Regulation of Ribosomal Protein Operons rplM-rpsI , rpmB-rpmG , and rplU-rpmA at the Transcriptional and Translational Levels

Abstract

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...

Pervasive Regulatory Functions of mRNA Structure Revealed by High-Resolution SHAPE Probing

“…6C). We hypothesized that this structure constituted a novel RARE and, indeed, a contemporaneous study found that L13 translationally represses the rplM - rpsI operon in vivo (Aseev et al, 2016). No RNA structure or mechanistic information has been reported for the putative L13-binding motif.…”

“…Thus, L13 binds RNA containing the H1–H5 RNA, and likely inhibits rplM translation by stabilizing H4 and occluding access to the RBS. L13 has also been shown to negatively regulate translation of the downstream rpsI gene (Aseev et al, 2016). Our structure models revealed that rpsI is structurally linked to rplM (Fig.…”

“…6E). Prior studies failed to observe autoregulation of the rpmB-rpmG operon by L28 or L33, but the potential involvement of L9 was not explored (Aseev et al, 2016; Maguire and Wild, 1997). The rpmB-rpmG 5′ UTR folds into several conformations at high salt concentrations, as visualized by non-denaturing gel electrophoresis, one of which has exceptionally slow mobility suggestive of a defined tertiary structure (Fig.…”

Discovery of 20 novel ribosomal leaders in bacteria and archaea

“…Because of the potential for incorrect ligand hypotheses, we list additional genes associated with the various motifs (Table 1 and Additional file 2). An additional point is that it is possible that some RNAs that regulate r-proteins in cis do not function by binding any r-protein [33].…”

Role of DNA Methylation in Persister Formation in UropathogenicE. coli

“…Persisters are phenotypic variants that are formed through epigenetic changes that involve multiple redundant pathways to survive lethal antibiotic stress. These include toxin-antitoxin (TA) modules, stringent response (ppGpp) 14, 15 , DNA repair, metabolism 16 , energy production, protein degradation pathway (trans-translation), signaling pathways, antioxidant system, and efflux and transporters 17 18 . To better understand persister cells development and functioning, classical genetic processes is not sufficient, the epigenetic controlling gene expression is urgently required.…”