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Molecular Biology > Chapter 6: Mechanism of Prokaryotic Transcription > Flashcards

Chapter 6: Mechanism of Prokaryotic Transcription Flashcards

1
Q

elongation model 1

A

DNA polymerase moves around the DNA template and wraps RNA around the template that would prevent DNA twisting or supercoiling

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1
Q

ID’d 4 key regions of sigma protein involved in core and DNA binding

A

Helmann & Chamberlin

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2
Q

binds strongly to DNA downstream of active site, helps orient enzyme, and provides strong DNA binding/sliding clamp with beta during elongation

A

Beta‘ subunit

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2
Q

discovered the Rho protein depresses RNA elongation but not initiation by studying phage DNA and RNA synthesis in vitro w/ GTP (initiation) and UTP (labels uracil)

A

J. Roberts et al.

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3
Q
  1. intrinsic (rho-independent)

simplest, don’t require external proteins, composed of inverted repeat in new RNA strandn (intramolecular base–pairing/hairpin loop) followed by T-rich region

  1. rho-dependent

Rho=protein, depresses RNA elongation by binding transcript 60-100 nt upstream of termination site (rho loading site)

A

Terminator: 2 kinds

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5
Q

virus composition

A

protein coat (capsid) and nucleic acid genome

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5
Q

Discovered 6-7 bp region in E. coli and phages 10bp upsteam of trasnscription start site called “-10 box” or Pribnow box (AT-rich)

A

David Pribnow

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6
Q

RNA polymerase alpha subunit

A

recognizes UP elements; 2 major domains separated by narrow linker

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6
Q

infectous particles with a genome; obligate intracellular parasites

A

Viruses

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7
Q

Sigma region 1

A

prevents sigma from binding DNA by itself

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7
Q
  1. close association between sigma & B/B’ subunits
  2. narrow channel requiring sigma to open the enzyme
  3. sigma loop controlling RNA transcript release & bond formation
A

crystal structure of *Thermus aquaticus *in 2002 revealed 3 keys features

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8
Q

elongation model 2

A

RNA polymerase moves in a straight line unwinding the DNA in front and behind of the enzyme creating a supercoiling strain on the DNA that is relaxed by topoisomerase

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8
Q

rho loading site

A

place where rho binds 60-100 nt upsteam of termination site

cytosine-rich

Rho becomes catalytically active after binding and moves along RNA to “catch” polymerase, where it releases RNA and polymerase

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8
Q

allows for strong association between polymerase & promoter, and strong transcription

A

RNA polymerase C-terminal domain (CTD)

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9
Q

RNA polymerase enzymes first ID’d in…

A

First ID’d in bacteria and their infections particles, bacteriophages (T4)

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9
Q

core polymerase

A

lacks sigma subunit; unable to transcribe DNA by itself

c/clam/clamp-shaped w/ catalytic center and channel for DNA

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9
Q

Requires viral DNA replication and viral proteins

blocked by: DNA or protein synthesis inhibitors

Time: 10-25min

A

Late phase

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10
Q

3 phases of viral transcription in prokaryotes

A

immediate early, delayed early, and late phase

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11
Q

-10 and -35 boxes; essential for gene expression

A

core promoter elements

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11
Q

DNA polymerase moves around the DNA template and wraps RNA around the template that would prevent DNA twisting or supercoiling

A

elongation model 1

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12
Q

small, RNA viruses

non-polio=2nd most common (10-15million/year)

Found in respiratory secretions, nasal mucus, and stool

A

Enteroviruses

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13
Q

Heil & Zillig

A

used a reconstitution assay & antibiotics rifampin and streptolydigin to block transcription and elongation, respectively

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14
Q

4 steps of transcription initiation

A
  1. formation of the closed promoter complex
  2. coversion of closed to open complex
  3. synthesis of the first several nucleotides
  4. promoter clearance & nucleotide stabilization/hybridization that allows the polymerase complex to shift to an elongation confirmation & loss of sigma factor
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14
Q

Nadler et al

A

proposed 2 major sites of DNA interaction with RNA polymerase: downstream hydrophobic site & upstream electrostatic site

also demoed that B subunit binds near melting/catalytic site of polymerase

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15
Q

RPo (promoter open)

A

DNA slightly bent, promoter & template strand entered channel/catalytic center, associated with sigma region 2, rudder splits melted DNA and holds nontemplate strand apart

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16
Q

Sigma region 4

A

broken into 2 parts; 4.2 binds -35 box

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17
Q

demonstrated that the sigma subunit was the essential specificity factor for transcription

Observed that the holoenzyme of E. coli could transcribe viral (T4) immediate early genes, but not the core polymerase alone

Also demoed that holoenzyme could complete immediate early gene transcription of T4 and is highly specific, but that core enzyme lacked specificity & abnormally transcribed both DNA strands

A

Buatz et al.

