Transcript Document 7780786

Transcription
Chapter 8
The Problem
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Information must be transcribed from DNA
in order function further.
REMEMBER:
DNARNAProtein
Tanscription in Prokaryotes
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Polymerization catalyzed by RNA
polymerase
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Can initiate synthesis
Uses rNTPs
Requires a template
Unwinds and rewinds DNA
4 stages
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Recognition and binding
Initiation
Elongation
Termination and release
RNA Polymerase
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5 subunits, 449 kd (~1/2 size of DNA pol
III)
Core enzyme
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2  subunits---hold enzyme together
--- links nucleotides together
’---binds templates
---recognition
Holoenzyme= Core + sigma
RNA Polymerase Features
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Starts at a promoter sequence, ends
at termination signal
Proceeds in 5’ to 3’ direction
Forms a temporary DNA:RNA hybrid
Has complete processivity
RNA Polymerase
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X-ray studies reveal a
“hand”
Core enzyme closed
Holoenzyme open
Suggested mechanism
NOTE: when sigma
unattached, hand is
closed
RNA polymerase stays on
DNA until termination.
Recognition
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Template strand
Coding strand
Promoters
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Core promoter elements for E. coli
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Binding sites for RNA pol on template strand
~40 bp of specific sequences with a specific
order and distance between them.
-10 box (Pribnow box)
-35 box
Numbers refer to distance from
transcription start site
Template and Coding Strands
Sense (+) strand
DNA coding strand
Non-template strand
5’–TCAGCTCGCTGCTAATGGCC–3’
3’–AGTCGAGCGACGATTACCGG–5’
transcription
DNA template strand
antisense (-) strand
5’–UCAGCUCGCUGCUAAUGGCC–3’
RNA transcript
Typical Prokaryote Promoter
Consensus sequences
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Pribnow box located at –10 (6-7bp)
-35 sequence ~(6bp)
Consensus sequences: Strongest
promoters match consensus
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Up mutation: mutation that makes
promoter more like consensus
Down Mutation: virtually any mutation that
alters a match with the consensus
In Addition to Core Promoter Elements
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UP (upstream promoter) elements
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Gene activator proteins
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Ex. E. coli rRNA genes
Facilitate recognition of weak promoter
E. coli can regulate gene expression
in many ways
Stages of Transcription
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Template recognition
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Initiation
Elongation
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RNA pol binds to DNA
DNA unwound
RNA pol moves and synthesizes
RNA
Unwound region moves
Termination
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RNA pol reaches end
RNA pol and RNA released
DNA duplex reforms
Transcription Initiation
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Steps
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Formation of closed promoter (binary)
complex
Formation of open promoter complex
Ternary complex (RNA, DNA, and enzyme),
abortive initiation
Promoter clearance (elongation ternary
complex)
First rnt becomes unpaired
 Polymerase loses sigma
 NusA binds
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Ribonucleotides added to 3’ end
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Holoenzyme
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Core + 
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Closed (Promoter)
Binary Complex
Open binary complex
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Ternary complex
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Promoter clearance
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Back
Sigma () Factor
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Essential for recognition of promoter
Stimulates transcription
Combines with holoenzyme
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“open hand” conformation
Positions enzyme over promoter
Does NOT stimulate elongation
Falls off after 4-9 nt incorporated
“Hand” closes
Variation in Sigma
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Variation in promoter sequence affects
strength of promoter
Sigmas also show variability
Much less conserved than other RNA pol
subunits
Several variants within a single cell. EX:
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E. coli has 7 sigmas
B. subtilis has 10 sigmas
Different  respond to different promoters
Sigma Variability in E. coli
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Sigma70
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(-35)CTGGCAC
(-10)TTGCA
alternative sigma factor involved in transcribing nitrogenregulated genes (among others).
Sigma32
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(-10)TATAAT
Primary sigma factor, or housekeeping sigma factor.
Sigma54
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(-35)TTGACA
heat shock factor involved in activation of genes after
heat shock.
SigmaS (sigma38)
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stationary phase sigma factor.
Activates genes involved in long term survival, eg.
peroxidase.
