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

2. RNA Polymerase
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. Differences in DNA and RNA Polymerases
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. Mitochondria and Chloroplasts
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. Bacterial RNA polymerase
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. E. coli RNA polymerase Core enzyme Holoenzyme Five subunits:
2 a subunits
1 b subunit
1 b’ subunit
1  subunit
σ factor
Core
enzyme
Holoenzyme

8. E. coli RNA polymerase 2α, 1β, 1β’, 1 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
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.
 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. E. coli RNA polymerase
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. σ (sigma) Factor 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
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
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. 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

18. Referencec : www.slideshare.com