Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria

Abstract

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.

An S6:S18 complex inhibits translation of E. coli rpsF

“…Unlike many regulators of ribosomal protein synthesis, the rpsF_leader does not respond to a primary rRNA-binding protein but rather a complex of secondary rRNA-binding proteins. This situation is relatively rare, as most characterized regulators to date interact with primary rRNA-binding proteins, although there are a few exceptions (e.g., S2, L25; Aseev et al 2008Aseev et al , 2015. The closest comparison to this situation is the rplJrplL regulator that interacts with either L10 or the L10 (L12) 4 complex (Yates et al 1981).…”

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

“…The strategy to generate specialized strains in which expression of the chromosomal lacZ gene would be governed by the transcriptional and translational regions of the unrelated gene has been described previously (22)(23)(24). Based on this approach, we created rplU=-=lacZ, rplM=-=lacZ, and rpmB=-=lacZ chromosomal fusions.…”

“…For the rplU-rpmA operon, we also created a rplU-rpmA=-=lacZ fusion, taking into account that a presumable regulatory site might be situated at the beginning of the second cistron. The resulting plasmids were checked by sequencing and then used for transformation of E. coli strain ENS0 to generate Lac ϩ clones by homologous recombination (22)(23)(24)(25). The strains obtained were named (Table 1).…”

“…The procedure for the induction of a stringent response and isolation of total RNA was carried out essentially as described previously (24). Strains were grown in LB medium at 37°C with vigorous shaking.…”

“…To study whether the rplM-rpsI, rpmB-rpmG, and rplUrpmA operons are stringently regulated by ppGpp/DksA during amino acid starvation, we quantified the corresponding transcripts in total RNA preparations isolated before and 15 min after SHX treatment of the wildtype, ppGpp 0 (relA::kan spoT::Cm), and dksA::tet cells. For this purpose, we exploited the reverse transcription-quantitative PCR (RT-qPCR) approach based on using the external RNA standard (24,27). To generate RNA standards for the calibration curves, DNA templates were prepared by PCR with the transcript-specific primers ( Table 2).…”