Chapter 6: Mechanism of Prokaryotic Transcription Flashcards
elongation model 1
DNA polymerase moves around the DNA template and wraps RNA around the template that would prevent DNA twisting or supercoiling
ID’d 4 key regions of sigma protein involved in core and DNA binding
Helmann & Chamberlin
binds strongly to DNA downstream of active site, helps orient enzyme, and provides strong DNA binding/sliding clamp with beta during elongation
Beta‘ subunit
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)
J. Roberts et al.
- 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
- rho-dependent
Rho=protein, depresses RNA elongation by binding transcript 60-100 nt upstream of termination site (rho loading site)
Terminator: 2 kinds
virus composition
protein coat (capsid) and nucleic acid genome
Discovered 6-7 bp region in E. coli and phages 10bp upsteam of trasnscription start site called “-10 box” or Pribnow box (AT-rich)
David Pribnow
RNA polymerase alpha subunit
recognizes UP elements; 2 major domains separated by narrow linker
infectous particles with a genome; obligate intracellular parasites
Viruses
Sigma region 1
prevents sigma from binding DNA by itself
- close association between sigma & B/B’ subunits
- narrow channel requiring sigma to open the enzyme
- sigma loop controlling RNA transcript release & bond formation
crystal structure of *Thermus aquaticus *in 2002 revealed 3 keys features
elongation model 2
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
rho loading site
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
allows for strong association between polymerase & promoter, and strong transcription
RNA polymerase C-terminal domain (CTD)
RNA polymerase enzymes first ID’d in…
First ID’d in bacteria and their infections particles, bacteriophages (T4)
core polymerase
lacks sigma subunit; unable to transcribe DNA by itself
c/clam/clamp-shaped w/ catalytic center and channel for DNA
Requires viral DNA replication and viral proteins
blocked by: DNA or protein synthesis inhibitors
Time: 10-25min
Late phase
3 phases of viral transcription in prokaryotes
immediate early, delayed early, and late phase
-10 and -35 boxes; essential for gene expression
core promoter elements
DNA polymerase moves around the DNA template and wraps RNA around the template that would prevent DNA twisting or supercoiling
elongation model 1
small, RNA viruses
non-polio=2nd most common (10-15million/year)
Found in respiratory secretions, nasal mucus, and stool
Enteroviruses
Heil & Zillig
used a reconstitution assay & antibiotics rifampin and streptolydigin to block transcription and elongation, respectively
4 steps of transcription initiation
- formation of the closed promoter complex
- coversion of closed to open complex
- synthesis of the first several nucleotides
- promoter clearance & nucleotide stabilization/hybridization that allows the polymerase complex to shift to an elongation confirmation & loss of sigma factor
Nadler et al
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
RPo (promoter open)
DNA slightly bent, promoter & template strand entered channel/catalytic center, associated with sigma region 2, rudder splits melted DNA and holds nontemplate strand apart
Sigma region 4
broken into 2 parts; 4.2 binds -35 box
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
Buatz et al.
Helmann & Chamberlin
ID’d 4 key regions of
David Pribnow
Discovered 6-7 bp region in E. coli and phages 10bp upsteam of trasnscription start site called “-10 box” or Pribnow box (AT-rich)
broken into 4 parts; 2.4 binds the -10 box
Sigma region 2
found that sigma stimulates initiation but not elongation
demoed that sigma was recycled and cycles from one core to another (sigma cycle)
Travers & Burgess
Sigma region 2
broken into 4 parts; 2.4 binds the -10 box
First ID’d in bacteria and their infections particles, bacteriophages (T4)
RNA polymerase enzymes first ID’d in…
prevents sigma from binding DNA by itself
Sigma region 1
core promoter elements
-10 and -35 boxes; essential for gene expression
requires host proteins
not blocked by anything
time: 0-2min
immediate early
form of DNA/polymerase complex in “open state” of transcription (Darst et al)
- entire promoter region spans mostly where sigma is (not core)
- sigma 2.4 region binds -10 box w/ 2 key amino acids: Gln 260 & ASN 263
- aromatic amino acids Phe 248, Tyr 253, and Trp 256 may participate in DNA melting & bind the single-stranded DNA
RF complex (3 key features)
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)
Hinkle and Chamberlin (x3)
Hinkle and Chamberlin (x3)
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)
RF complex (3 key features)
form of DNA/polymerase complex in “open state” of transcription (Darst et al)
- entire promoter region spans mostly where sigma is (not core)
- sigma 2.4 region binds -10 box w/ 2 key amino acids: Gln 260 & ASN 263
- aromatic amino acids Phe 248, Tyr 253, and Trp 256 may participate in DNA melting & bind the single-stranded DNA
Buatz et al.
