryptophan operon?

A
  • 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
Q

how are anti-terminators generated?

A

using sequences 2 and 3

terminator generated using 3 and 4 on emerging mRNA

82
Q

what are the properties of proteins?

A
  • 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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