Transcript E. coli RNA polymerase

E. coli RNA Polymerase
M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar
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Catalyzes the formation
of the phosphodiester
bonds between the
nucleotides (sugar to
phosphate)
Uncoils the DNA, adds
the nucleotide one at a
time in the 5’ to 3’
fashion
Uses the energy
trapped in the
nucleotides themselves
to form the new bonds
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RNA polymerase adds ribonucleotides (rNTPs) not
deoxynucleotides (dNTPs)
RNA polymerase does not have the ability to
proofread what they transcribe
RNA polymerase can work without a primer
RNA will have an error 1 in every 10,000 nt (DNA is
1 in 10,000,000 nt)
Forms of RNA polymerases (RNAPs)
Bacteriophages
- large, single subunit RNA polymerases
- make specificity factors that alter the promoter
recognition of host bacterial enzymes
Bacteria
- 4 or more “core” subunits, with exchangeable
specificity factors (“sigmas”) E. coli has β, β’, α2, ω, σ
Archaea
- multiple subunits related to both bacteria and
eukaryotic
Eukaryotes
Three RNA polymerases; many with subunits
pol I - only the large ribosomal RNA subunit precursors
pol II - all pre-mRNAs, some small nuclear RNAs
(snRNAs), most small nucleolar RNAs (snoRNAs)
used in rRNA processing
pol III - tRNAs, 5S rRNA, U6 snRNA, 7SL RNA (in SRP),
and other small functional RNAs
Mitochondria and Chloroplasts
- combination of phage-like (single subunit) and
bacterial-like (multi-subunit)
Eukaryotic viruses
- can take over host RNAP or encode own in some
large viruses (e.g. vaccinia)
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Isolated in bacterial extracts in 1960 by
independent groups – Samuel Weiss and
Jerard Hurwitz
Responsible for synthesis of all 3 types of RNA
species: mRNA, rRNA and tRNA
RNAP is a huge enzyme (460 kD) made of five
subunits
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Five subunits:
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2 a subunits
1 b subunit
1 b’ subunit
1  subunit
σ factor
Core
enzyme
Holoenzyme
E. coli RNA polymerase
2α, 1β, 1β’, 1 and σ factor
Required for
polymerization
activity
Required for correct
initiation of transcription:
binding to promoter
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α subunit: Mol wt is 36.5 kDa, encoded by
rpoA gene. Required for core protein
assembly, and also play a role in promoter
recognition. Assembly of β and β’.
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β subunit: Mol wt is 151 kDa, encoded by
rpoB gene. DNA-binding active center.
Rifampicin is shown to bind to the β subunit
and inactivates.
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β’ subunit: Mol wt is 155 kDa, encoded by
rpoC gene. Responsible for binding to the
template DNA. Uses 2 Mg2+ ions for catalytic
function of the enzyme.
 subunit: Mol wt 91 kDa, encoded by rpoZ
gene. restores denatured RNA polymerase
to its functional form in vitro. It has been
observed to offer a protective/chaperone
function to the β' subunit in
Mycobacterium smegmatis.
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The processivity of E. coli RNA polymerase is
around 40 nt/sec at 37ºC, and requires Mg2+
(RNA polymerase of T3 and T7 are single
polypeptides with a processivity of 200 nt/sec)
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The enzyme has a nonspherical structure with
a projection flanking a cylindrical channel
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The size of the channel suggests that it can
bind directly to 16 bp of DNA
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The enzyme binds over a region of DNA
covering around 60 bp
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Binds the core enzyme to convert it to the holoenzyme
It is encoded by rpoD gene (σ70 )
It has a critical role in promoter recognition, but is not
required for transcription elongation
It recognizes the correct promoter site by decreasing
the affinity of the enzyme at the nonspecific DNA
sequences
The amount is only 30% to amount of the enzyme
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Each σ factor recognizes a particular
sequence of nucleotides upstream from the
gene
σ70 looks for -35 sequence TTGACA and
-10 sequence TATAAT
Other σ factors look for other sequences
The match need not always be exact
The better the match, the more likely transcription
will be initiated
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Alternative sigma factors can be classified into
two structurally unrelated families:
◦ σ70 and σ54
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Although no sequence conservation exists
between σ70 and σ54–like family members,
both types bind to core RNA polymerase.
Promoter structures recognized by σ54–RNAP
differ from those recognized by σ70–RNAP.
◦ σ54 –RNAP recognizes -24 and -12
◦ σ70 –RNAP recognizes -35 and -10
Sigma factors have four main regions that are
generally conserved:
N-terminus --------------------- C-terminus
1.1 2 3 4
The regions are further subdivided
(e.g. 2 includes 2.1, 2.2, etc.)
The exception to this organization is
in σ54-type sigma factors.
Proteins homologous to σ54/RpoN are
functional sigma factors, but they have
significantly different primary amino acid
sequences.
E. coli Sigma Factors
σ70 (RpoD) - the "housekeeping" sigma factor or also called as primary
sigma factor, transcribes most genes in growing cells. Makes the proteins
necessary to keep the cell alive.
σ54 (RpoN) - the nitrogen-limitation sigma factor
σ38 (RpoS) - the starvation/stationary phase sigma factor
σ32 (RpoH) - the heat shock sigma factor, it is turned on when exposed to
heat
σ28 (RpoF) - the flagellar sigma factor
σ24 (RpoE) - the extracytoplasmic/extreme heat stress sigma factor
σ19 (FecI) - the ferric citrate sigma factor, regulates the fec gene for iron
transport