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
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
elongation model 2
binds the cord
Sigma region 3
Sigma region 3
binds the cord
recognizes UP elements; 2 major domains separated by narrow linker
RNA polymerase alpha subunit
UP elements
contained by some core promoters, upstream
attract RNA polymerase more strongly
Beta‘ subunit
binds strongly to DNA downstream of active site, helps orient enzyme, and provides strong DNA binding/sliding clamp with beta during elongation
- formation of the closed promoter complex
- coversion of closed to open complex
- synthesis of the first several nucleotides
- promoter clearance & nucleotide stabilization/hybridization that allows the polymerase complex to shift to an elongation confirmation & loss of sigma factor
4 steps of transcription initiation
immediate early
requires host proteins
not blocked by anything
time: 0-2min
requires at least one viral protein
Blocked by: protein synthesis inhibitors
Time: 2-10min
delayed early
crystal structure of *Thermus aquaticus *in 2002 revealed 3 keys features
- close association between sigma & B/B’ subunits
- narrow channel requiring sigma to open the enzyme
- sigma loop controlling RNA transcript release & bond formation
protein coat (capsid) and nucleic acid genome
virus composition
RPc(promoter closed)
DNA is straight, unmelted, bound to UP element, B subunit=close state
immediate early, delayed early, and late phase
3 phases of prokarotic transcription
used a reconstitution assay & antibiotics rifampin and streptolydigin to block transcription and elongation, respectively
Heil & Zillig
DNA is straight, unmelted, bound to UP element, B subunit=close state
RPc(promoter closed)
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
RNA polymerase B subunit
broken into 2 parts; 4.2 binds -35 box
Sigma region 4
lacks sigma subunit; unable to transcribe DNA by itself
c/clam/clamp-shaped w/ catalytic center and channel for DNA
core polymerase
Viruses
infectous particles with a genome; obligate intracellular parasites
RNA polymerase C-terminal domain (CTD)
allows for strong association between polymerase & promoter, and strong transcription
Terminator: 2 kinds
- 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
- rho-dependent
Rho=protein, depresses RNA elongation by binding transcript 60-100 nt upstream of termination site (rho loading site)
Travers & Burgess
found that sigma stimulates initiation but not elongation
demoed that sigma was recycled and cycles from one core to another (sigma cycle)
J. Roberts et al.
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)
discovered -35box upstream of transcription start site
Mark Ptaschne
DNA slightly bent, promoter & template strand entered channel/catalytic center, associated with sigma region 2, rudder splits melted DNA and holds nontemplate strand apart
RPo (promoter open)
Mark Ptaschne
discovered -35box upstream of transcription start site
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
Nadler et al
delayed early
requires at least one viral protein
Blocked by: protein synthesis inhibitors
Time: 2-10min
contained by some core promoters, upstream
attract RNA polymerase more strongly
UP elements
Late phase
Requires viral DNA replication and viral proteins
blocked by: DNA or protein synthesis inhibitors
Time: 10-25min
RNA polymerase B subunit
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
Enteroviruses
small, RNA viruses
non-polio=2nd most common (10-15million/year)
Found in respiratory secretions, nasal mucus, and stool
4 subunits of RNA polymerase
B’ (160kD), B (150kD), sigma (70kD), alpha (40kD)
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
rho loading site
B’ (160kD), B (150kD), sigma (70kD), alpha (40kD)
4 subunits of RNA polymerase