E. coli RNA Polymerase

M.Prasad NaiduMSc Medical Biochemistry,Ph.D.Research Scholar

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

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)

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

Five subunits:◦ 2 subunits◦ 1 subunit◦ 1 ’ subunit◦ 1 subunit◦ σ factor

Coreenzyme

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

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 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.

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

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

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 sequencesThe match need not always be exactThe better the match, the more likely transcription

will be initiated

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

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.

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 factorσ32 (RpoH) - the heat shock sigma factor, it is turned on when exposed to heatσ28 (RpoF) - the flagellar sigma factorσ24 (RpoE) - the extracytoplasmic/extreme heat stress sigma factorσ19 (FecI) - the ferric citrate sigma factor, regulates the fec gene for iron transport

E. coli Sigma Factors