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18
Q

Helmann & Chamberlin

A

ID’d 4 key regions of

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19
Q

David Pribnow

A

Discovered 6-7 bp region in E. coli and phages 10bp upsteam of trasnscription start site called “-10 box” or Pribnow box (AT-rich)

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20
Q

broken into 4 parts; 2.4 binds the -10 box

A

Sigma region 2

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21
Q

found that sigma stimulates initiation but not elongation

demoed that sigma was recycled and cycles from one core to another (sigma cycle)

A

Travers & Burgess

23
Q

Sigma region 2

A

broken into 4 parts; 2.4 binds the -10 box

24
Q

First ID’d in bacteria and their infections particles, bacteriophages (T4)

A

RNA polymerase enzymes first ID’d in…

24
Q

prevents sigma from binding DNA by itself

A

Sigma region 1

26
Q

core promoter elements

A

-10 and -35 boxes; essential for gene expression

27
Q

requires host proteins

not blocked by anything

time: 0-2min

A

immediate early

27
Q

form of DNA/polymerase complex in “open state” of transcription (Darst et al)

  1. entire promoter region spans mostly where sigma is (not core)
  2. sigma 2.4 region binds -10 box w/ 2 key amino acids: Gln 260 & ASN 263
  3. aromatic amino acids Phe 248, Tyr 253, and Trp 256 may participate in DNA melting & bind the single-stranded DNA
A

RF complex (3 key features)

28
Q

performed filter T7 H3-DNA-binding assays with holoenzyme and core enzyme: mixed enzymes w/ H3-DNA and purified on filters; more radioactive filters=more DNA polymerase in complex (occured with holoenzyme, not core polymerase)

Also demoed holoenzyme binding is temp-dependent (optimal=37)

Created model: RNA polymerase binds loosely to DNA at first in closed promoter complex, then DNA opens and binds tightly to holoenzyme to form open promoter complex (requires sigma)

A

Hinkle and Chamberlin (x3)

30
Q

Hinkle and Chamberlin (x3)

A

performed filter T7 H3-DNA-binding assays with holoenzyme and core enzyme: mixed enzymes w/ H3-DNA and purified on filters; more radioactive filters=more DNA polymerase in complex (occured with holoenzyme, not core polymerase)

Also demoed holoenzyme binding is temp-dependent (optimal=37)

Created model: RNA polymerase binds loosely to DNA at first in closed promoter complex, then DNA opens and binds tightly to holoenzyme to form open promoter complex (requires sigma)

31
Q

RF complex (3 key features)

A

form of DNA/polymerase complex in “open state” of transcription (Darst et al)

  1. entire promoter region spans mostly where sigma is (not core)
  2. sigma 2.4 region binds -10 box w/ 2 key amino acids: Gln 260 & ASN 263
  3. aromatic amino acids Phe 248, Tyr 253, and Trp 256 may participate in DNA melting & bind the single-stranded DNA
33
Q

Buatz et al.

A

demonstrated that the sigma subunit was the essential specificity factor for transcription

Observed that the holoenzyme of E. coli could transcribe viral (T4) immediate early genes, but not the core polymerase alone

Also demoed that holoenzyme could complete immediate early gene transcription of T4 and is highly specific, but that core enzyme lacked specificity & abnormally transcribed both DNA strands

34
Q

RNA polymerase moves in a straight line unwinding the DNA in front and behind of the enzyme creating a supercoiling strain on the DNA that is relaxed by topoisomerase

A

elongation model 2

36
Q

binds the cord

A

Sigma region 3

38
Q

Sigma region 3

A

binds the cord

39
Q

recognizes UP elements; 2 major domains separated by narrow linker

A

RNA polymerase alpha subunit

41
Q

UP elements

A

contained by some core promoters, upstream

attract RNA polymerase more strongly

42
Q

Beta‘ subunit

A

binds strongly to DNA downstream of active site, helps orient enzyme, and provides strong DNA binding/sliding clamp with beta during elongation

