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BIOL 3421 - molecular biochem > transcription > Flashcards

transcription Flashcards

1
Q

list 3 ways that RNA polymerases are different from DNA polymerases

A

RNA pol adds ribonucleotides (U instead of T), doesn’t require a primer, lacks 3’-5’ exonuclease proofreading

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2
Q

T or F: RNA pol requires a primer to work

A

false; it doesn’t need a primer

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3
Q

T or F: the polymerization method is the same for DNA pol and RNA pol

A

true

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4
Q

describe the polymerization mechanism for RNA pol

A

nucleophilic attack by the 3’ OH on the phosphorous of an incoming NTP. Pyrophosphate exits as NMP is incorporated into the growing RNA strand. Mg2+ facilitates the nucleophilic attack + the other helps displace the leaving PPi

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5
Q

in which direction is the template strand copied

A

3’-5’

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6
Q

describe the general shape of the bacterial RNA pol

A

5 core subunits in a claw-like shape

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7
Q

what does the sixth subunit of bacterial RNA pol do + what is it’s name

A

sigma subunit: binds transiently to the core and helps direct it to specific DNA binding sites

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8
Q

T or F: RNA pol only binds to DNA in the presence of the sigma subunit

A

true

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9
Q

list the components of the holoenzyme

A

core + sigma subunit + DNA

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10
Q

where on the DNA will the sigma subunit bind during bacterial initiation

A

it binds to promoters upstream of the genes that need to be transcribed

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11
Q

which two promoter sequences does the sigma subunit bind to in bacterial initiation

A

-10 and -35

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12
Q

why are the -10 and -30 called consensus sequences (ie why consensus specifically)

A

bc these sites are usually similar across different promoters

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13
Q

describe why promoters establish a basal level of gene expression for every gene that can be upregulated or downregulated

A

mutations in promoters can greatly lower RNA pol binding and transcript initiation

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14
Q

what happens to the DNA when sigma binds to the consensus sequences in the promoter in bacterial initiation

A

DNA bends

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15
Q

what happens once sigma binds to promoter and DNA bends? (ie what is the result of this)

A

allows the RNA pol to interact with both the -10 and -35 regions at the same time

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16
Q

in bacterial initiation, describe what occurs once RNA pol is bound to both -10 and -35

A

the upstream promoter (UP) binds to the core RNA pol

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17
Q

the UP is rich in which bases

A

AT

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18
Q

T or F: in bacterial initiation, the upstream promoter (UP) binds to the sigma subunit

A

false; it only binds to the core RNA pol, not the sigma subunit

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19
Q

bacterial initiation: describe the events once everything is bound (consensus sequences, RNA pol, UP)

A

sigma binds to promoter and brings the polymerase core with it. First a closed complex is formed where DNA is still wound, then an open complex is formed when DNA begins to unwind near -10

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20
Q

bacterial initiation: where does DNA begin to unwind

A

-10

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21
Q

when bacterial initiation has started via conformational changes in RNA pol, what happens once RNA pol clears the promoter

A

sigma dissociates and NusA protein binds in its place

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22
Q

bacterial elongation: once sigma dissociates, which protein binds in its place

A

NusA

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23
Q

which step in the process signifies the start of bacterial elongation

A

once RNA pol has cleared the promoter, we’re now in the elongation stage

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24
Q

why is bacterial regulation tightly regulated

A

if RNA pol falls off too early, it has to restart the transcript all the way back at the promoter

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25
Q

what two types of bacterial termination are there

A

rho-dependent and rho-independent

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26
Q

in rho dependent termination, what is the name of the sequence that RNA has

A

RUT sequence

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27
Q

what does RUT stand for

A

Rho utilization element

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28
Q

in rho dependent termination, describe how the rho protein interacts with the RUT sequence on RNA

A

it binds to the RUT and travels 5’-3’ down the RNA

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29
Q

is RUT on RNA or DNA

A

RNA

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30
Q

which way does rho travel down the RNA after binding to RUT

A

5’-3’

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31
Q

which bases is RUT rich in?

