Autogenous regulation is a general strategy of balancing ribosomal protein synthesis in bacteria. Control mechanisms have been studied in detail for most of ribosomal protein operons, except for rpsB-tsf encoding essential r-protein S2 and elongation factor Ts, where even the promoter has remained unknown. By using single-copy translational fusions with the chromosomal lacZ gene and Western-blot analysis, we demonstrate here that S2 serves as a negative regulator of both rpsB and tsf expression in vivo, acting at a single target within the rpsB 59-untranslated region (59-UTR). As determined by primer extension, transcription of the Escherichia coli rpsB-tsf operon starts 162 nucleotides upstream of the rpsB initiation codon at a single promoter TGTGGTATAAA belonging to the extended À10 promoter class. Both the promoter signature and the 59-UTR structure of the rpsB gene appear to be highly conserved in g-proteobacteria. Deletion analysis of the rpsB 59-UTR within rpsB9-9lacZ fusions has revealed that an operator region involved in the S2 autoregulation comprises conserved structural elements located upstream of the rpsB ribosome binding site. The S2-mediated autogenous control is impaired in rpsB mutants and, more surprisingly, in the rpsA mutant producing decreased amounts of truncated r-protein S1 (rpsATIS10), indicating that S2 might act as a repressor in cooperation with S1.
Autogenous regulation in vivo of the rpmE gene encoding ribosomal protein L31 (bL31), a key component of the protein-protein intersubunit bridge B1b.
Ribosomal protein (r-protein) L25 is one of the three r-proteins (L25, L5, L18) that interact with 5S rRNA in eubacteria. Specific binding of L25 with a certain domain of 5S r-RNA, a so-called loop E, has been studied in detail, but information about regulation of L25 synthesis has remained totally lacking. In contrast to the rplE (L5) and rplR (L18) genes that belong to the polycistronic spc-operon and are regulated at the translation level by r-protein S8, the rplY (L25) gene forms an independent transcription unit. The main goal of this work was to study the regulation of the rplY expression in vivo. We show that the rplY promoter is down-regulated by ppGpp and its cofactor DksA in response to amino acid starvation. At the level of translation, the rplY expression is subjected to the negative feedback control. The 5 ′ -untranslated region of the rplY mRNA comprises specific sequence/structure features, including an atypical SD-like sequence, which are highly conserved in a subset of gammaproteobacterial families. Despite the lack of a canonical SD element, the rplY'-'lacZ single-copy reporter showed unusually high translation efficiency. Expression of the rplY gene in trans decreased the translation yield, indicating the mechanism of autogenous repression. Site-directed mutagenesis of the rplY 5 ′ UTR revealed an important role of the conserved elements in the translation control. Thus, the rplY expression regulation represents one more example of regulatory pathways that control ribosome biogenesis in Escherichia coli and related bacteria.
It is widely assumed that in the best-characterized model bacterium Escherichia coli, transcription units encoding ribosomal proteins (r-proteins) and regulation of their expression have been already well defined. However, transcription start sites for several E. coli r-protein operons have been established only very recently, so that information concerning the regulation of these operons at the transcriptional or posttranscriptional level is still missing. This paper describes for the first time the in vivo regulation of three r-protein operons, rplM-rpsI, rpmB-rpmG, and rplU-rpmA. The results demonstrate that transcription of all three operons is subject to ppGpp/DksA-dependent negative stringent control under amino acid starvation, in parallel with the rRNA operons. By using single-copy translational fusions with the chromosomal lacZ gene, we show here that at the translation level only one of these operons, rplM-rpsI, is regulated by the mechanism of autogenous repression involving the 5= untranslated region (UTR) of the operon mRNA, while rpmB-rpmG and rplU-rpmA are not subject to this type of regulation. This may imply that translational feedback control is not a general rule for modulating the expression of E. coli r-protein operons. Finally, we report that L13, a primary protein in 50S ribosomal subunit assembly, serves as a repressor of rplM-rpsI expression in vivo, acting at a target within the rplM translation initiation region. Thus, L13 represents a novel example of regulatory r-proteins in bacteria. IMPORTANCEIt is important to obtain a deeper understanding of the regulatory mechanisms responsible for coordinated and balanced synthesis of ribosomal components. In this paper, we highlight the major role of a stringent response in regulating transcription of three previously unexplored r-protein operons, and we show that only one of them is subject to feedback regulation at the translational level. Improved knowledge of the regulatory pathways controlling ribosome biogenesis may promote the development of novel antibacterial agents. Ribosomal protein (r-protein) operons rplM-rpsI, rplU-rpmA, and rpmB-rpmG encode r-proteins L13-S9, L21-L27, and L28-L33, respectively. Despite long-term studies of the regulation of expression of ribosomal components (1-5), these three operons have never been explored and their regulation remains largely unexplained. At the same time, the products of the operons play noticeable roles in ribosome assembly and functioning, which stimulates investigation into the control mechanisms of their expression, given the importance of knowledge about ribosome biogenesis and its control.Proteins L21 and L27 encoded by rplU-rpmA are bacterium specific (6). L27 was suggested to contribute to the function of the ribosomal peptidyl transferase center (PTC) via interactions of its N-terminal tail with both the A-site and P-site tRNAs, facilitating their accommodation in the PTC (7, 8). The L27-lacking strain has severe growth defects and is deficient in both the assembly and funct...
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