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
14%
10%
High
mRNA
3%
32%
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 a2,b, b’, w
• The b’ subunit is
involved in DNA
binding
• The b subunit contains
the polymerase active
site
• The a 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 s-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 s-factors for specific classes of
genes
General Gene Structure
5’
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 s70-factor binds.
Other s-Factors
• Standard genes – s70
• Nitrogen regulated genes – s54
• Heat shock regulated genes – s32
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 nonspecifically 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 PPi)
• 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 ATPdependent 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
Eukaryotic RNA
Polymerases
type
Location
Products
RNA polymerase I
Nucleolus
rRNA
RNA polymerase
II
Nucleoplasm
mRNA
RNA polymerase
rRNA, tRNA,
Nucleoplasm
III
others
Mitochondrial RNA
Mitochondrial gene
Mitochondria
polymerase
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 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 b and b'
• 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
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 holoenzyme complex
• TFIID – Initiation factor,
contains TATA binding
protein (TBP) subunit.
TATA box recognition.
• TFIIF – (RAP30/74)
decrease affinity to nonpromoter 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.