Download RNA synthesis/Transcription I Biochemistry 302

RNA synthesis/Transcription I
Biochemistry 302
February 4, 2005
Bob Kelm
RNA metabolism: major and minor
classes of RNA
•
Messenger RNA (mRNA)
– Relatively short half-life (∼3
min in E. coli, ∼30 min in
eukaryotic cells)
•
Ribosomal RNA (rRNA)
– Major structural components
of the ribosome
•
Transfer RNA (tRNA)
– Adaptor molecules allowing
physical linkage between
mRNA and amino acids
•
“Small” RNAs
– snRNAs (splicing)
– Components of RNP
enzymes (e.g. RNase P)
– miRNAs (micro RNAs
involved in PTGS)
Overview of RNA polymerases
• Prokaryotes
– Single processive RNA
polymerase (technically,
primase is a RNAP too).
– Inhibited by rifampicin
(binds RNAP β subunit &
blocks path of RNA chain
elongation)
• Eukaryotes
– Three processive RNAPs
– Differential sensitivity to
inhibition by α-amanitin
• RNA Pol I (resistant)
→ rRNA
• RNA Pol II (low conc)
→ mRNA
• RNA Pol III (high conc)
→ tRNA plus 5S rRNA
Fig. 26.4
Note: α-amanitin, a non-competitive
inhibitor, stops the translocation of
RNAP along the DNA template after
the formation of the first phosphodiester bond.
Features of RNA vs DNA synthesis
• Similarities to DNA synthesis
–
–
–
–
Synthesis of ribonucleotide chain is template-dependent.
Substrates are nucleoside triphosphates (rNTPs).
Direction of chain growth is 5′→3′.
Same chemical mechanism applies (base-pairing of incoming
rNTP, 3′ OH attack, loss of PPi).
– Highly processive enzyme
• Differences from DNA synthesis
– One DNA strand is transcribed per gene w/o a primer.
– Only certain genes are transcribed at any given time.
– Kinetics favor “slow” transcription of multiple genes. (Vmax ∼50
nt/s for RNA Pol vs ∼103/s for DNA Pol III; ∼3000 RNA Pol/cell vs
∼10 DNA Pol III complexes/cell)
– Less accurate ∼10-5 vs 10-10
– Cofactor-mediated proofreading
Anatomy,chemistry, and nomenclature
of RNAP-mediated transcription in E. coli
~17 bp
NTP
~35 bp for RNAP
“footprint”
Lehninger Principles of Biochemistry, 4th ed., Ch 26
Biochemical features of E. coli RNAP
Core RNAP
holoenzyme assembly
contains part of active site
*
•
•
•
•
sliding clamp
450 kDa enzyme containing six subunits
Two Mg2+ and one Zn2+ required (chemistry and clamping)
No independent 3′→5′ exonuclease activity but may have
kinetic proofreading capabilities
Two binding sites for ribonucleotides
– Initiation site binds only purine rNTPs (GTP or ATP) with Kd =
100 µM…most mRNAs start with purine on 5′ end.
– Elongation site binds any of 4 rNTPs with Kd = 10 µM.
σ factors: regulatory factors which
direct transcription of certain genes
• Assist RNAP in binding DNA at the proper site for
initiation of transcription – the promoter.
• Different sigma factors orchestrate transcription
of different classes of genes.
– Heat shock (σ35)
– Other stress responses
– Metabolic enzymes (σ70, most abundant)
• Not required for core RNA polymerase activity.
Transcription like replication can be
construed to occur in distinct steps
• Initiation (requires special signals)
– RNAP recognizes the promoter, binds to DNA,
and starts transcription.
– Highly regulated
• Elongation
– RNAP tracks down the length of the gene
synthesizing RNA along the way.
• Termination (requires special signals)
– Transcription stops then RNAP and the
nascent mRNA dissociate.
Features of initiation phase in E. coli
1:RNAP binding and sliding
(electrostatic interaction)
Signal for specific
DNA-binding seen
by σ factor
2:Formation of closed complex
(–55 to –5, Ka∼107-108 M−1,T½~10 s)
3:Formation of open complex
(–10 to –1, Ka∼1012 M−1, T½~15s to
20 min), temp-dependent, stable
4:Mg2+-dependent conformational
change (–12 to +2), add 1st nt
5:Promoter clearance: RNAP
moves away from promoter
6:Release of σ after first 8-9 nts &
continuation of elongation (now
cannot be inhibited by rifampicin)
Fig. 26-6
7,8:Pausing → Termination
Transcription initiation: key role of the
gene promoter
• RNAP binding sequence: −70 to +30 in E. coli
• DNA sequence specifying start site and basal rate
of transcription
– Constitutive: Specify that a gene product will be
transcribed at a constant rate (e.g. genes involved in
metabolic control)
– Inducible or regulated: Specify transcription of certain
genes in response to external signals (requires
additional protein-DNA interactions)
• Promoter recognition by RNAP: rate limiting for
transcription (structure → frequency of initiation)
• Promoters: exhibit certain consensus sequences
Sequence conservation of core
promoter elements (RNAP-σ70)
•Variations in sequence and
core element position account
for differences in frequency of
initiation.
