Dissecting the extended "-10" Escherichia coli rpsB promoter activity and regulation in vivo.

As we have shown previously, transcription of the rpsB-tsf operon encoding essential components of the translation machinery, a ribosomal protein S2 and an elongation factor Ts, is driven by a single promoter PrpsB, which is highly conserved among γ-proteobacteria. PrpsB belongs to the extended "-10" promoter class; it comprises a TGTG-extension upstream of the "-10" hexamer TATAAA, a suboptimal "-35" region TTGGTG, and a GC-rich discriminator GCGCGC that separates the "-10" element from the transcription start site. In this work, we examined an impact of site-directed mutations in the rpsB promoter region on expression of the reporter gene PrpsB-lacZ within the E. coli chromosome as well as promoter regulation by transcription factors ppGpp and DksA upon amino acid starvation. The results show that the transcription level largely depends on both the TGTG-extension and the TTG-element in the "-35" region, as mutations in these sequences dramatically decrease the activity of the promoter. Upon induction of amino acid starvation, the rpsB promoter is negatively regulated by ppGpp due to the presence of the GC-rich discriminator, whose substitution for the AT-rich element abolished stringent control. These and other data obtained demonstrate the necessity of a natural combination of all the conserved promoter elements for efficient and regulated transcription of the essential rpsB-tsf operon.

[Extraribosomal functions of bacterial ribosomal proteins].

Though their primary role in a cell is to serve as integral components of protein synthesis machinery, the ribosome, many of them have functions beyond the ribosome (the phenomenon known as moonlighting), acting either as individual regulatory proteins or in complexes with other cellular components. Extraribosomal activities of some ribosomal proteins have been observed as early as in the 1970-1980s. During the last years both a list of r-proteins-moonlighters and the repertoire of their additional functions beyond the ribosome have been greatly expanded, mainly due to newly developed techniques for dissecting RNA/DNA-protein or protein-protein interactions within functional complexes involved in various cellular processes. In this review, we surveyed information on the experimentally proven as well as on presumptive extraribosomal functions which may be performed by bacterial r-proteins in a cell.

[Conservation of the regulatory elements implicated in the control of the rpsB-tsf operon expression in gamma-proteobacteria].

In eubacteria, the rpsB-tsf operon encodes two essential components of translational apparatus, ribosomal protein (r-protein) S2 and elongation factor Ts. Recently, we located the promoter region of the Escherichia coli rpsB-tsf operon and demonstrated that both rpsB and tsf genes are negatively regulated by r-protein S2 at the translational level. In this paper, we present data of phylogenetic analysis showing high conservation of both the promoter signature and the structure of the 5'-untranslated region (5'-UTR) of the rpsB mRNA in gamma-proteobacteria. Despite the difference in length and overall primary structure of the rpsB 5'-UTRs for various representatives of this bacterial phylum, several short regions within the 5'-UTRs appeared to be universally conserved, implying their participation in the expression regulation. Phylogenetic predictions have been experimentally confirmed. We show here that the presumable rpsB promoter regions from Yersinia pestis, Haemophilus influenzae and Pseudomonas aeruginosa are able to drive transcription of the lacZ -reporter in E. coli and that the corresponding rpsB 5'-UTRs are subjected to autogenous repression by r-protein S2 in vivo.

[An Escherichia coli strain producing a leaderless mRNA from the chromosomal lac promoter].

A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding the ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens possibilities for investigation of the specific conditions for leaderless mRNA translation in vivo using molecular genetic approaches.