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RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

ABSTRACT: Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within the leader region, but a 64-nucleotide sequence is required for the bona fide interaction. Binding to the S10 leader mRNA is competed by the 23 S rRNA L4 binding site. Although the secondary structures of the mRNA and rRNA binding sites appear different, phosphorothioate footprinting of the L4-RNA complexes reveals close structural similarity in three dimensions. Mutational analysis of the mRNA binding site is compatible with the structural model. In vitro binding of L4 induces structural changes of the S10 leader RNA, providing a first clue for how protein L4 may provoke transcription termination.

SUBMITTER: Stelzl U

PROVIDER: S-EPMC4692380 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

Stelzl Ulrich U Zengel Janice M JM Tovbina Marina M Walker Marquis M Nierhaus Knud H KH Lindahl Lasse L Patel Dinshaw J DJ

The Journal of biological chemistry 20030508 30

Ribosomal protein L4 regulates the 11-gene S10 operon in Escherichia coli by acting, in concert with transcription factor NusA, to cause premature transcription termination at a Rho-independent termination site in the leader sequence. This process presumably involves L4 interaction with the leader mRNA. Here, we report direct, specific, and independent binding of ribosomal protein L4 to the S10 mRNA leader in vitro. Most of the binding energy is contributed by a small hairpin structure within th ...[more]

PMID: 12738792

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Project description:Escherichia coli ribosomal protein (r-protein) L4 has extraribosomal biological functions. Previously, we described L4 as inhibiting RNase E activity through protein-protein interactions. Here, we report that from stabilized transcripts regulated by L4-RNase E, mRNA levels of tnaA (encoding tryptophanase from the tnaCAB operon) increased upon ectopic L4 expression, whereas TnaA protein levels decreased. However, at nonpermissive temperatures (to inactivate RNase E), tnaA mRNA and protein levels both increased in an rne temperature-sensitive [rne(Ts)] mutant strain. Thus, L4 protein fine-tunes TnaA protein levels independently of its inhibition of RNase E. We demonstrate that ectopically expressed L4 binds with transcribed spacer RNA between tnaC and tnaA and downregulates TnaA translation. We found that deletion of the 5' or 3' half of the spacer compared to the wild type resulted in a similar reduction in TnaA translation in the presence of L4. In vitro binding of L4 to the tnaC-tnaA transcribed spacer RNA results in changes to its secondary structure. We reveal that during early stationary-phase bacterial growth, steady-state levels of tnaA mRNA increased but TnaA protein levels decreased. We further confirm that endogenous L4 binds to tnaC-tnaA transcribed spacer RNA in cells at early stationary phase. Our results reveal the novel function of L4 in fine-tuning TnaA protein levels during cell growth and demonstrate that r-protein L4 acts as a translation regulator outside the ribosome and its own operon.IMPORTANCE Some ribosomal proteins have extraribosomal functions in addition to ribosome translation function. The extraribosomal functions of several r-proteins control operon expression by binding to own-operon transcripts. Previously, we discovered a posttranscriptional, RNase E-dependent regulatory role for r-protein L4 in the stabilization of stress-responsive transcripts. Here, we found an additional extraribosomal function for L4 in regulating the tna operon by L4-intergenic spacer mRNA interactions. L4 binds to the transcribed spacer RNA between tnaC and tnaA and alters the structural conformation of the spacer RNA, thereby reducing the translation of TnaA. Our study establishes a previously unknown L4-mediated mechanism for regulating gene expression, suggesting that bacterial cells have multiple strategies for controlling levels of tryptophanase in response to varied cell growth conditions.

Project description:Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Dataset Information

RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

Publications

RNA-structural mimicry in Escherichia coli ribosomal protein L4-dependent regulation of the S10 operon.

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