Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 s ribosomal RNA

Pagel, Frances T.; Zhao, Song Q; Hijazi, Kathryn A.; Murgola, Emanuel J.

doi:10.1006/jmbi.1997.0943

Cited by 23 publications

(25 citation statements)

References 45 publications

(69 reference statements)

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“…This bias seems to be due to the use of XL1-Red, because transversions engineered at several of these positions confer strong suppressor phenotypes that would have been easily detected in the screens (see below). Of the 34 mutations recovered, at least six (G886A, U911C, C1054U, C1200U, C1469U, and G1491A) were shown previously to influence the fidelity of elongation (Allen and Noller 1991;Gregory and Dahlberg 1995;Murgola et al 1995;Lodmell and Dahlberg 1997;Pagel et al 1997;Velichutina et al 2000).…”

Section: Resultsmentioning

confidence: 93%

“…The ability of mutations at position 1054 to increase read-through of stop codons is well established (Hanfler et al 1990;Moine and Dahlberg 1994;Gregory and Dahlberg 1995;Murgola et al 1995;Chernoff et al 1996;Arkov et al 1998), but whether these A-site mutations increase the frequency of the missense errors has been less clear. Murgola and colleagues failed to see effects of these mutations on misreading of several codons in trpA (Pagel et al 1997). However, the relationship between the misreading error rate and the concentration of competing cognate aa-tRNA (Kramer and Farabaugh 2007) was unappreciated at that time, and the ability of ribosomal mutations to suppress those particular missense mutations was not demonstrated.…”

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

“…However, the relationship between the misreading error rate and the concentration of competing cognate aa-tRNA (Kramer and Farabaugh 2007) was unappreciated at that time, and the ability of ribosomal mutations to suppress those particular missense mutations was not demonstrated. Murgola and coworkers did see effects of 1054 mutations on the activities of several suppressor tRNAs that varied depending on the ribosomal mutation, suppressor tRNA, and/or trpA mutation (Pagel et al 1997). Those experiments, involving missense suppression in the presence of a suppressor tRNA, essentially assess a competition between two cognate tRNAs and, hence, speak little to the question of decoding fidelity.…”

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

“…3E). Two of these (C1200U and G1491A) were isolated in previous screens for nonsense suppressors (Gregory and Dahlberg 1995;Murgola et al 1995;Pagel et al 1997) and conferred larger effects on UGA read-through than on GAU miscoding (Figs. 1, 2).…”

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

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Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

McClory¹,

Leisring²,

Qin³

et al. 2010

RNA

126

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The molecular basis of the induced-fit mechanism that determines the fidelity of protein synthesis remains unclear. Here, we isolated mutations in 16S rRNA that increase the rate of miscoding and stop codon read-through. Many of the mutations clustered along interfaces between the 30S shoulder domain and other parts of the ribosome, strongly implicating shoulder movement in the induced-fit mechanism of decoding. The largest subset of mutations mapped to helices h8 and h14. These helices interact with each other and with the 50S subunit to form bridge B8. Previous cryo-EM studies revealed a contact between h14 and the switch 1 motif of EF-Tu, raising the possibility that h14 plays a direct role in GTPase activation. To investigate this possibility, we constructed both deletions and insertions in h14. While ribosomes harboring a 2-base-pair (bp) insertion in h14 were completely inactive in vivo, those containing a 2-bp deletion retained activity but were error prone. In vitro, the truncation of h14 accelerated GTP hydrolysis for EF-Tu bearing near-cognate aminoacyl-tRNA, an effect that can largely account for the observed miscoding in vivo. These data show that h14 does not help activate EF-Tu but instead negatively controls GTP hydrolysis by the factor. We propose that bridge B8 normally acts to counter inward rotation of the shoulder domain; hence, mutations in h8 and h14 that compromise this bridge decrease the stringency of aminoacyl-tRNA selection.

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Section: Resultsmentioning

confidence: 93%

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

Section: Elements Of 16s Rrna Involved In the Fidelity Of Translationmentioning

confidence: 99%

See 2 more Smart Citations

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

McClory¹,

Leisring²,

Qin³

et al. 2010

RNA

126

View full text Add to dashboard Cite

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“…In both bacteria and yeast, substitutions of C1054 cause variable suppression phenotypes, depending on the particular reporter constructs employed (Hanfler et al 1990;Prescott et al 1991;Prescott and Kornau 1992;Chernoff et al 1996;Pagel et al 1997;Konstantinidis et al 2006;McClory et al 2010McClory et al , 2011. The basis of this "phenotypic heterogeneity" has been a long-standing puzzle in the field.…”

Section: Discussionmentioning

confidence: 99%

Distinct functional classes oframmutations in 16S rRNA

McClory

Devaraj

Fredrick

2014

RNA

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During decoding, the ribosome selects the correct (cognate) aminoacyl-tRNA (aa-tRNA) from a large pool of incorrect aa-tRNAs through a two-stage mechanism. In the initial selection stage, aa-tRNA is delivered to the ribosome as part of a ternary complex with elongation factor EF-Tu and GTP. Interactions between codon and anticodon lead to activation of the GTPase domain of EFTu and GTP hydrolysis. Then, in the proofreading stage, aa-tRNA is released from EF-Tu and either moves fully into the A/A site (a step termed "accommodation") or dissociates from the ribosome. Cognate codon-anticodon pairing not only stabilizes aa-tRNA at both stages of decoding but also stimulates GTP hydrolysis and accommodation, allowing the process to be both accurate and fast. In previous work, we isolated a number of ribosomal ambiguity (ram) mutations in 16S rRNA, implicating particular regions of the ribosome in the mechanism of decoding. Here, we analyze a representative subset of these mutations with respect to initial selection, proofreading, RF2-dependent termination, and overall miscoding in various contexts. We find that mutations that disrupt inter-subunit bridge B8 increase miscoding in a general way, causing defects in both initial selection and proofreading. Mutations in or near the A site behave differently, increasing miscoding in a codon-anticodon-dependent manner. These latter mutations may create spurious favorable interactions in the A site for certain near-cognate aa-tRNAs, providing an explanation for their context-dependent phenotypes in the cell.

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Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene

Way

Hosking

Braun

et al. 2000

Journal of Molecular Biology

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Phenotypic heterogeneity of mutational changes at a conserved nucleotide in 16 s ribosomal RNA

Cited by 23 publications

References 45 publications

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

Missense suppressor mutations in 16S rRNA reveal the importance of helices h8 and h14 in aminoacyl-tRNA selection

Distinct functional classes oframmutations in 16S rRNA

Mot protein assembly into the bacterial flagellum: a model based on mutational analysis of the motB gene

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