Bacillus subtilis mutants with alterations in ribosomal protein S4

Henkin, Tina M.; Chambliss, Glenn H.; Grundy, Frank J.

doi:10.1128/jb.172.11.6380-6385.1990

Cited by 7 publications

(8 citation statements)

References 33 publications

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“…In S. enterica, selections for compensatory mutations that reversed the streptomycin dependence or slow-growth phenotypes conferred by altered S12 proteins allowed the isolation of S4 proteins with changes at residues D49, Y50, Q53, A78, N84, T85, I199, V200, E201, Y203, S204, and K205 (8,33). The same selections in B. subtilis led to the isolation of S4 proteins carrying substitutions at E54, as well as insertions or deletions at residues 74 to 78 (34,35). Early work with E. coli led to the isolation of S5 ram mutants carrying alterations at G103 or R111 (36).…”

Section: Discussionmentioning

confidence: 94%

Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

et al. 2015

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bRibosomal proteins S4 and S5 participate in the decoding and assembly processes on the ribosome and the interaction with specific antibiotic inhibitors of translation. Many of the characterized mutations affecting these proteins decrease the accuracy of translation, leading to a ribosomal-ambiguity phenotype. Structural analyses of ribosomal complexes indicate that the tRNA selection pathway involves a transition between the closed and open conformations of the 30S ribosomal subunit and requires disruption of the interface between the S4 and S5 proteins. In agreement with this observation, several of the mutations that promote miscoding alter residues located at the S4-S5 interface. Here, the Escherichia coli rpsD and rpsE genes encoding the S4 and S5 proteins were targeted for mutagenesis and screened for accuracy-altering mutations. While a majority of the 38 mutant proteins recovered decrease the accuracy of translation, error-restrictive mutations were also recovered; only a minority of the mutant proteins affected rRNA processing, ribosome assembly, or interactions with antibiotics. Several of the mutations affect residues at the S4-S5 interface. These include five nonsense mutations that generate C-terminal truncations of S4. These truncations are predicted to destabilize the S4-S5 interface and, consistent with the domain closure model, all have ribosomal-ambiguity phenotypes. A substantial number of the mutations alter distant locations and conceivably affect tRNA selection through indirect effects on the S4-S5 interface or by altering interactions with adjacent ribosomal proteins and 16S rRNA. C ellular protein synthesis systems translate mRNAs quickly and with high accuracy. The accuracy of the decoding process can, however, be altered by agents such as the antibiotic streptomycin that promote miscoding and by mutations in rRNA, ribosomal proteins, or translation factors (1). Among the first such accuracy mutants to be characterized were some of the streptomycin-resistant Escherichia coli strains carrying alterations in ribosomal protein S12 (2). Subsequently, E. coli mutants carrying altered ribosomal protein S4 or S5 were isolated that supported increased levels of miscoding (3-5). Since those early studies, many other mutants have been isolated that affect the accuracy of decoding and carry alterations in different components of the translation machinery (1, 4).The determination of high-resolution structures of ribosomes has offered structural interpretations of the effects of some accuracy-altering ribosomal mutations (6). X-ray crystallography of ribosomal complexes has shown that conserved RNA elements of the decoding center use a shape-sensing mechanism to monitor base pairing between the A site codon and the anticodon of incoming aminoacyl-tRNA. Successful interaction of a ternary complex of EF-Tu-GTP-aminoacyl-tRNA with mRNA in the decoding center triggers a series of conformational rearrangements of the head and shoulder domains of the 30S subunit, ultimately resulting in the formation of a ...