A

C and A

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32
Q

what type of activity does rho have

A

helicase

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33
Q

T or F: rho is ATP-dependent

A

true

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34
Q

at which point will rho separate DNA/RNA

A

when it runs into the elongating polymerase

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35
Q

describe how rho-independent termination occurs in bacteria

A

a region of DNA produces an RNA transcript that has self-complimentary sequences hairpins with itself = strain. This disrupts base pairing in the RNA-DNA hybrid section and RNA pol can no longer bind properly to the RNA so it falls off

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36
Q

in rho independent termination, where is the hairpin located in regards to the desired termination site

A

10-20 nucleotides before

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37
Q

in rho dependent termination, describe the structure of the hairpin near the 3’ end

A

has 3 highly conserved U residues

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38
Q

list 3 key differences between pro and eu transcription

A
  1. the 3 RNA pol have similar subunits but each is recruit to different types of promoters
  2. eu promoters have much greater diversity and only two conserved promoter sequences (TATA box)
  3. eu has no sigma molecule: instead basal TFs bind to the promoter
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39
Q

what is the position of the TATA box

A

-30

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40
Q

what does CTD stand for

A

carboxyl terminal domain

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41
Q

which molecule is the CTD located on

A

RNA pol II

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42
Q

which RNA pol is the CTD located on

A

RNA pol II

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43
Q

describe the structure/orientation of the CTD on RNA pol II

A

it projects away from the remainder of the complex and is spatially separated from it by an intrinsically disordered linker

the tail includes a 7 aa sequence that repeats 25+ times

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44
Q

which in the first TF that arrives at the promoter

A

TFIID

45
Q

structure of TFIID?

A

multisubunit complex, one of which is TATA-binding protein (TBP)

46
Q

what does TBP stand for

A

TATA-binding protein

47
Q

on which TF is TBP located

A

TFIID

48
Q

the whole TFIID complex reaches across ___ elongated base pairs

A

70

49
Q

what does TBP do

A

binds to the TATA box

50
Q

result of TBP binding to the TATA box?

A

this positions the initiator sequence in the center of the elongated DNA with TFIID straddling it on either side

51
Q

what happens after TBP is bound to the TATA box and the initiatior sequence is in the center of the elongated DNA

A

TFIIB binds to TBP

52
Q

what does TFIIB bind to

A

TBP

53
Q

what does TFIIB recruit

A

RNA pol II

54
Q

where does TFIIB recruit RNA pol II to?

A

to the promoter

55
Q

which TF is RNA pol II tightly associated with

A

TFIIF

56
Q

once RNA pol II and TFIIF are recruited to the promoter, what happens

A

TFIIE binds to pol II

57
Q

what does TFIIE recruit

A

TFIIH

58
Q

what is the name for the complex once all TFs and RNA pol II have bound to the promoter

A

the closed preinitiation complex

59
Q

which TF has helicase activity to start unwinding DNA

A

TFIIH

60
Q

eu: near which site is DNA unwound

A

near the initiator site

61
Q

what is the name of the structure that forms after TFIIH unwinds DNA

A

the transcription bubble

62
Q

what is the name of the complex of unwound DNA + TFs + RNA pol II

A

open initiation complex

63
Q

which event starts transcription

A

TFIIH kinase activity: phosphorylated CTD of RNA pol II and the promoter is cleared

64
Q

once the promoter is cleared in eu initiation/elongation, which TFs leave the complex?

A

TFIIH and TFIIE leave

65
Q

which TF stays tightly associated with pol II during elongation

A

TFIIF

66
Q

binding of which type of molecules prevent pol II arrest in eu elongation?

A

elongation factors

67
Q

describe what happens at the end of eu transcription

A

elongation factors leave and termination factors bind

68
Q

what happens to RNA pol II after eu transcription is over

A

it’s dephosphorylated and recycled

69
Q

what happens if the eu transcription machinery halts at certain DNA lesions

A

TFIIH can recruit NER machinery (nucleotide excision repair)

70
Q

TFIIH mutations can cause which disease

A

xeroderma pigmentosum

71
Q

T or F: the phosphorylation status of CTD changes throughout transcription

A

true

72
Q

treatment of tuberculosis involved what type of actions from an antibiotic

A

an antibiotic can stall the RNA pol II at the stage of promoter clearance, preventing it from ever entering elongation

73
Q

what molecule is the 5’ cap

A

7-methylguanosine

74
Q

purpose of the 5’ cap?