42
Q
  1. formation of the closed promoter complex
  2. coversion of closed to open complex
  3. synthesis of the first several nucleotides
  4. promoter clearance & nucleotide stabilization/hybridization that allows the polymerase complex to shift to an elongation confirmation & loss of sigma factor
A

4 steps of transcription initiation

44
Q

immediate early

A

requires host proteins

not blocked by anything

time: 0-2min

45
Q

requires at least one viral protein

Blocked by: protein synthesis inhibitors

Time: 2-10min

A

delayed early

47
Q

crystal structure of *Thermus aquaticus *in 2002 revealed 3 keys features

A
  1. close association between sigma & B/B’ subunits
  2. narrow channel requiring sigma to open the enzyme
  3. sigma loop controlling RNA transcript release & bond formation
48
Q

protein coat (capsid) and nucleic acid genome

A

virus composition

49
Q

RPc(promoter closed)

A

DNA is straight, unmelted, bound to UP element, B subunit=close state

51
Q

immediate early, delayed early, and late phase

A

3 phases of prokarotic transcription

52
Q

used a reconstitution assay & antibiotics rifampin and streptolydigin to block transcription and elongation, respectively

A

Heil & Zillig

53
Q

DNA is straight, unmelted, bound to UP element, B subunit=close state

A

RPc(promoter closed)

54
Q

found to be the essential part of the enzyme that synthesizes phosphodiester bonds and is closest to active site of bond formation; also essential for DNA binding

contains rifampicin-binding site

A

RNA polymerase B subunit

55
Q

broken into 2 parts; 4.2 binds -35 box

A

Sigma region 4

56
Q

lacks sigma subunit; unable to transcribe DNA by itself

c/clam/clamp-shaped w/ catalytic center and channel for DNA

A

core polymerase

57
Q

Viruses

A

infectous particles with a genome; obligate intracellular parasites

58
Q

RNA polymerase C-terminal domain (CTD)

A

allows for strong association between polymerase & promoter, and strong transcription

59
Q

Terminator: 2 kinds

A
  1. intrinsic (rho-independent)

simplest, don’t require external proteins, composed of inverted repeat in new RNA strandn (intramolecular base–pairing/hairpin loop) followed by T-rich region

  1. rho-dependent

Rho=protein, depresses RNA elongation by binding transcript 60-100 nt upstream of termination site (rho loading site)

61
Q

Travers & Burgess

A

found that sigma stimulates initiation but not elongation

demoed that sigma was recycled and cycles from one core to another (sigma cycle)

62
Q

J. Roberts et al.

A

discovered the Rho protein depresses RNA elongation but not initiation by studying phage DNA and RNA synthesis in vitro w/ GTP (initiation) and UTP (labels uracil)

63
Q

discovered -35box upstream of transcription start site

A

Mark Ptaschne

64
Q

DNA slightly bent, promoter & template strand entered channel/catalytic center, associated with sigma region 2, rudder splits melted DNA and holds nontemplate strand apart

A

RPo (promoter open)

65
Q

Mark Ptaschne

A

discovered -35box upstream of transcription start site

66
Q

proposed 2 major sites of DNA interaction with RNA polymerase: downstream hydrophobic site & upstream electrostatic site

also demoed that B subunit binds near melting/catalytic site of polymerase

A

Nadler et al

68
Q

delayed early

A

requires at least one viral protein

Blocked by: protein synthesis inhibitors

Time: 2-10min

69
Q

contained by some core promoters, upstream

attract RNA polymerase more strongly

A

UP elements

70
Q

Late phase

A

Requires viral DNA replication and viral proteins

blocked by: DNA or protein synthesis inhibitors

Time: 10-25min

71
Q

RNA polymerase B subunit

A

found to be the essential part of the enzyme that synthesizes phosphodiester bonds and is closest to active site of bond formation; also essential for DNA binding

contains rifampicin-binding site

72
Q

Enteroviruses

A

small, RNA viruses

non-polio=2nd most common (10-15million/year)

Found in respiratory secretions, nasal mucus, and stool

73
Q

4 subunits of RNA polymerase

A

B’ (160kD), B (150kD), sigma (70kD), alpha (40kD)

75
Q

place where rho binds 60-100 nt upsteam of termination site

cytosine-rich

Rho becomes catalytically active after binding and moves along RNA to “catch” polymerase, where it releases RNA and polymerase

A

rho loading site

76
Q

B’ (160kD), B (150kD), sigma (70kD), alpha (40kD)

A

4 subunits of RNA polymerase

Molecular Biology (13 decks)

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