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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
(-35)TNNCNCNCTTGAA (-10)CCCATNT
heat shock factor involved in activation of genes after
heat shock.
POINT: gives E. coli flexibility in responding to
different conditions
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
transcript
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?
Coding strand
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
tRNA and
rRNA
Processing
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Both are
excised from
large primary
transcripts
1º transcript
may contain
several tRNA
molecules, tRNA
and rRNA
rRNAs simply
excised from
larger transcript
tRNAs modified
extensively
5. Base
modifications
Examples of Covalent Modification of Nucleotides in tRNA
CH3
H
H
CH3
N
N
6
N
H
N
N
6
N
N
N
C
C
CH3
CH2
O
N
N
N6-Methyladenylate
N6-Isopentenyladenylate
(m6A)
(i6A)
O
H
H
H
H
NH
HN 3
4
N
O
Dihydrouridylate
(D)
2
1
5
C
H2C
NH
O
N
O
Uridylate
5-oxyacetic acid
(cmo5U)
Pseudouridylate (Ψ)
(ribose at C-5)
N
N
3
N
CH3
6
CH2
5
NH
NH
5
NH2
7-Methylguanylate
(m7G)
O
O
N
N
O
3-Methylcytidylate
(m3C)
O
NH2
H3C
N
N
O
C
NH
7
Inosinate
(I)
HO
O
N
NH
N
N
O
CH3
O
5-Methylcytidylate
(m5C)
Base
O
H
H
O
H
O CH3
H
1'
2'
2’-O-Methylated nucleotide
(Nm)
Modifications are
shown in blue.
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 Polymerase I Promoters
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RNA Polymerase I
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Transcribes rRNA
Sequence not well conserved
Two elements
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Core element- surrounds the transcription
start site (-45 to + 20)
Upstream control element- between -156 and
-107 upstream
Spacing affects strength of transcription
Eukaryotic Polymerase II Promoters
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Much more complicated
Two parts
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Core promoter
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Core promoter
Upstream element
TATA box at ~-30 bases
Initiator—on the transcription start site
Downstream element-further downstream
Many natural promoters lack recognizable
versions of one or more of these sequences
TATA-less Promoters
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Some genes transcribed by RNA pol II lack the
TATA box
Two types:
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Housekeeping genes ( expressed constitutively). EX
Nucleotide synthesis genes
Developmentally regulated genes. EX Homeotic genes
that control fruit fly development.
Specialized (luxury) genes that encode cell-type
specific proteins usually have a TATA-box
mRNA Processing in Eukaryotes
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Primary transcript much larger than
finished product
Precursor and partially processed RNA
called heterogeneous nuclear RNA
(hnRNA)
Processing occurs in nucleus
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Splicing
Capping
Polyadenylation
Capping mRNA
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5’ cap is a reversed
guanosine residue so
there is a 5’-5’ linkage
between the cap and
the first sugar in the
mRNA.
Guanosine cap is
methylated.
First and second
nucleosides in mRNA
may be methylated
BACK
Polyadenylation
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Polyadenylation occurs on the 3’ end of
virtually all eukaryotic mRNAs.
Occurs after capping
Catalyzed by polyadenylate polymerase
Polyadenylation associated with mRNA
half-life
Histones not polyadenylated
Introns and Exons
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Introns--Untranslated
intervening
sequences in mRNA
Exons– Translated
sequences
Process-RNA splicing
Heterogeneous
nuclear RNA
(hnRNA)-Transcript
before splicing is
complete
Splicing Overview
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Occurs in the nucleus
hnRNAs complexed with specific proteins,
form a ribonucleoprotein particle (RNP)
Primary transcripts assembled into hnRNP
Splicing occurs on spliceosomes consist of
Small nuclear ribonucleoproteins (SnRNPs)
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components of spliceosomes
Contain small nuclear RNA (snRNA)
Many types of snRNA with different functions
in the splicing process
Spliceosome
Splice Site Recognition
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Introns contain invariant 5’-GU and 3’-AG sequences at their
borders (GU-AG Rule)
Recognized by small nuclear ribonucleoprotein particles
(snRNPs) that catalyze the cutting and splicing reactions.
Internal intron sequences are highly variable even between
closely related homologous genes.
Alternative splicing allows different proteins from a single
original transcript
Simplified Splicing Mechanism
Close-up of Internal A
Alternative
Splicing I
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Exon
removed
with intron
Alternative
Splicing II
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Multiple 3’
cleavage
sites
EX. AG
found at 5’
end of
exon 2
and inside
exon 2
RNA pol III
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Precursors to tRNAs,5SrRNA, other small RNAs
Promoter Type I
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Lies completely within the transcribed region
5SrRNA promoter split into 3 parts
tRNA promoters split into two parts
Polymerase II-like promoters
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EX. snRNA
Lack internal promoter
Resembles pol II promoter in both sequence and
position
DNAse
Footprinting
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Protected region
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Use: promoter
ID
End Label
template strand
Add DNA
binding protein
Digest with
DNAse I
Remove protein
Separate on gel
siRNA and microRNA
Maxam-Gilbert
Sequencing
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Prep ssDNA
End label
Treat with different
reagents specific
for each nt
RNA Splicing
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RNA splicing is the
removal of
intervening
sequences (IVS)
that interrupt the
coding region of a
gene
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Excision of the IVS
(intron) is
accompanied by the
precise ligation of
the coding regions
(exons)
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
mRNA-monocistronic
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
Methods for Studying Regulatory DNA
DNAse footprinting: (aka DNAse protection
assay) identifies the sequence of DNA
bound by a transcription factor
protein binding prevents DNA from being
attacked by DNAse I
otherwise DNAse I cuts random sequences
so that bands of many sizes are found on a
gel everywhere EXCEPT where the
transcription factor protects the DNA
DNA Footprinting
What are the roles of TAFs?
TAFIIs help TBP with transcirption from promoters with initiators an downstream elemens
• Prokaryotes
• rrn operons
•The bacterium have several rrn operons that
contain rRNA genes.
tRNA tRNA
tRNA
•Transcription of the rrnD operon yields a 30S
precursor, which must be cut up to release the
three rRNA and three tRNAs.
•rRNA processing
Formation of a cap at
the 5’ end of a
eukaryotic mRNA
precursor
1
2
4
3
DNA footprinting
short segment of 32P end-labeled dsDNA
1. Unprotected
control DNA
2. Protected by
DNA binding protein
Partial digestion
with DNase
1
Gelelectrophoresis
and autoradiography
32P
end-labeled fragments
2
Facts to remember
DNA-dependent RNA synthesis:
1.
2.
3.
4.
5.
6.
7.
8.
starts at a promoter sequence, ends at
termination signal
the first 5’-triphosphate is NOT cleaved
proceeds in 5’ to 3’ direction
new residues are added to the 3’ OH
the template is copied in the 3’ – 5’ direction
forms a temporary DNA:RNA hybrid
transcription rate ( 50 to 90 nts/sec)
RNA polymerase has complete processivity
Rho (protein) dependent termination
Is Rho a termination factor?
 Rho affects chain elongation, but not
initiation.
 Rho causes production of shorter
transcripts.
 Rho release transcripts from the DNA
template.
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1.
2.
DNA termination site:
sites on DNA with specific structure
DNA template contains inverted repeats
6 to 8 A sequence on the DNA template that codes for
U