A

help protect mRNA from ribonuclease degradation in the cytosol

75
Q

by which linkage is the 5’ cap attached to mRNA

A

5’-5’ triphosphate linkage

76
Q

describe the 5’-5’ triphosphate linkage that links the 5’ cap to the mRNA

A

condensation of GTP at the exposed 5’ phosphate end of mRNA. two 5’ carbons are thus involved in the linkage with 3 P’s in the middle

77
Q

when is the 5’ cap added

A

in early transcription: after the first 20-30 nucleotides

78
Q

what two complexes are required for the addition of the 5’ cap

A

cap-synthesizing complex and cap-binding complex

79
Q

T or F: both the cap-synthesizing and cap-binding complex can interact with the CTD

A

true

80
Q

T or F: the protection provided by the 5’ cap is permanent

A

false; it’s not permanent

81
Q

describe why the 5’ cap’s protection isn’t permanent

A

the cytosol has cellular decapping enzymes, so eventually the cap will be removed and the transcript will be vulnerable to 5’-3’ exonucleases

82
Q

what are the 3 splicing mechanisms

A

group 1, group 2, spliceosomal

83
Q

describe group 1 splicing

A

free guanosine 2’ or 3’ OH nucleophilic attack on the phosphorous between exon and intron. The 3’ OH of the displaced exon then does a nucleophilic attack on the other end of the intron

84
Q

describe group 2 splicing

A

the nucleophile for the first attack is an adenosine WITHIN the intron. This forms an intermediate lariat structure

85
Q

T or F: group 1 and 2 introns are self splicing (no protein or ATP required)

A

true

86
Q

T or F: spliceosomal introns require large RNA-protein spliceosome complex

A

true

87
Q

T or F: ATP is not required to splice spliceosomal introns

A

false; ATP is required for spliceosome assembly

88
Q

describe the structure of the spliceosome

A

made up of snRNPs

89
Q

describe the structure of snRNPs

A

each one has a small nuclear RNA (snRNA) and many proteins

90
Q

list the five snRNAs that are involved in splicing

A

u1, u2, u4, u5, u6

91
Q

T or F: the snRNAs have intrachain binding and complimentary regions to parts of the mRNA transcript

A

true

92
Q

which sites on the mRNA mark splicing sites

A

GU at the 5’ end of an intron and AG at the 3’ end of an intron

93
Q

which snRNA binds to the 5’ GU of an intron

A

U1

94
Q

which snRNA binds to internal A of an intron

A

U2

95
Q

how is the inactive spliceosome formed

A

u1 binds to GU, u2 binds to internal A, then u4-u6 dimer and u5 join to make the inactive spliceosome

96
Q

what occurs once the inactive spliceosome is formed

A

spliceosome is rearrnaged to displace u1 and u4. It also places u6 at the 5’ intron end to make the active spliceosome

97
Q

describe the structure of the active spliceosome

A

u1 and u4 have been displaced, u6 is at the 5’ end of the intron and the internal A and GU are super close together now

98
Q

what events occur once the active spliceosome has been formed

A

u2 which is bound to the internal A and u6 brings the A close enough for nucleophilic attack on the GU (via u6)

99
Q

which snRNA is the catalytic one

A

u6

100
Q

what happens once u6 does nucleophilic attack on GU

A

lariat formation + intron release

101
Q

which two snRNAs are tethered to the CTD on RNA pol at the end of splicing

A

u2 and u1

102
Q

where does the intron go after being spliced

A

stays in the nucleus

103
Q

what happens to the intron in the nucleus after being spliced

A

it’s degraded

104
Q

which conserved sequence is found at the end of a transcript

A

AAUAAA

105
Q

describe the addition of the poly A tail

A

RNA pol II synthesizes RNA beyond the AAUAAA sequence. Endonucleases cleave RNA downstream of AAUAAA, then adenylate polymerase adds 80-250 As

106
Q

in poly A tail addition, where do the endonuclease + polyadenylate polymerase come from?

A

AAUAAA is bound by a protein complex bound to RNA pol II CTD that includes these two enzymes

107
Q

benefit of alternative splicing?

A

more than one protein can be produced from the same primary transcript

108
Q

describe polyA site choice

A

poly A tails can form at different sites

109
Q

list some ways you can get alternative splicing

A
  1. alternative exons, 5’ sites or 3’ sites
  2. mutually exclusive alternative exons, alternative promoters and first exons, alternative poly A site and terminal exon

BIOL 3421 - molecular biochem (20 decks)

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