E. coli RNA Polymerase

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. 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 • Five subunits: • 2 a subunits • 1 b subunit • 1 b’ subunit • 1  subunit • σ factor Core enzyme Holoenzyme

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

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 βandβ’. • β subunit: Mol wt is 151 kDa, encoded by rpoBgene. 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 wt91 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 σfactor 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: • σ70andσ54 • Although no sequence conservation exists betweenσ70andσ54–like family members, both types bind to core RNA polymerase. • Promoter structures recognized byσ54–RNAP differ from those recognized byσ70–RNAP. • σ54 –RNAP recognizes -24 and -12 • σ70 –RNAPrecognizes -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. 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