Autogenous regulation in vivo of the rpmE gene encoding ribosomal protein L31 (bL31), a key component of the protein-protein intersubunit bridge B1b.

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...

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.

The autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. In this work, taking the rpsO gene as a classic example, we addressed for the first time the in vivo regulation of r-protein synthesis in the mycobacteria M. smegmatis (Msm) and M. tuberculosis (Mtb). We used a strategy based on chromosomally integrated reporters under the control of the rpsO regulatory regions and the ectopic expression of Msm S15 to measure its impact on the reporter expression. Because the use of E. coli as a host appeared inefficient, a fluorescent reporter system was developed by inserting Msm or Mtb rpsO-egfp fusions into the Msm chromosome and expressing Msm S15 or E. coli S15 in trans from a novel replicative shuttle vector, pAMYC. The results of the eGFP expression measurements in Msm cells provided evidence that the rpsO gene in Msm and Mtb was feedback-regulated at the translation level. The mutagenic analysis showed that the folding of Msm rpsO 5′UTR in a pseudoknot appeared crucial for repression by both Msm S15 and E. coli S15, thus indicating a striking resemblance of the rpsO feedback control in mycobacteria and in E. coli.

Autogenous regulation of ribosomal protein (r-protein) synthesis plays a key role in maintaining the stoichiometry of ribosomal components in bacteria. Our main goal was to develop techniques for investigating the r-protein synthesis regulation in mycobacteria, Gram-positive organisms with a high GC-content, which has never been addressed. We started with the rpsO gene known to be autoregulated by its product, r-protein S15, in a broad range of bacterial species. To study the in vivo regulation of rpsO from Mycobacterium smegmatis (Msm), we first applied an approach based on chromosomally integrated Msm rpsO'-'lacZ reporters by using E. coli as a surrogate host. The β-galactosidase assay has shown that mycobacterial rpsO expression is feedback regulated at the translation level in the presence of Msm S15 in trans, like in E. coli. Next, to overcome difficulties caused by the inefficiency of mycobacterial gene expression in E. coli, we created a fluorescent reporter system based on M. smegmatis. To this end, the integrative shuttle plasmid pMV306 was modified to provide insertion of the Msm or Mtb (M. tuberculosis) rpsO-egfp reporters into the Msm chromosome, and a novel E. coli- mycobacteria replicative shuttle vector, pAMYC, a derivative of pACYC184, was built. Analysis of the eGFP expression in the presence of the pAMYC derivative expressing MsmrpsO vs an empty vector confirms the autogenous regulation of the rpsO gene in mycobacteria. Additionally, we have revealed that the mycobacterial rpsO core promoters are rather weak and require upstream activating elements to enhance their strength.