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Page 1: Chapter 21 (part 1)

Chapter 21 (part 1)

Transcription

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Central Dogma

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Genes

• Sequence of DNA that is transcribed.• Encode proteins, tRNAs, rRNAs, etc..• “Housekeeping” genes encode

proteins or RNAs that are essential for normal cellular activity.

• Simplest bacterial genomes contain 500 to 600 genes.

• Mulitcellular Eukaryotes contain between 15,000 and 50,000 genes.

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Types of RNAs

• tRNA, rRNA, and mRNA• rRNA and tRNA very abundant

relative to mRNA.• But mRNA is transcribed at

higher rates than rRNA and tRNA

• Abundance is a reflection of the relative stability of the different forms of RNA

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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

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Phases of Transcription

• Initiation: Binding of RNA polymerase to promoter, unwinding of DNA, formation of primer.

• Elongation: RNA polymerase catalyzes the processive elongation of RNA chain, while unwinding and rewinding DNA strand

• Termination: termination of transcription and disassemble of transcription complex.

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E. Coli RNA Polymerase• RNA polymerase core

enzyme is a multimeric protein ’

• The ’ subunit is involved in DNA binding

• The subunit contains the polymerase active site

• The subunit acts as scaffold on which the other subunits assemble.

• Also requires -factor for initiation –forms holo enzyme complex

Site of DNA binding and

RNA polymerization

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-factor• The -factor is required for binding of the RNA

polymerase to the promoter

• Association of the RNA polynerase core complex w/ the -factor forms the holo-RNA polymerase complex

• W/o the -factor the core complex binds to DNA non-specifically.

• W/ the -factor, the holo-enzyme binds specifically with high affinity to the promoter region

• Also decreases the affinity of the RNA polymerase to non-promoter regions

• Different -factors for specific classes of genes

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General Gene Structure

• Promoter – sequences recognized by RNA polymerase as start site for transcription.

• Transcribed region – template from which mRNA is synthesized

• Terminator – sequences signaling the release of the RNA polymerase from the gene.

5’ 3’Transcribed region terminatorPromoter

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Gene Promoters• Site where RNA polymerase binds and

initiates transcription.• Gene that are regulated similarly contain

common DNA sequences (concensus sequences) within their promoters

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Important Concensus Sequences

• Pribnow Box – position –10 from transcriptional start

• -35 region – position –35 from transcriptional start.

• Site where -factor binds.

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Other -Factors

• Standard genes – 70

• Nitrogen regulated genes – 54

• Heat shock regulated genes – 32

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How does RNA polymerase finds the

promoter?• RNA polymerase does not disassociate

from DNA strand and reassemble at the promoter (2nd order reaction – to slow)

• RNA polymerase holo-enzyme binds to DNA and scans for promoter sequences (scanning occurs in only one dimension, 100 times faster than diffusion limit)

• During scanning enzyme is bound non-specifically to DNA.

• Can quickly scan 2000 base pairs

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Transcriptional Initiation

• Rate limiting step of trxn.• Requires unwinding of DNA and

synthesis of primer.• Conformational change occurs after DNA

binding of RNA polymerase holo-enzyme.• First RNA Polymerase binds to DNA

(closed-complex), then conformational change in the polymerase (open complex) causes formation of transcription bubble (strand separation).

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Initiation of Polymerization

• RNA polymerase has two binding sites for NTPs

• Initiation site prefers to binds ATP and GTP (most RNAs begin with a purine at 5'-end)

• Elongation site binds the second incoming NTP

• 3'-OH of first attacks alpha-P of second to form a new phosphoester bond (eliminating PPi)

• When 6-10 unit oligonucleotide has been made, sigma subunit dissociates, completing "initiation“

• NusA protein binds to core complex after disassociation of -factor to convert RNA polymerase to elongation form.

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Transcriptional Initiation

Closed complex

Open complex

Primer formation

Disassociation of -factor

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Chain Elongation Core polymerase - no sigma

• Polymerase is accurate - only about 1 error in 10,000 bases

• Even this error rate is OK, since many transcripts are made from each gene

• Elongation rate is 20-50 bases per second - slower in G/C-rich regions (why??) and faster elsewhere

• Topoisomerases precede and follow polymerase to relieve supercoiling

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Transcriptional Termination

• Process by which RNA polymerase complex disassembles from 3’ end of gene.

• Two Mechanisms – Pausing and “rho-mediated” termination

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Pausing induces termination

• RNA polymerase can stall at “pause sites”

• Pause sites are GC rich (difficult to unwind)

• Can decrease trxn rates by a factor of 10 to 100.

• Hairpin formation in RNA can exaggerate pausing

• Hairpin structures in transcribed RNA can destabilize DNA:RNA hybrid in active site

• Nus A protein increases pausing when hairpins form.

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

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Rho Dependent Termination

• rho is an ATP-dependent helicase

• it moves along RNA transcript, finds the "bubble", unwinds it and releases RNA chain

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Eukaryotic Transcription

• Similar to what occurs in prokaryotes, but requires more accessory proteins in RNA polymerase complex.

• Multiple RNA polymerases

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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

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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 and ' in E.coli RNA polymerase, so catalytic site may be conserved

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Eukaryotic Gene Promoters• Contain AT rich concensus sequence

located –19 to –27 bp from transcription start (TATA box)

• Site where RNA polymerase II binds

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RNA Polymerase II • Most interesting because it

regulates synthesis of mRNA • Yeast Pol II consists of 10 different

peptides (RPB1 - RPB10) • RPB1 and RPB2 are homologous to E.

coli RNA polymerase and ' • RPB1 has DNA-binding site; RPB2 binds

NTP • RPB1 has C-terminal domain (CTD) or

PTSPSYS • 5 of these 7 have -OH, so this is a

hydrophilic and phosphorylatable site

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More RNA Polymerase II

• CTD is essential and this domain may project away from the globular portion of the enzyme (up to 50 nm!)

• Only RNA Pol II whose CTD is NOT phosphorylated can initiate transcription

• TATA box (TATAAA) is a consensus promoter

• 7 general transcription factors are required

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Transcription Factors • Polymerase I, II, and III do not bind

specifically to promoters• They must interact with their

promoters via so-called transcription factors

• Transcription factors recognize and initiate transcription at specific promoter sequences

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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

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Eukaryotic Transcription

• Once initiation complex assembles process similar to bacteria (closed complex to open complex transition, primer formation)

• Once elongation phase begins most transcription factor disassociate from DNA and RNA polymerase II (but TFIIF may remain bound).

• TFIIS – Elongation factor binds at elongation phase. May also play analogous role to NusA protein in termination.


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