Chapter 6: Mechanism of Prokaryotic Transcription

Question 1

elongation model 1

Answer 1

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

Question 1

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

Answer 1

Helmann & Chamberlin

Question 2

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

Answer 2

Beta‘ subunit

Question 2

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)

Answer 2

J. Roberts et al.

Question 3

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

2. rho-dependent

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

Answer 3

Terminator: 2 kinds

Question 5

virus composition

Answer 5

protein coat (capsid) and nucleic acid genome

Question 5

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

Answer 5

David Pribnow

Question 6

RNA polymerase alpha subunit

Answer 6

recognizes UP elements; 2 major domains separated by narrow linker

Question 6

infectous particles with a genome; obligate intracellular parasites

Answer 6

Viruses

Question 7

Sigma region 1

Answer 7

prevents sigma from binding DNA by itself

Question 7

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

Answer 7

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

Question 8

elongation model 2

Answer 8

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

Question 8

rho loading site

Answer 8

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

Question 8

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

Answer 8

RNA polymerase C-terminal domain (CTD)

Question 9

RNA polymerase enzymes first ID’d in…

Answer 9

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

Question 9

core polymerase

Answer 9

lacks sigma subunit; unable to transcribe DNA by itself

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

Question 9

Requires viral DNA replication and viral proteins

blocked by: DNA or protein synthesis inhibitors

Time: 10-25min

Answer 9

Late phase

Question 10

3 phases of viral transcription in prokaryotes

Answer 10

immediate early, delayed early, and late phase

Question 11

-10 and -35 boxes; essential for gene expression

Answer 11

core promoter elements

Question 11

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

Answer 11

elongation model 1

Question 12

small, RNA viruses

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

Found in respiratory secretions, nasal mucus, and stool

Answer 12

Enteroviruses

Question 13

Heil & Zillig

Answer 13

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

Question 14

4 steps of transcription initiation

Answer 14

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

Question 14

Nadler et al

Answer 14

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

Question 15

RP_o (promoter open)

Answer 15

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

Question 16

Sigma region 4

Answer 16

broken into 2 parts; 4.2 binds -35 box

Question 17

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

Answer 17

Buatz et al.

Question 18

Helmann & Chamberlin

Answer 18

ID'd 4 key regions of

Question 19

David Pribnow

Answer 19

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

Question 20

broken into 4 parts; 2.4 binds the -10 box

Answer 20

Sigma region 2

Question 21

found that sigma stimulates initiation but not elongation demoed that sigma was recycled and cycles from one core to another (_sigma cycle_)

Answer 21

Travers & Burgess

Question 23

Sigma region 2

Answer 23

broken into 4 parts; 2.4 binds the -10 box

Question 24

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

Answer 24

RNA polymerase enzymes first ID'd in...

Question 24

prevents sigma from binding DNA by itself

Answer 24

Sigma region 1

Question 26

core promoter elements

Answer 26

-10 and -35 boxes; essential for gene expression

Question 27

requires host proteins not blocked by anything time: 0-2min

Answer 27

immediate early

Question 27

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

Answer 27

RF complex (3 key features)

Question 28

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)

Answer 28

Hinkle and Chamberlin (x3)

Question 30

Hinkle and Chamberlin (x3)

Answer 30

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)

Question 31

RF complex (3 key features)

Answer 31

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

Question 33

Buatz et al.

Answer 33

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

Question 34

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

Answer 34

elongation model 2

Question 36

binds the cord

Answer 36

Sigma region 3

Question 38

Sigma region 3

Answer 38

binds the cord

Question 39

recognizes UP elements; 2 major domains separated by narrow linker

Answer 39

RNA polymerase alpha subunit

Question 41

UP elements

Answer 41

contained by some core promoters, upstream attract RNA polymerase more strongly

Question 42

Beta**'** subunit

Answer 42

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

Question 42

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

Answer 42

4 steps of transcription initiation

Question 44

immediate early

Answer 44

requires host proteins not blocked by anything time: 0-2min

Question 45

requires at least one viral protein Blocked by: protein synthesis inhibitors Time: 2-10min

Answer 45

delayed early

Question 47

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

Answer 47

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

Question 48

protein coat (capsid) and nucleic acid genome

Answer 48

virus composition

Question 49

RP_c (promoter closed)

Answer 49

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

Question 51

immediate early, delayed early, and late phase

Answer 51

3 phases of prokarotic transcription

Question 52

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

Answer 52

Heil & Zillig

Question 53

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

Answer 53

RP_c (promoter closed)

Question 54

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

Answer 54

RNA polymerase B subunit

Question 55

broken into 2 parts; 4.2 binds -35 box

Answer 55

Sigma region 4

Question 56

lacks sigma subunit; unable to transcribe DNA by itself c/clam/clamp-shaped w/ catalytic center and channel for DNA

Answer 56

core polymerase

Question 57

Viruses

Answer 57

infectous particles with a genome; obligate intracellular parasites

Question 58

RNA polymerase C-terminal domain (CTD)

Answer 58

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

Question 59

Terminator: 2 kinds

Answer 59

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

Question 61

Travers & Burgess

Answer 61

found that sigma stimulates initiation but not elongation demoed that sigma was recycled and cycles from one core to another (_sigma cycle_)

Question 62

J. Roberts et al.

Answer 62

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)

Question 63

discovered -35box upstream of transcription start site

Answer 63

Mark Ptaschne

Question 64

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

Answer 64

RP_o (promoter open)

Question 65

Mark Ptaschne

Answer 65

discovered -35box upstream of transcription start site

Question 66

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

Answer 66

Nadler et al

Question 68

delayed early

Answer 68

requires at least one viral protein Blocked by: protein synthesis inhibitors Time: 2-10min

Question 69

contained by some core promoters, upstream attract RNA polymerase more strongly

Answer 69

UP elements

Question 70

Late phase

Answer 70

Requires viral DNA replication and viral proteins blocked by: DNA or protein synthesis inhibitors Time: 10-25min

Question 71

RNA polymerase B subunit

Answer 71

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

Question 72

Enteroviruses

Answer 72

small, RNA viruses non-polio=2nd most common (10-15million/year) Found in respiratory secretions, nasal mucus, and stool

Question 73

4 subunits of RNA polymerase

Answer 73

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

Question 75

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

Answer 75

rho loading site

Question 76

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

Answer 76

4 subunits of RNA polymerase