Monoclonal antibodies inhibiting RNA polymerase from Escherichia coli

Monoclonal hybridoma antibodies directed against RNA polymerase from E. coli have been obtained. Only a few have been found to inhibit the enzyme activity. Antibodies produced by two clones, PYN-1 and PYN-2, inhibit RNA polymerase at the stage of RNA chain elongation. The PYN-1 antibodies react with the beta'-subunit of the enzyme. The PYN-2 antibodies react with the beta-subunit and with its 130 kDa amber fragment.     

2-Ketobutyrate: a putative alarmone of Escherichia coli

2-ketobutyrate is synthesized from threonine by threonine deaminase (dehydratase) in E. coli. The effects of 2-ketobutyrate as a regulatory metabolite were studied in vivo. 2-ketobutyrate was shown to inhibit the phosphoenolpyruvate (PEP): sugar phosphotransferase system resulting in aspartate starvation, elevation of ppGpp endogenous pools, and cessation of growth in E. coli grown in glucose and related carbon sources. Accordingly, we propose that 2-ketobutyrate might serve as an alarmone whose concentration precisely governs the shift from anaerobic growth to aerobic growth in E. coli. Such shifts are common phenomena among the Enterobacteriaceae.     

Revisiting the stringent response, ppGpp and starvation signaling

Microbial adaptation to environmental stress plays an important role in survival. It is necessary to understand the mechanisms underlying the survival of microbes under stress, as they may eventually aid in the successful control of the growth and persistence of these organisms. During nutrient starvation, Escherichia coli elicits a stringent response to conserve energy. The hallmark of the stringent response is the accumulation of guanosine tetra- (ppGpp) and pentaphosphates (pppGpp), which probably bind RNA polymerase to regulate gene expression at certain promoters. Recently, there has been renewed interest in the stringent responses of other microbes, with a view to correlating it with sporulation, virulence and long-term persistence.     

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.     

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 sigma70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to sigma70. 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-sigma70 association unlike the omega subunit. The omega subunit may therefore be needed for stabilization of the structure of RNA polymerase.