Construction of Escherichia coli amber suppressor tRNA genes

Kleina, Lynn G.; Masson, Jean-Michel; Normanly, Jennifer; Abelson, John; Miller, Jeffrey H

doi:10.1016/s0022-2836(05)80257-8

Cited by 148 publications

(81 citation statements)

References 66 publications

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“…The temperature-resistant revertant su+2UGA C39U70 suppresses at 30°C and 37°C (Table 1), whereas su+2UGA C39 suppresses at 42°C as well. As discussed below, the role of this mutation is to increase suppressor efficiency, consistent with previous results for mutations outside the anticodon (30,31). Mutations outside the anticodon of su+2 amber suppressor have also been found that probably increase suppressor efficiency (32,33) and are not mischarged by the DHFR assay (34).…”

Section: Resultssupporting

confidence: 88%

Switching tRNA(Gln) identity from glutamine to tryptophan.

Rogers

Adachi

Inokuchi

et al. 1992

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

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The middle base (U35) of the anticodon of tRNAGII is a major element ensuring the accuracy of aminoacylation by Escherichia coUl glutaminyl-tRNA synthetase (GlnRS). An opal suppressor of tRNAGI, (su+2UGA) containing C35 (anticodon UCA) was isolated by genetic selection and mutagenesis. Suppression of a UGA mutation in the E. colifol gene followed by N-terminal sequence analysis of purified dihydrofolate reductase showed that this tRNA was an efficient suppressor that inserted predominantly tryptophan. Mutations of the 3-70 base pair (U70 and A3U70) were made.

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Section: Resultssupporting

confidence: 88%

Switching tRNA(Gln) identity from glutamine to tryptophan.

Rogers

Adachi

Inokuchi

et al. 1992

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

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“…All but one of the substitutions made by the amber suppression method have a rate that is at least 25% of the control. These rates are about what is expected for Kdp, given that the efficiency of suppression ranges from about 10 to 70% (20). The relatively low rates of the Lys substitutions are consistent with the relatively low efficiency of this suppressor.…”

Section: Resultssupporting

confidence: 69%

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Dorus¹,

Mimura²,

Epstein

2001

Journal of Biological Chemistry

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The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K ؉ with high affinity and specificity. ؉ binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K ؉ binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K ؉ affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K ؉ , an indication that a residue is in or very near a K ؉ binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K ؉ and thus reflects the effective distance between a residue and the site of binding or passage of K ؉ in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K ؉ as it moves through the KdpA subunit to cross the membrane.

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“…Further experimentation led to the conclusion that the lack of strong suppressor activity was attributable to a defect in codon recognition or tRNA-ribosome interaction, rather than to a defect in aminoacylation [7]. Similar conclusions were reached during analysis of amber suppressor derivatives of tRNA ~u containing a C34/A36 double mutation [8,9].…”

Section: Introductionmentioning

confidence: 70%

“…The alternative explanation, that the U36 mutant is completely unrecognized by GluRS, is less appealing and is inconsistent with the observed (albeit low level) production of fl-galactosidase activity in CSH106. Previous studies with tRNAG~UA36(SuUAA/G) [8,9], tRNAG1uC34/A36(SuUAG) (amber suppressor) [7], and tRNAC~UG37 [20], demonstrated that at least some alterations in the anticodon permit recognition by GIuRS. Mischarging by LysRS is also unlikely since tRNA Glu contains several negative determinants for lysine identity [20].…”

Section: Discussionmentioning

confidence: 99%

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Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli

Gregory¹,

Dahĺberg²

1995

FEBS Letters

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Mutations in the anticodon of tRNA Clu (UUC) were isolated or constructed and characterized for their ability to suppress cognate nonsense or missense mutations in vivo. The C36-to-A36 transversion mutation was isolated as an ochre and an amber suppressor, while the G36 transversion was selected as a CAG missense suppressor, tRNA G~u suppressors of an AAG missense mutation could not be isolated, and a 1536 transition mutation introduced into tRNA G~u in vitro conferred no suppressor phenotype. Over-expression of glutamyl-tRNA synthetase did not increase the activity of the U36 mutant tRNA clu, suggesting a defect at the level of translation rather than at the level of synthetase recognition.Key words: Suppressor tRNA; Translational efficiency; tRNAG~U; Anticodon a GAG(glu) to AAG(lys) missense mutation at the same position in lacZ were unsuccessful. A tRNA c~u allele containing a C-to-U transition at position 36 constructed by oligonucleotide-directed mutagenesis failed to suppress the same lacZ AAG missense mutation. The activity of the U36 mutant tRNA ~u, measured as fl-galactosidase activity, was not increased by over-expression of glutamyl-tRNA synthetase (GluRS). While defects in aminoacylation cannot be excluded, our observations are consistent with previous findings [6,7] and indicate that mutations at position 36 in the anticodon of tRNA Clu fail to produce efficient translational suppressors due primarily to a defect in codon recognition or tRNA-ribosome interaction. Materials and methods

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Construction of Escherichia coli amber suppressor tRNA genes

Cited by 148 publications

References 66 publications

Switching tRNA(Gln) identity from glutamine to tryptophan.

Switching tRNA(Gln) identity from glutamine to tryptophan.

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli

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Construction of Escherichia coli amber suppressor tRNA genes

Cited by 148 publications

References 66 publications

Switching tRNA(Gln) identity from glutamine to tryptophan.

Switching tRNA(Gln) identity from glutamine to tryptophan.

Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

Effects of mutations at position 36 of tRNAGlu on missense and nonsense suppression in Escherichia coli

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Effects of mutations at position 36 of tRNA^Glu on missense and nonsense suppression in Escherichia coli