Transcription

By- Dr. Armaan SinghBy- Dr. Armaan Singh

Central Dogma

RNA Content of E. coli Cells

typeSteady State Levels

Synthetic Capacity Stability

rRNA 83% 58% High

tRNA 14% 10% High

mRNA 3% 32% Very Low

E. Coli RNA Polymerase• RNA polymerase core enzyme is a multimeric protein α 2,β, β ’, ω• The β ’ subunit is involved in DNA binding• The β subunit contains the polymerase active site• The α subunit acts as scaffold on which the other subunits assemble.• Also requires σ-factor for initiation –forms holo enzyme complex

Site of DNA binding and RNA polymerization

General Gene Structure5’ 3’Transcribed region terminatorPromoter

Gene Promoters

Other σ-Factors

• Standard genes – σ 70

• Nitrogen regulated genes – σ 54

• Heat shock regulated genes – σ 32

Transcriptional Initiation

Closed complex

Open complex

Primer formation

Disassociation of σ-factor

Pausing induces termination

3’end tends to be AU rich easily to disrupt during pausing. Leads to disassembly of RNA polymerase complex

Rho Dependent Termination• rho is an ATP-dependent helicase • it moves along RNA transcript,

finds the "bubble", unwinds it and releases RNA chain

Eukaryotic RNA Polymerases

type Location Products

RNA polymerase I Nucleolus rRNA

RNA polymerase II

Nucleoplasm mRNA

RNA polymerase III

Nucleoplasm rRNA, tRNA, others

Mitochondrial RNA polymerase

Mitochondria Mitochondrial gene transcripts

Chloroplast RNA polymerase

Chloroplast Chloroplast gene transcripts

Eukaryotic RNA Polymerases

• RNA polymerase I, II, and III

• All 3 are big, multimeric proteins (500-700 kD)

• All have 2 large subunits with sequences similar to β and β' in E.coli RNA polymerase, so catalytic site may be conserved

Eukaryotic Gene Promoters• Contain AT rich concensus sequence located –19 to –27 bp from transcription start (TATA box)• Site where RNA polymerase II binds

Transcription Factors• TFAIIA, TFAIIB – components of RNA polymerase II holo-enzyme complex• TFIID – Initiation factor, contains TATA binding protein (TBP) subunit. TATA box recognition. • TFIIF – (RAP30/74) decrease affinity to non-promoter DNA