chapter 21 (part 1) transcription. central dogma
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TRANSCRIPT
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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.