Sigma and Phage SP01
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Early promoter—recognized by bacterial
sigma factor. Transcription includes
product, gp28.
gp28 recognizes a phage promoter for
expression of mid-stage genes, including
gp33/34, which recognizes promoters for
late gene expression.
Promoter Clearance and Elongation
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Occurs after 4- 10 nt are added
First rnt becomes unpaired from antisense
(template) strand.DNA strands re-anneal
Polymerase loses sigma, sigma recycled
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Result “Closed hand” surrounds DNA
NusA binds to core polymerase
As each nt added to 3’, another is melted
from 5’, allowing DNA to re-anneal.
RNA pol/NusA complex stays on until
termination. Rate=20-50nt/second.
Termination
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Occurs at specific sites on template strand
called Terminators
Two types of termination
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Intrinsic terminators
Rho () dependent treminators
Sequences required for termination are in
transcribed region
Variation in efficiencies.
Intrinsic Terminators
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DNA template contains inverted repeats (G-C rich)
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Can form hairpins
6 to 8 A sequence on the DNA template that codes
for U
Consequences of poly-U:poly-A stretch?
Intrinsic Termination
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RNA pol passes over
inverted repeats
Hairpins begin to form
in the transcript
Poly-U:poly-A stretch
melts
RNA pol and transcript
fall off
UUUUU
Rho () Dependent Terminators
rho factor is ATP dependent helicase
 catalyses unwinding of RNA: DNA
hybrid
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Rho
Dependent
Termination
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rho factor is
ATP
dependent
helicase
catalyzes
unwinding
of RNA:
DNA hybrid
50~90
nucleotides/
sec
(17 bp)
Rho:
Mechanism
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Rho binds to transcript
at  loading site (up
stream of terminator)
Hairpin forms, pol stalls
Rho helicase releases
transcript and causes
termination
hexamer
Abortive
initiation,
elongation
mRNA
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Function—Transcribe message from DNA to
protein synthesis machinery
Codons
Bacterial—polycistronic
Eukaryotic– monocistronic
Leader sequence—non-translated at 5’ end
 May contain a regulatory region (attenuator)
Also untranslated regions at 3’ end.
Spacers (untranslated intercistronic sequences)
Prokaryotic mRNA—short lived
Eukaryotic mRNA-can be long lived
Stable RNA
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rRNA -Structural component of ribosomes
tRNA-Adaptors, carry aa to ribosome
Synthesis
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Promoter and terminator
Post-transcriptional modification (RNA
processing)
Evidence
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Both have 5’ monophospates
Both much smaller than primary transcript
tRNA has unusual bases. EX pseudouridine
Eukaryotic
Transcription
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3 classes RNA pol (I-III)
Many mRNA long lived
5’ and 3’ ends of mRNA
modified. EX.
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5’ cap
Poly-A tail
Primary mRNA transcript
large, introns removed
Monocistronic
Eukaryotic Transcription
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Regulation very complex
Three different pols distinguished by amanitin sensitivity
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Pol I—rRNA, least sensitive
Pol II– mRNA, most sensitive
Pol III– tRNA and 5R RNA moderately
sensitive
Each polymerase recognizes a distinct
promoter
Eukaryotic RNA Polymerases
RNA Pol. Location Products
-Amanitin Promoter
Sensitivity
I
Nucleolus Large rRNAs
(28S, 18S,
5.8S)
II
Nucleus
Pre-mRNA,
some snRNAs
Highly
sensitive
III
Nucleus
tRNA, small
rRNA (5S),
snRNA
Intermediate
sensitivity
Insensitive
bipartite
promoter
Upstream
Internal
promoter and
terminator
Eukaryotic RNA Polymerases
RNA Pol.
I
II
III
Location
Products
-Amanitin
sensitivity
Nucleolus
Large rRNAs
(28S, 18S, 5.8S)
Insensitive
Nucleus
Pre-mRNA, some
snRNAs,
snoRNAs
Highly
sensitive
Nucleus
tRNA, small
rRNA (5S),
snRNA
Intermediate
sensitivity