RNA polymerase and its accessory factors in Escherichia coli

RNA polymerase is one of the most important enzymes in living organizms as it controls the synthesis of the key intermediate RNA from DNA message. Regulation of gene expression is primarily governed by the interplay among various protein factors and this enzyme. In this review we tried to emphasize and catalogue all the factors that are known till-date in prokaryotes, taking E., coli as a model. Structural requirements for intricate protein-protein and protein-DNA interaction are also discussed

Studies on the ω subunit of Escherichia coli RNA polymerase: its role in the recovery of denatured enzyme activity

Highly purified Escherichia coli RNA polymerase contains a small subunit termed ω that has a molecular mass of 10105 Da and is comprised of 91 amino acids. To elucidate the function of ω, whose role is as yet undefined, the subunit was purified to over 95% purity from an overproducing strain [BL21 (pGP1-2, pE3C-2)]. Purified ω was then reconstituted with RNA polymerase isolated from an ω-less mutant. Externally added ω inhibited promoter-specific transcriptional activity at all promoters tested. Renaturation of fully denatured ω-less RNA polymerase in the presence of excess ω yielded maximum recovery of activity suggesting a structural rather than functional role for ω

Alteration in template recognition by Escherichia coli RNA polymerase lacking the \omega subunit: A mechanistic analysis through gel retardation and foot-printing studies

The \omega subunit of Escherichia coli RNA polymerase is a 91 amino acid polypeptide which co-purifies with the enzyme and is thought to help in maturing the rest of the enzyme to its full functionality. Purified \omega when added externally was found to inhibit general transcriptional activity of \omega-less RNA polymerase as well as promoter-specific single-round transcriptional activity at all the promoters tested. In this study we have tried to analyse the observed inhibition of transcription using gel retardation assays and KMnO4 foot- printing. Further, through protein foot-printing we have attempted to identify alterations in the interaction of the \omega-less core enzyme with the s70 subunit. Our results suggest that the \omega-less holoenzyme has lesser affinity towards the DNA template and external addition of \omega destabilizes the open complex for both the wild-type and \omega- less enzyme. The \omega-less core enzyme interacts with the s70 subunit to expose the - 35 recognition domain (domain 4.2) unlike that observed in the wild-type interaction. Thus the absence of the omega subunit leads to the formation of an enzyme which has altered DNA binding and s70 binding properties. Circular dichroic measurements also indicate a major conformational alteration of both hole and core RNA polymerase in the presence and absence of the \omega subunit

GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the \omega subunit in vivo

Highly purified Escherichia coli RNA polymerase contains a small subunit termed \omega that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking \omega (\omega-less) are viable, but exhibit a slow-growth phenotype. Renaturation of RNA polymerase isolated from an \omega-less mutant, in the presence of \omega, resulted in maximum recovery of activity. The \omega-less RNA polymerase from \omega-less strains recruits the chaperonin, GroEL (unlike the wild-type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL-containing core RNA polymerase interacts efficiently with \sigma^7^0 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to \sigma^7^0. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of \omega or GroEL. GroES was found to have an inhibitory effect on the core-\sigma^7^0 association unlike the \omega subunit. The \omega subunit may therefore be needed for stabilization of the structure of RNA polymerase

Peptide Deformylase Inhibitors as Potent Antimycobacterial Agents

Peptide deformylase (PDF) catalyzes the hydrolytic removal of the N-terminal formyl group from nascent proteins. This is an essential step in bacterial protein synthesis, making PDF an attractive target for antibacterial drug development. Essentiality of the def gene, encoding PDF from Mycobacterium tuberculosis, was demonstrated through genetic knockout experiments with Mycobacterium bovis BCG. PDF from M. tuberculosis strain H37Rv was cloned, expressed, and purified as an N-terminal histidine-tagged recombinant protein in Escherichia coli. A novel class of PDF inhibitors (PDF-I), the N-alkyl urea hydroxamic acids, were synthesized and evaluated for their activities against the M. tuberculosis PDF enzyme as well as their antimycobacterial effects. Several compounds from the new class had 50% inhibitory concentration (IC(50)) values of <100 nM. Some of the PDF-I displayed antibacterial activity against M. tuberculosis, including MDR strains with MIC(90) values of <1 μM. Pharmacokinetic studies of potential leads showed that the compounds were orally bioavailable. Spontaneous resistance towards these inhibitors arose at a frequency of ≤5 × 10(−7) in M. bovis BCG. DNA sequence analysis of several spontaneous PDF-I-resistant mutants revealed that half of the mutants had acquired point mutations in their formyl methyltransferase gene (fmt), which formylated Met-tRNA. The results from this study validate M. tuberculosis PDF as a drug target and suggest that this class of compounds have the potential to be developed as novel antimycobacterial agents

Peptide deformylase inhibitors of Mycobacterium tuberculosis: synthesis, structural investigations, and biological results.

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported