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Bacteria are known to be constantly adapting to become resistant to antibiotics. Currently, efficient antibacterial compounds are still available; however, it is only a matter of time until these compounds also become inefficient. Ribonucleases are the enzymes responsible for the maturation and degradation of RNA molecules, and many of them are essential for microbial survival. Members of the PNPase and RNase II families of exoribonucleases have been implicated in virulence in many pathogens and, as such, are valid targets for the development of new antibacterials. In this paper, we describe the use of virtual high-throughput screening (vHTS) to identify chemical compounds predicted to bind to the active sites within the known structures of RNase II and PNPase from Escherichia coli. The subsequent in vitro screening identified compounds that inhibited the activity of these exoribonucleases, with some also affecting cell viability, thereby providing proof of principle for utilizing the known structures of these enzymes in the pursuit of new antibacterials. [ABSTRACT FROM AUTHOR]

The coordinated regulation of gene expression is required for homeostasis, growth and development in all organisms. Such coordination may be partly achieved at the level of messenger RNA stability, in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors. RNase E cleaves RNA internally, but its catalytic power is determined by the 5' terminus of the substrate, even if this lies at a distance from the cutting site. Here we report crystal structures of the catalytic domain of RNase E as trapped allosteric intermediates with RNA substrates. Four subunits of RNase E catalytic domain associate into an interwoven quaternary structure, explaining why the subunit organization is required for catalytic activity. The subdomain encompassing the active site is structurally congruent to a deoxyribonuclease, making an unexpected link in the evolutionary history of RNA and DNA nucleases. The structure explains how the recognition of the 5' terminus of the substrate may trigger catalysis and also sheds light on the question of how RNase E might selectively process, rather than destroy, specific RNA precursors.

Experimental studies on 10-13 nucleotides which have the RNase E-cleaved sequence of RNA I but lack the stem-loop indicate that the nucleotides are cleaved at the same phosphodiester bond as RNA I and the cleavage is 20 times more efficient than that of RNA I. RNase E does not enter via stem-loops, which merely restrict cleavage at susceptible sites. Mutations in a non-adjacent stem-loop restrict cleavage, indicating that enzyme access is affected by distant conformational changes.

The temperature sensitive mutations ams-1 and rne-3071 are consequences of a G to A transition at position 736 in the nucleotide sequence and a C to T transition at position 742 respectively. An altered Ams domain results from certain modifications in the ams (altered mRNA stability) gene. Glycine is substituted by serine as a result of ams-1 mutation, and leucine is substituted by phenyalanines as a result of rne-3071 mutation. Th Ams domain is homologous to a protein responsible for conferring bacterial shape. In addition, there are other regions in the ams protein which share homology with cytoskeltetal proteins.

The processing activity of the endoribonuclease, RNase E, is mediated by a conserved catalytic domain, which is a homotetramer. Results show that the tetramer binds up to four molecules of specific RNA substrate analogue and that the tetramer possesss D(sub)2 dihedral symmetry. Data point out that the S1-RNA binding domains of RNase E lies on the periphery of the tetramer.

The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators ( CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin ( C-1027), a clinically relevant antitumor agent, by S treptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S . globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S . coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S . globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes. [ABSTRACT FROM AUTHOR]

S treptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. E scherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E . coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S . coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E . coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression. [ABSTRACT FROM AUTHOR]