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Section: Discussionmentioning

confidence: 94%

Modulation of Decoding Fidelity by Ribosomal Proteins S4 and S5

et al. 2015

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“…Mutations which result in two different alterations in the electrophoretic mobility of S4 protein were * Corresponding author. mapped to 2630, distant from the site of the a operon (14,15). Together, these results indicate that the a operon genes in E. coli and B. subtilis differ in the location of the gene which in E. coli encodes the autogenous regulatory protein for the operon.…”

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confidence: 60%

“…In addition, it appears that the infA and rpmJ genes, located upstream of the S13 gene, are also a part of the a operon transcriptional unit in B. subtilis (3). Mutations conferring alterations in the electrophoretic mobility of protein S4 were localized to a position at 2630 on the B. subtilis map (14,15), distant from the major ribosomal gene cluster. The locus defined by these mutations was presumed to be the structural gene for S4 but could have been a gene involved in posttranslational modification of S4.…”

Section: Resultsmentioning

confidence: 99%

“…Mutations that result in alterations in the electrophoretic mobility of S4 were previously isolated and mapped to a position on the B. subtilis chromosomal map between argGH (2600) and aroG (2640; 28), at 2630 (14,15).…”

Section: Resultsmentioning

confidence: 99%

“…Mutations which result in two different alterations in the electrophoretic mobility of S4 protein were * Corresponding author. mapped to 2630, distant from the site of the a operon (14,15 Cloning procedures. Restriction endonucleases and DNAmodifying enzymes were purchased from New England BioLabs (Beverly, Mass.)…”

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Cloning and analysis of the Bacillus subtilis rpsD gene, encoding ribosomal protein S4

Grundy

Henkin

1990

J Bacteriol

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The rpsD gene, encoding ribosomal protein S4, was isolated from Bacillus subtilis by hybridization with oligonucleotide probes derived from the S4 amino-terminal protein sequence. Sequence analysis of the cloned DNA indicated that rpsD is likely to be monocistronic, in contrast to Escherichia coli rpsD, which is located in the a operon and is the translational regulator for a operon ribosomal protein gene expression in E. coli. The cloned gene was shown to map at position 2630 on the B. subtilis chromosome, at the position to which mutations conferring alterations in the electrophoretic mobility of protein S4 were localized. A promoter was identified upstream of the rpsD coding sequence; initiation of transcription at this promoter would result in a transcript containing a leader region 180 bases in length. One of the best-characterized E. coli ribosomal protein operons is the ot operon, which includes the genes for ribosomal proteins S13, S11, S4, and L17 and for the at subunit of RNA polymerase, in the order S13-S11-S4-a-L17 (2). S4 is the translational regulator for this operon and binds to a target site on the ot operon mRNA near the start of the S13-coding region (9, 19). Binding of S4 to the mRNA blocks translation of S13 and also inhibits translation of S11 and S4, probably by a translational coupling mechanism (35). Expression of rpoA, which encodes the a subunit of RNA polymerase, is not controlled by S4, and there is evidence that a second S4-binding site on the mRNA upstream from the L17-coding region is necessary for S4 regulation of L17 expression (24). The ot operon of B. subtilis was recently isolated, and its DNA sequence was determined (3, 33). The most striking feature of the B. subtilis a operon is the absence of the S4-coding region. The at operon is located in the major cluster of ribosomal protein genes, at 120 on the 3600 B.subtilis map (33). Mutations which result in two different alterations in the electrophoretic mobility of S4 protein were * Corresponding author. mapped to 2630, distant from the site of the a operon (14,15). Together, these results indicate that the a operon genes in E. coli and B. subtilis differ in the location of the gene which in E. coli encodes the autogenous regulatory protein for the operon. It is therefore of interest to examine regulation of S4 and at operon gene expression in B subtilis. We report here a first step toward this goal, which is the cloning and characterization of the rpsD gene of B. subtilis, encoding ribosomal protein S4. We find that the rpsD gene is apparently monocistronic in B. subtilis. Also, a gene homologous to tyrosyltRNA synthetases from Bacillus stearothermophilus (39), Bacillus caldotenax (21), and E. coli (1) was found to be located immediately downstream of rpsD but in the opposite orientation.MATERIALS AND METHODS Bacterial strains, plasmids, and growth conditions. The B.subtilis strains used in this study are BR151 (lys-3 metB10 trpC2; obtained from G. Chambliss) and 1A92 [arg(GH)2 aroG932 bioB141 sacA321; obtained from the Bacil...

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