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1. E. coli RNA Polymerase
M.Prasad Naidu
MSc Medical Biochemistry,
Ph.D.Research Scholar

2.  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

3.  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)

4. 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

5. 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)

6.  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

7.  Five subunits:
◦ 2 α subunits
◦ 1 β subunit
◦ 1 β’ subunit
◦ 1 ω subunit
◦ σ factor
Core
enzyme
Holoenzyme

8. E. coli RNA polymerase
2α, 1β, 1β’, 1ω and σ factor
Required for
polymerization
activity
Required for correct
initiation of transcription:
binding to promoter

9.  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’.
 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.
 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.
• ω 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.

10.  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)
 The enzyme has a nonspherical structure with
a projection flanking a cylindrical channel
 The size of the channel suggests that it can
bind directly to 16 bp of DNA
 The enzyme binds over a region of DNA
covering around 60 bp

13.  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

14.  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

15.  Alternative sigma factors can be classified into
two structurally unrelated families:
◦ σ70
and σ54
 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

16. 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.

17. 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
E. coli Sigma Factors