A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA

Conrad, Richard C.; Craven, Gary R.

doi:10.1093/nar/15.24.10331

Cited by 12 publications

(12 citation statements)

References 25 publications

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“…The Bacillus and E. coli S4 proteins are approximately 50% identical, and conservation is especially high in the region of the protein shown for E. coli S4 to interact with 16S rRNA (4,5). The proteins are likely to play very similar roles in ribosomal structure and function; the region of 16S rRNA with which S4 interacts is highly conserved in eubacteria, and active ribosomal subunits could be assembled in heterologous reconstitution experiments mixing E. coli and B. stearothermophilus components (16,22).…”

Section: Resultsmentioning

confidence: 99%

“…E. coli JM103 and DH5a were used for propagation of bacteriophage M13 and plasmids, respectively; strain RZ1032 was used for preparation of uracil-containing M13 template DNA for oligonucleotide mutagenesis. Antibiotics were used at the following concentrations: ampicillin, 50 ,ug/ml; chloramphenicol, 5 ,ug/ml; neomycin, 5 p.g/ml; erythromycin, 1 ,ug/ml; and lincomycin, 25 ,ug/ml. Chloramphenicol and erythromycin were used at 0.1 ,ug/ml for induction of the cat and erm genes, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of the Bacillus subtilis rpsD regulatory target site

Grundy

Henkin

1992

J Bacteriol

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The BaciUus subtilis rpsD gene, which encodes ribosomal protein S4, is subject to autogenous regulation.Repression of rpsD expression by excess S4 protein was previously shown to be affected by mutations in the leader region of the gene. A large number of deletion and point mutations in the leader region were generated, and their effect on repression by S4 in vivo was tested. These studies indicated that the required region was within positions +30 to +190 relative to the transcription start point. Replacement of the rpsD promoter with a lac promoter derivative which is expressed in B. subtUlis had no effect, indicating that repression by S4 occurs at a level subsequent to transcription initiation. The rpsD leader region was isolated from several Bacilus species. Members of the B. subtilis group, as defined by analysis of 16S rRNA sequence, contained a leader region target site very closely related in structure to that of B. subtilis, despite considerable primary sequence variation; the B. brevis rpsD leader contained some but not all of the structural features found in the regulatory target sites of the other Bacilus species. Very little similarity to the Escherichia coli a operon S4 target site was found at either the primary-sequence or the secondary-structure level. Mutagenic and phylogenetic data indicate that the secondary structure of the leader region regulatory target site contains two large stem-loop domains. The first of these helices has a side loop which is essential for autoregulation, is highly conserved among Bacilus rpsD genes, and is similar to a region of 16S rRNA important in S4 binding.Little information concerning the mechanisms responsible for control of ribosome biosynthesis in gram-positive bacteria is currently available. The Bacillus subtilis rpsD gene, which encodes ribosomal protein S4, was chosen for initial analysis because it was found to be located in a region of the chromosome distant from the major cluster of ribosomal protein genes. In Escherichia coli, rpsD is in the a operon, which contains the genes for ribosomal proteins S13, Sil, S4, and L17 and for the a subunit of RNA polymerase (2). S4 acts as a translational repressor to regulate a operon expression, binding to a target site at the start of the S13 coding sequence (6,18,25). In B. subtilis, the genes for S13, S11, a, and L17 are located within a large transcriptional unit in the major ribosomal gene cluster at 120 on the 3600 map (3), while rpsD is located at 2630 (14) and is monocistronic (10).It was therefore of interest to determine whether this difference in gene organization was associated with a difference in gene regulation.Transcription 26). Elements conserved with sequences within the region of 16S rRNA with which S4 is proposed to interact (24, 28) were also identified.In an effort to elucidate the basic structure of the B. subtilis rpsD regulatory target site and to identify sequence and structural elements critical for autogenous regulation, we have employed phylogenetic analysis of the rpsD gene isolated ...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Characterization of the Bacillus subtilis rpsD regulatory target site

Grundy

Henkin

1992

J Bacteriol

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“…The rpsDl and rpsD2 mutations result in the duplication or deletion of four amino acids within a region of the protein showing high conservation with the E. coli S4 protein (12). This region of the protein has been shown to be essential for binding of protein S4 to 16S rRNA in Escherichia coli (6,7). It was therefore of interest to examine the effect of the rpsD mutations on the growth properties of the cell.…”

Section: Resultsmentioning

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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“…Not only was S4:Δ41 unable to bind the 5WJ in vitro (Fig. 1B), previous reconstitution experiments (Changchien and Craven 1976;Conrad and Craven 1987) showed that complexes containing S4:Δ41 lack a subset of secondary and tertiary binding proteins and were therefore nonfunctional. Thus the intrinsically disordered N-terminal extension of bacterial S4 contributes essential contacts for RNA recognition and viability.…”

Section: The δ41 Mutation Is Lethalmentioning

confidence: 76%

“…Early studies demonstrated that an S4 fragment missing the N-terminal flexible extension (S4:Δ41) (Fig. 1A, red) bound 16S rRNA with low micromolar affinity Craven 1976, 1978;Conrad and Craven 1987) and does not significantly rearrange its structure upon binding (Newberry et al 1977). Ribosome reconstitution experiments by Changchien and Craven (1976) and, later, Conrad and Craven (1987) showed that the S4 fragment could incorporate into ribosomal particles, although these particles were missing a subset of secondary and tertiary binding proteins.…”

Section: Resultsmentioning

confidence: 99%

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

Mayerle

Woodson

2013

RNA

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Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5 ′ domain of the 16S rRNA. In vitro, transformation of initial S4-rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4-rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein-RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4-RNA interactions guide rRNA folding and impact late steps of 30S assembly.

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A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA

Cited by 12 publications

References 25 publications

Characterization of the Bacillus subtilis rpsD regulatory target site

Characterization of the Bacillus subtilis rpsD regulatory target site

Bacillus subtilis mutants with alterations in ribosomal protein S4

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

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