Transcript Chapter 21 (part 1) - University of Nevada, Reno
Chapter 21 (part 1)
Transcription
Central Dogma
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.
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
RNA Content of E. coli Cells
type Steady State Levels Synthetic Capacity Stability rRNA 83% 58% High tRNA mRNA 14% 3% 10% 32% High Very Low
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.
E. Coli RNA Polymerase
• RNA polymerase core
enzyme is a multimeric protein
a 2 ,b, b
’
, w • The • The b
’ subunit is involved in DNA binding site
• The b a
subunit contains the polymerase active subunit acts as scaffold on which the other subunits assemble.
• Also requires s
-factor for initiation –forms holo enzyme complex Site of DNA binding and RNA polymerization
s
-factor
• The s
-factor is required for binding of the RNA polymerase to the promoter
• Association of the RNA polynerase core complex
w/ the
s
-factor forms the holo-RNA polymerase complex
• W/o the s
-factor the core complex binds to DNA non-specifically.
• W/ the
region
s
-factor, the holo-enzyme binds specifically with high affinity to the promoter
• Also decreases the affinity of the RNA
polymerase to non-promoter regions
• Different s
-factors for specific classes of genes
5’
General Gene Structure
Promoter Transcribed region • 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.
terminator 3’
Gene Promoters
• Site where RNA polymerase binds and initiates
transcription.
• Gene that are regulated similarly contain
common DNA sequences (concensus sequences) within their promoters
Important Concensus Sequences
• Pribnow Box – position –10 from
transcriptional start
• -35 region – position –35 from
transcriptional start.
• Site where s 70
-factor binds.
Other
s
-Factors
• Standard genes – s
70
• Nitrogen regulated genes – s • Heat shock regulated genes –
54
s
32
How does RNA polymerase finds the promoter?
• RNA polymerase does not disassociate
from DNA strand and reassemble at the promoter (2 nd 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
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).
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 PP i )
• When 6-10 unit oligonucleotide has been made, sigma
subunit dissociates, completing "initiation“
• NusA protein binds to core complex after
disassociation of
s
-factor to convert RNA polymerase to elongation form.
Transcriptional Initiation
Closed complex Open complex Primer formation Disassociation of
s
-factor
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
Transcriptional Termination
• Process by which RNA polymerase
complex disassembles from 3’ end of gene.
• Two Mechanisms – Pausing and
“rho-mediated” termination
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
Rho Dependent Termination
• rho is an ATP-
dependent helicase
• it moves along RNA
transcript, finds the "bubble", unwinds it and releases RNA chain
Eukaryotic Transcription
• Similar to what occurs in
prokaryotes, but requires more accessory proteins in RNA polymerase complex.
• Multiple RNA polymerases
type
Eukaryotic RNA Polymerases
Location Products RNA polymerase I RNA polymerase II RNA polymerase III Mitochondrial RNA polymerase Chloroplast RNA polymerase Nucleolus Nucleoplasm Nucleoplasm Chloroplast rRNA mRNA rRNA, tRNA, others Chloroplast gene transcripts
II, and III
Eukaryotic RNA
• All 3 are big,
(500-700 kD) subunits with
Polymerases
• RNA polymerase I,
multimeric proteins
• All have 2 large
sequences similar to
b
and
b
' 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
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 PTSPSYS
b
and
b
'
• RPB1 has DNA-binding site; RPB2 binds NTP • RPB1 has C-terminal domain (CTD) or • 5 of these 7 have -OH, so this is a
hydrophilic and phosphorylatable site
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
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
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
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.