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Multiple defects in translation associated with altered ribosomal protein L4.

ABSTRACT: The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribosomes carrying the altered L4 protein support increased levels of frameshifting, missense decoding and readthrough of stop codons during the elongation phase of protein synthesis and stimulate utilization of non-AUG codons and mutant initiator tRNAs at initiation. L4 mutant ribosomes are also altered in their interactions with a range of 30S-targeted antibiotics. In contrast, the L22 mutant is relatively unaffected in both decoding activities and antibiotic interactions. These results suggest that mutations in the large subunit protein L4 not only alter the structure of the 50S subunit, but upon subunit association, also affect the structure and function of the 30S subunit.

SUBMITTER: O'Connor M

PROVIDER: S-EPMC528798 | biostudies-literature | 2004

REPOSITORIES: biostudies-literature

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Multiple defects in translation associated with altered ribosomal protein L4.

O'Connor Michael M Gregory Steven T ST Dahlberg Albert E AE

Nucleic acids research 20041027 19

The ribosomal proteins L4 and L22 form part of the peptide exit tunnel in the large ribosomal subunit. In Escherichia coli, alterations in either of these proteins can confer resistance to the macrolide antibiotic, erythromycin. The structures of the 30S as well as the 50S subunits from each antibiotic resistant mutant differ from wild type in distinct ways and L4 mutant ribosomes have decreased peptide bond-forming activity. Our analyses of the decoding properties of both mutants show that ribo ...[more]

PMID: 15509870

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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.

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Multiple defects in translation associated with altered ribosomal protein L4.

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Multiple defects in translation associated with altered ribosomal protein L4.

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