A mutation suppressing streptomycin dependence

Brownstein, Barbara L.; Lewandowski, Leon J.

doi:10.1016/0022-2836(67)90281-1

Cited by 58 publications

(13 citation statements)

References 12 publications

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“…Because the ram strains were isolated as suppressors of the SmD phenotype (typically exhibited by the most restrictive ribosomes) (Brownstein and Lewandowein, 1967), the initial prediction was that ribosomes from the ram strain would inversely affect the same steps of the tRNA selection pathway as the restrictive ribosomes. In contrast, we observe disparate kinetic effects on the pathway conferred by these mutations in the S4 and S12 proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Most of these mutations in S12 are located at the interface with h27/h44 of the 16S rRNA, likely destabilizing interactions that are important for closure (and the completion of tRNA selection). Revertants of the streptomycin-dependent (SmD) phenotype (typically displayed by the most restrictive variants) were found to carry compensatory mutations in either protein S4 or S5 (Birge and Kurland, 1970; Brownstein and Lewandowein, 1967; Stoffler et al, 1971). On their own, these mutations typically confer an error-prone phenotype and are referred to as ribosomal ambiguity ( ram ) mutants (Rosset and Gorini, 1969).…”

Section: Introductionmentioning

confidence: 99%

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Hyperaccurate and Error-Prone Ribosomes Exploit Distinct Mechanisms during tRNA Selection

2010

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Summary Escherichia coli strains displaying hyper-accurate (restrictive) and ribosomal ambiguity (ram) phenotypes have long been associated with alterations in rpsL and rpsD/rpsE, respectively. Crystallographic evidence shows the ribosomal proteins S12 and S4/S5 (corresponding to these genes) to be located in separate regions of the small ribosomal subunit that are important for domain-closure thought to take place during tRNA selection. Mechanistically, the process of tRNA selection is separated into two distinct phases, initial selection and proofreading. Here, we explore the effects of mutations in rpsL and rpsD on these steps. Surprisingly, both restrictive and ram ribosomes primarily affect tRNA selection through alteration of the off-rates of the near-cognate tRNA species, but during distinct phases of the overall process (proofreading and initial selection, respectively). These observations suggest that closure interfaces (S12/h27/h44 versus S4/S5) in two distinct regions of the small ribosomal subunit function independently to promote high-fidelity tRNA selection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Hyperaccurate and Error-Prone Ribosomes Exploit Distinct Mechanisms during tRNA Selection

2010

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“…Over the last several decades, there have been significant efforts to elucidate the roles of ribosomal proteins in translation, and numerous mutations that confer diverse phenotypes and antibiotic resistance in E. coli have been isolated 15; 16; 17; 18 . For instance, restrictive mutations in S12, originally identified in streptomycin-resistant/dependent strains, confer a hyperaccurate phenotype (where frequency of miscoding and stop-codon readthrough are decreased) to the ribosome 19; 20 , while ram mutations in S4 and S5, identified in revertants of streptomycin-dependence, give rise to an error-prone phenotype (where frequency of miscoding and stop-codon readthrough are increased) 21; 22 . Additionally, some restrictive mutations in S12 (R86S and P91Q), which are known to slow the rate of ribosome translation, can give rise to increased folding and expression of aggregation-prone eukaryotic proteins in E. coli 23 .…”

Section: Introductionmentioning

confidence: 99%

Systematic Chromosomal Deletion of Bacterial Ribosomal Protein Genes

Shoji

Dambacher

Shajani

et al. 2011

Journal of Molecular Biology

101

121

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Detailed studies of ribosomal proteins, essential components of the protein biosynthetic machinery, have been hampered by the lack of readily accessible chromosomal deletions of the corresponding genes. Here, we report the systematic genomic deletion of 41 individual ribosomal protein genes in Escherichia coli, which are not included in the Keio collection. Chromosomal copies of these genes were replaced by an antibiotic resistance gene in the presence of an inducible, easy-to-exchange plasmid-born allele. Using this knockout collection, 9 ribosomal proteins (L15, L21, L24, L27, L29, L30, L34, S9 and S17) were found nonessential for survival under induction conditions at various temperatures. Taken together with previous results, this analysis revealed that 22 of the 54 E. coli ribosomal protein genes can be individually deleted from the genome. These strains also allow expression of truncated protein variants to probe the importance of RNA-protein interactions in functional sites of the ribosome. This set of strains should enhance in vivo studies of ribosome assembly/function, and may ultimately allow systematic substitution of ribosomal proteins with RNA.

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“…RAM MUTANTS Mutants with decreased translational accuracy were isolated owing to their property of reversing the effect of restrictive mutations (62,63). As second-site mutations, these so-called ram (ribosomal ambiguity) mutations remove the streptomycin-dependent phenotype in the restrictive background mentioned in the preceding paragraph (57,64).…”

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confidence: 99%

Structural Insights Into Translational Fidelity

Ogle

Ramakrishnan

2005

Annu. Rev. Biochem.

551

538

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■ Abstract The underlying basis for the accuracy of protein synthesis has been the subject of over four decades of investigation. Recent biochemical and structural data make it possible to understand at least in outline the structural basis for tRNA selection, in which codon recognition by cognate tRNA results in the hydrolysis of GTP by EF-Tu over 75Å away. The ribosome recognizes the geometry of codonanticodon base pairing at the first two positions but monitors the third, or wobble position, less stringently. Part of the additional binding energy of cognate tRNA is used to induce conformational changes in the ribosome that stabilize a transition state for GTP hydrolysis by EF-Tu and subsequently result in accelerated accommodation of tRNA into the peptidyl transferase center. The transition state for GTP hydrolysis is characterized, among other things, by a distorted tRNA. This picture explains a large body of data on the effect of antibiotics and mutations on translational fidelity. However, many fundamental questions remain, such as the mechanism of activation of GTP hydrolysis by EF-Tu, and the relationship between decoding and frameshifting. CONTENTS

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A mutation suppressing streptomycin dependence

Cited by 58 publications

References 12 publications

Hyperaccurate and Error-Prone Ribosomes Exploit Distinct Mechanisms during tRNA Selection

Hyperaccurate and Error-Prone Ribosomes Exploit Distinct Mechanisms during tRNA Selection

Systematic Chromosomal Deletion of Bacterial Ribosomal Protein Genes

Structural Insights Into Translational Fidelity

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