• 1975, David Pribnow and
Heinz Schaller independently
defined consensus promoter
sequences, the –10 region or
Pribnow box (TATAAT) and
the –35 region (TTGACA).
• Among 114 E. coli promoters
studied, 6/12 nucleotides in the
two consensus elements
found in 75% of promoters.
Fig. 26-11
Transcription start site
Genetic evidence for functional
importance of core promoter elements
(naturally-occurring and site-directed mutations)
• The more closely core
elements resemble the
consensus, the more
efficient the promoter at
initiating transcription.
• ↑Mutations: those toward
the consensus sequence.
• ↓Mutations: those away
from the consensus
sequence.
• Spacing (optimal 17 bp)
between core consensus
sequences is important.
Fig. 26-12
Biochemical evidence of RNAP binding
to lac promoter: Footprint analysis
Lehninger Principles of Biochemistry, 4th ed., Ch 26
Putative structure of E. coli core RNA
polymerase during elongation phase
β and β′ subunits: light gray and white, α subunits shades of red, ω
subunit on other side not visible.
Note circuitous route taken by the DNA and RNA through the complex.
Transcription elongation: a detailed view
• Elongation complexes are
stabilized by contact between
specific regions/residues of β/β′
and the growing RNA chain
(RBS), heteroduplex (HBS), or
“downstream” DNA (DBS).
• Core RNAP moves along the
DNA template simultaneously
unwinding DNA ahead and
rewinding the template behind.
Zn2+-binding domain of β′
subunit is the sliding clamp.
RNAP activity requires Mg2+.
Formation of 5′ RNA hairpin may
be a signal for termination.
Fig. 26-9
But elongation of ternary complex
often proceeds discontinuously….
• Transcription “bubble”
model implies continuous
movement but RNAP may
pause at difficult to “read”
sites (e.g. high G/C content).
“backtracked” RNAP
Fig. 26-10
• Resolution of pause sites
may involve backtracking to
create a RNA 3′ end which is
displaced from the active
site.
• GreA and GreB bind
transiently to RNAP active
site and stimulate its
intrinsic transcript (i.e. RNA)
hydrolysis activity creating
a new base-paired 3′ end.
Donation of catalytic residues to RNAP
by GreB (RNAP in hydrolysis mode)
Sosunova et al. PNAS
100:15469, 2003
GreB
turned
120°
relative to
RNAP β′
Termination of transcription: another
process controlled by signals in DNA
• Termination signals are similar to signals
that promote pausing
– High G/C content (tend to form stem-loop structure)
– Palindromic sequences that de-stabilize the DNA/RNA
heteroduplex
• Two types of termination mechanisms
– Factor independent: Dyad symmetry followed by
poly A sequence - intrastrand stem loop followed by
rU:dA that destabilizes RNA/template
– Factor (ρ, rho) dependent: Rho protein (RNA-
dependent ATPase) destabilizes the RNA-DNA duplex.
Rho factor-independent (or sequencedependent) termination
a: RNAP pauses when it reaches
G:C sequence that enzyme finds
hard to unwind.
b: Pausing allows time for selfcomplementary regions of RNA
transcript to bp. This displaces
some RNA from DNA & RNAP RBS.
c: Unstable A-U bonds cannot hold
weakened ternary complex (RNAP +
RNA + DNA) together. RNAP and
mRNA dissociate from the DNA
template.
Note: Actual mechanism is more
complex and requires additional
signals both 5′ and 3′.
Fig. 26-15
Rho-dependent termination…less frequent
and more complex
1: Rho (ρ) protein binds as a
homohexamer to RNA at a
CA-rich site (rut for rho
utilization) near 3′ end and
slides toward paused RNAP.
2: RNA-DNA helicase and
ATPase activity of Rho
unwinds RNA away from
template DNA.
3: Template and transcript
dissociate.
Note: An additional protein,
NusA, may be required for
RNAP pausing. NusA binds to
core RNAP after σ has
dissociated.
Fig. 26-16
NusA = N utilization substance