Transcription: similar to replication but different 1.RNA polymerases use ribonucleotides (NTPs), and Uracil instead of Thymine. 2.Produces single-stranded molecules that are complementary to one strand of the DNA template (or non-coding strand). 3.Only part of the genome is transcribed – “functional genome” or “transcriptome”
Transcription: Basic Enzymology 1.DNA-dependent RNA Polymerases (RNAPs) 2.RNAPs polymerize in the 5’ to 3’ direction (rNTP added only to the 3’ end). 3.3’-OH of chain reacts with the phosphate of incoming rNTP, liberating pyrophosphate. 4.Order of added ribonucleotides follows Watson- Crick pairing rules, determined by template strand. 5.RNAPs do not need a primer, but do need DS DNA.
E. coli RNA polymerase (RNAP) - 2 copies - 1 copy ’ - 1 copy - 1 copy Core holoenzyme Cells have multiple forms of (sigma), which binds the promoter.
Transcription in 3 Steps 1.Initiation 2.Elongation 3.Termination
1. Initiation Occurs at promoters: – the -10, -35 or σ 70 promoters are most common type – recognized by σ 70 subunit – E. coli has at least 2 other promoter types recognized by other factors
1. Initiation in steps 1.RNAP is bound tightly to promoter, “Closed Complex” 2.RNAP unwinds 12-17 bp DNA (includes bp +3 to -9) forming “Open Complex ” 3.RNAP synthesizes a short (8-10 nt) RNA 4.RNAP clears promoter Fig. 6.9
Estimating the size and location of the DNA melted by the RNAP Discuss 2 ways and 2 experiments: 1.DMS methylation (also used for footprinting) of melted DNA 2.Melting-induced supercoiling
Fig. 6.18 1.Bind the RNAP to SV40, which has a promoter for E. coli. 2. Relax supercoils outside of the bound RNAP. 3. Knock off the RNAP. 4. DNA responds to the underwound region by changing overall conformation to resemble supercoiled DNA. Graph is degree of superhelicity per RNAP added. Slope = 1.6 superhelical turns per RNAP. 1.6 turns ~ 17 bp
3. Elongation (by the core RNAP) A.After ~10 nucleotides have been added, 5’-terminal nucleotide unpairs from template. B.But an RNA-DNA hybrid of ~8 bp (-1 to -8) is maintained during elongation. C.The subunit is not needed for elongation, and it may dissociate from core and cycle to new RNAP cores. Similar to Fig. 6.12
4. Termination Two types of termination mechanisms in E. coli –Rho Independent –Rho Dependent Rho Independent also called Intrinsic –triggered by an Inverted Repeat (IR) downstream of Rho-Independent genes –IR followed by an A-rich region
Stem-loop formation competes with the RNA-DNA hybrid (Open Complex). Causing DNA helix to reform (Closed Complex).
Rho-Dependent Termination Rho is a RNA helicase (and ATPase) 1.Travels along RNA chasing polymerase. 2.When polymerase stalls at an IR (w/o the A- T rich region), Rho dissociates the RNA- DNA hybrid. 3.DNA helix closes.
Fig. 6.51 Rho in Action Rho binds transcripts at stretches of ~100 nt free of 2 nd structure and rich in cytosines. Rho is a hexamer like DnaB except prefers RNA. Can unwind RNA-DNA hybrids.
Does the RNAP Slide and/or Hop? Promoter binding by RNAP too fast for random collision (diffusion) theory “Facilitated diffusion” theory (2 models) –Sliding? (not much evidence for it) –Hopping? Could be used on supercoiled, compact molecules.
A closer look at the prokaryotic RNA polymerase : Sigma factors 7 sigma factors in E. coli ; differ in their affinity for the core enzyme. Recognize at least 3 different promoters. Sigma factors are themselves regulated: Example, σ 32 : – 32 activated by heat shock (42 C) –Displaces 70 from the core –Directs the RNAP to heat-shock promoters
2.1 - Binds core of RNAP 2.3 - DNA melting 2.4 - Recognition of -10 region 4.1 - Binds activators 4.2 - Recognition of -35 region Conserved regions of sigma factors Besides binding at promoters, sigma factors also reduce the non-specific DNA binding affinity of the core, thereby increasing specific binding. 3 - DNA & core binding
What are the functions of the other subunits of the E. coli RNAP? Identifying the active site.
Drugs that inhibit E. coli RNAP Rifampicin - blocks at initiation (Open Complex forms); rifampicin-resistance mutations map to the β subunit. Streptolydigin- inhibits elongation stage; resistance mutations also map to β subunit. What do initiation and elongation have in common? Phosphodiester bond formation. Suggests β subunit is the catalytic subunit.
Direct evidence for β active site by “Affinity Labeling”. (1) Reagent in (a) resembles ATP, but reacts with NH 2 groups when binds active site. (2) Then add radioactive α - 32 P-UTP, which is added (i.e., polymerized) onto reagent I. Fig. 6.29
(3) Separate enzyme subunits by SDS- polyacrylamide gel electrophoresis, and see which ones are radioactive. Only the β subunit is highly radioactive. β subunit must have active site. Fig. 6.30