Isolation of Temperature-sensitive Mutants of 16 S rRNA in Escherichia coli

Triman, Kathleen L.; Becker, Elisa; Dammel, Carol; Katz, John; Mori, Hiro; Douthwaite, Stephen; Yapijakis, Christos; Yoast, Sienna; Noller, Harry F.

doi:10.1016/0022-2836(89)92000-7

Cited by 74 publications

(61 citation statements)

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“…In a previous study, recessive temperature-sensitive mutants were localized to positions -13, -30, and -59 in the upstream precursor region (Mori et al 1989}. Because three of the suppressors isolated in the present work (C-5U, C -5 5 U , and G-114A} are Spc s under conditions where cold sensitivity is suppressed, the resulting double mutants ( U -5U23, U -55U23, and U -114U23] behave like recessive lethals.…”

Section: Suppressors Of Cold Sensitivitymentioning

confidence: 60%

“…Plasmid DNA was prepared from the candidates and shown to confer the cold-sensitive phenotype to newly transformed cells. The mutation responsible for the cold-sensitive phenotypes was localized by restriction fragment exchange and identified by DNA sequence analysis as described by Triman et al (1989}. Restriction fragment exchanges, between the four mutant plasmids and pSTL102 lwild typet, localized the site of the cold-sensitive mutation to the 5' end of the 16S rRNA gene and its upstream region, between nucleotide positions -358 and 80 (BstBI-HindlII fragment~ Table 1). DNA sequence analysis revealed a single C -> U nucleotide alteration at position 23 in each of the four in- dependently isolated mutants.…”

Section: A C--~ U M U T a T I O N At Position 23 Confers D O M I N A mentioning

confidence: 99%

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A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

Dammel¹,

Noller²

1993

Genes Dev.

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A C ---> U substitution at position 23 of 16S rRNA confers a dominant, cold-sensitive phenotype. The mutation changes the Gll-C23 base pair of the 5' terminal pseudoknot helix to a G-U pair, which is predicted to cause significant weakening of the helix. Ribosomes containing mutant RNA are impaired in assembly and function at low temperature. Cells expressing the C23U mutation have decreased polysome levels and accumulate free 30S and 50S subunits and particles that resemble those previously observed in cold-sensitive alleles of ribosomal protein $5 and in in vitro reconstitution of 30S subunits carried out at low temperature. Three second-site suppressor mutations suggest that cold sensitivity is caused by competition between the 5' helix and an alternative helix formed by base-pairing of the upstream precursor sequence with one strand of the mature helix. Cold sensitivity appears to be relieved by destabilization of the competing precursor helix relative to the mature helix.

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Section: Suppressors Of Cold Sensitivitymentioning

confidence: 60%

Section: A C--~ U M U T a T I O N At Position 23 Confers D O M I N A mentioning

confidence: 99%

A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

Dammel¹,

Noller²

1993

Genes Dev.

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“…There has been a general assumption that C1192U and A2058G are silent, and these mutations had been widely used as selectable markers for the isolation of mutants with detectable phenotypes prior to the development of the ⌬7rrn strain (Triman et al 1989;Powers and Noller 1990;Lodmell et al 1995;Rosendahl et al 1995). The results presented here indicate that the incompatibility of the H27 and r-protein mutations seen in the original study (Lodmell and Dahlberg 1997) was influenced by the presence of these two mutations.…”

Section: Viability Of An S5 Mutant Strain Containing the H27 Quadruplmentioning

confidence: 69%

“…The plasmids pKK3535 (Brosius et al 1981) and pSTL102 (Triman et al 1989) were used as rRNA expression vectors. Plasmid pKK3535 contains an intact wild-type rrnB operon and plasmid pSTL102 contains an rrnB operon with the C1192U spectinomycin-resistance mutation in the 16S rRNA gene and the A2058G erythromycin-resistance mutation in the 23S rRNA gene.…”

Section: Bacterial Strains and Plasmidsmentioning

confidence: 99%

Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model

Rodriguez-Correa

Dahĺberg²

2003

RNA

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A mechanistic understanding of ribosome function demands knowledge of the conformational changes that occur during protein synthesis. One current model proposes a conformational switch in Helix 27 (H27) of 16S rRNA involved in the decoding of mRNA. This model was based on the behavior of mutations in the 912 region of H27 of Escherichia coli 16S rRNA, which were predicted to stabilize the helix in either of two alternative conformations. This interpretation was supported by evidence from both genetics and structural biochemistry. However, recently published X-ray crystallographic structures of the Thermus thermophilus 30S subunit at different stages of tRNA selection have raised doubts regarding the validity of this model. We have therefore revisited the model genetically by constructing a H27 quadruple mutation (C912G, C910G, G885C, and G887C), which would create multiple mismatches in the proposed alternative conformation without perturbing the native H27 conformation seen in the crystal structures. Inconsistent with the H27 switch model, cells containing pure populations of quadruple mutant ribosomes grow at essentially wild-type rates. The mutants used to construct the H27 switch model all carried A2058G in 23S rRNA and C1192U in 16S rRNA as selectable markers. The quadruple mutant carrying these additional marker mutations is deleterious, and we conclude that they have a synergistic effect when combined with other mutations and are not phenotypically silent. Their presence confounded the interpretation of the original mutant phenotypes and, in light of the viability of the quadruple mutant, we conclude that the genetic evidence no longer supports the model.

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“…For example, a selection that involves unnatural substrates (that cannot be readily generated in vivo) could lead to the identification of ribosome variants capable of translating DNA or other unnatural polymers or capable of synthesizing altered polypeptide backbones. A selection where the temperature is substantially altered could allow for the identification of potentially mobile regions of the rRNA (4,5,48). Finally, a selection where specific translation components are omitted could shed light on the mechanism of the processes these factors promote, such as decoding and translocation.…”

Section: Discussionmentioning

confidence: 99%

Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

Cochella

Green

2004

Proc. Natl. Acad. Sci. U.S.A.

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Genetic, biochemical, and structural data support an essential role for the ribosomal RNA in all steps of the translation process. Although in vivo genetic selection techniques have been used to identify mutations in the rRNAs that result in various miscoding phenotypes and resistance to known ribosome-targeted antibiotics, these are limited because the resulting mutant ribosomes must be only marginally disabled if they are able to support growth of the cell. Furthermore, in vivo, it is not possible to control the environment in precise ways that might allow for the isolation of certain types of rRNA variants. To overcome these limitations, we have developed an in vitro selection system for the isolation of functionally competent ribosomal particles from populations containing variant rRNAs. Here, we describe this system and present an example of its application to the selection of antibiotic resistance mutations. From a pool of 4,096 23S rRNA variants, a double mutant (A2058U͞A2062G) was isolated after iteration of the selection process. This mutant was highly resistant to clindamycin in in vitro translation reactions and yet was not viable in Escherichia coli. These data establish that this system has the potential to identify mutations in the rRNA not readily accessed by comparable in vivo systems, thus allowing for more exhaustive ribosomal genetic screens.

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Isolation of Temperature-sensitive Mutants of 16 S rRNA in Escherichia coli

Cited by 74 publications

References 0 publications

A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

A cold-sensitive mutation in 16S rRNA provides evidence for helical switching in ribosome assembly.

Genetic evidence against the 16S ribosomal RNA helix 27 conformational switch model

Isolation of antibiotic resistance mutations in the rRNA by using an in vitro selection system

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