Purification and characterization of 30S ribosomal proteins from Bacillus subtilis: correlation to Escherichia coli 30S proteins

Higo, Kenichi; Otaka, Eiko; Osawa, Syozo

doi:10.1007/bf00330792

Cited by 48 publications

(28 citation statements)

References 15 publications

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“…subtilis and E. coli has been established for 30S proteins [16] but not for all 50S proteins). rpsD2 mutations both involve lesions in the structural gene for protein S4.…”

Section: Discussionmentioning

confidence: 99%

Bacillus subtilis mutants with alterations in ribosomal protein S4

1990

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Two mutants with different alterations in the electrophoretic mobility of ribosomal protein S4 were isolated as spore-plus revertants of a streptomycin-resistant, spore-minus strain of Bacillus subtilis. The mutations causing the S4 alterations, designated rpsDI and rpsD2, were located between the argGH and aroG genes, at 2630 on the B. subtilis chromosome, distant from the major ribosomal protein gene cluster at 120. The mutant rpsD alleles were isolated by hybridization using a wild-type rpsD probe, and their DNA sequences were determined. The two mutants contained alterations at the same position within the S4-coding sequence, in a region containing a 12-bp tandem duplication; the rpsDI allele corresponded to an additional copy of this repeated segment, resulting in the insertion of four amino acids, whereas the rpsD2 allele corresponded to deletion of one copy of this segment, resulting in the loss of four amino acids. The effects of these mutations, alone and in combination with streptomycin resistance mutations, on growth, sporulation, and streptomycin resistance were analyzed.A powerful strategy in characterization of the structure and function of the bacterial ribosome is the isolation and analysis of mutants with alterations in ribosomal components. In previous studies, we reported the isolation and characterization of a streptomycin-resistant (Strr)J, sporeminus (Spo-) mutant of Bacillus subtilis (4, 14) and secondsite revertants in which the Spo-phenotype was suppressed. Several of these revertants exhibited changes in the electrophoretic mobility of individual ribosomal proteins (13). The original Strr Spo-strain contains two mutations, one of which, designated rpsL2, affects the 30S subunit ribosomal protein S12; the second, designated strR, affects an unidentified 30S subunit component (14). One revertant strain contained an additional mutation that causes an alteration in the electrophoretic mobility of ribosomal protein S4. This mutation, designated rpsDl, maps to position 2630 on the B. subtilis chromosome, between argGH (2600) and aroG (264°; 23), distant from the major cluster of ribosomal protein genes at 120 (15). Although the rpsDl mutation was presumed to be a lesion in the structural gene encoding ribosomal protein S4, it was also possible that the mutation affected a gene involved in the posttranslational modification of S4.In this study, a second mutant with a different alteration in the electrophoretic mobility of protein S4 was isolated by using a similar selection procedure. Since this mutation presumably causes a different structural alteration in protein S4, analysis of the mutations at the primary structural level would provide strong evidence as to whether they affect the S4 structural gene. We report (i) the isolation and characterization of the second S4 mutant and (ii) the cloning and analysis of both mutant alleles. The results presented show that both mutations do in fact represent alterations in rpsD, the gene encoding S4, and provide information about the role of S4 in the...

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“…subtilis and E. coli has been established for 30S proteins [16] but not for all 50S proteins). rpsD2 mutations both involve lesions in the structural gene for protein S4.…”

Section: Discussionmentioning

confidence: 99%

Bacillus subtilis mutants with alterations in ribosomal protein S4

1990

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“…Cloning of rpsD used synthetic oligonucleotide probes designed on the basis of the B. subtilis S4 amino-terminal amino acid sequence (18). Three different oligonucleotide probes were used, all of which hybridized strongly to an EcoRI fragment approximately 12 kb in size, and to a 3.5-kb band in DNA digested with EcoRI and StuI, in Southern analyses of B. subtilis chromosomal DNA.…”

Section: Resultsmentioning

confidence: 99%

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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“…The predicted 131-residue product of the succeeding open reading frame had extensive homology to E. coli Sli (Fig. 4) (14). The first three residues of our sequence are missing from the mature sequence, perhaps because of proteolytic processing in vivo.…”

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confidence: 83%

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster

Suh¹,

Boylan²,

Price³

1986

J Bacteriol

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We isolated the gene encoding the alpha subunit of Bacillus subtilis RNA polymerase from a Agtll expression vector library by using anti-alpha antibody as a probe. Four unique clones were isolated, one carrying a lacZ-alpha gene fusion and three carrying the entire alpha coding region together with additional sequences upstream. The identity of the cloned alpha gene was confirmed by the size and immunological reactivity of its product expressed in Escherichia coli. Further, a partial DNA sequence found the predicted NH2 terminus of alpha homologous with E. coli alpha. By plasmid integration and PBS1 transduction, we mapped alpha near rpsE and within the major ribosomal protein gene cluster on the B. subtilis chromosome. Additional DNA sequencing identified rpsM (encoding S13) and rpsK (encoding Sll) upstream of alpha, followed by a 180-base-pair intercistronic region that may contain two alpha promoters. Although the organization of the alpha region resembles that of the alpha operon of E. coli, the putative promoters and absence of rpsD (encoding S4) immediately preceding the B. subtilis alpha gene suggest a different regulation.The function of the alpha subunit of the procaryotic DNA-dependent RNA polymerase is unclear. In the Escherichia coli system, there is insufficient genetic evidence to choose between a suggested structural role for alpha or a possible role in transcriptional selectivity (44). Four mutations have been identified in rpoA, the gene encoding E. coli alpha. The rpoA109 alteration prevents late gene expression of phage P2 but has no apparent effect on cell growth (8, 37). In contrast, the conditional rpoA101 and rpoA112 mutations prevent RNA synthesis in vivo at nonpermissive temperatures; these mutations also affect transcriptional fidelity in vitro but not in vivo (15). The phs alteration, which very likely lies within rpoA, has a pleiotropic phenotype which includes altered metabolism of melibiose, glutamate, sulfur, and arabinose, as well as a Cym auxotrophy (9, 32). Transcription of the araBAD operon is specifically affected in a phs background, leading Rowland et al. (32) to suggest that alpha is involved in transcriptional selectivity through interaction with either the promoter or with other transcriptional factors.We undertook a genetic analysis of the alpha gene of Bacillus subtilis, a procaryote which is evolutionarily distant from E. coli and which undergoes a simple developmental process, sporulation. By taking advantage of the available developmental phenotypes (26), we have additional means to characterize the interactions of alpha with other components of the B. subtilis transcriptase. We can also compare the organization and regulation of the B. subtilis rpoA region to that of E. coli, which comprises, in addition to alpha, at least 29 genes active in translation (23). E. coli rpoA lies in an operon with four ribosomal protein genes in the order promoter, rpsM (encoding ribosomal protein S13), rpsK (S11), rpsD (S4), rpoA, and rplQ (L17). Transcriptional and posttranscription...

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Purification and characterization of 30S ribosomal proteins from Bacillus subtilis: correlation to Escherichia coli 30S proteins

Cited by 48 publications

References 15 publications

Bacillus subtilis mutants with alterations in ribosomal protein S4

Bacillus subtilis mutants with alterations in ribosomal protein S4

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

Gene for the alpha subunit of Bacillus subtilis RNA polymerase maps in the ribosomal protein gene cluster

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