Mutant tRNAHis Ineffective in Repression and Lacking Two Pseudouridine Modifications

Singer, Clifford E; Smith, Gerald R.; Cortese, Riccardo; Ames, Bruce N.

doi:10.1038/newbio238072a0

Cited by 204 publications

(116 citation statements)

References 30 publications

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“…on reversed phase chromatography [2]. It will be interesting to learn whether the situation is similar in other cases where the tRNA's contain two pseudouridines in their anticodon region (e.g.…”

Section: Discussionmentioning

confidence: 97%

Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines

1972

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Section: Discussionmentioning

confidence: 97%

Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines

1972

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“…For example, because the suppressor tRNAs used are derived from tRNA of the same organism or a related organism, transcripts derived from the suppressor tRNA genes are likely to be processed well to yield functional suppressor tRNAs (34). Moreover, the suppressor tRNAs are likely to carry base modifications that are normally found in the tRNA and are, therefore, more likely to be optimally active in suppression (35). We have shown that the suppressor activity of the human initiator tRNA mutant in yeast is quite high.…”

Section: Discussionmentioning

confidence: 99%

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Kowal

Köhrer

RajBhandary

2001

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

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Two critical requirements for developing methods for the sitespecific incorporation of amino acid analogues into proteins in vivo are (i) a suppressor tRNA that is not aminoacylated by any of the endogenous aminoacyl-tRNA synthetases (aaRSs) and (ii) an aminoacyl-tRNA synthetase that aminoacylates the suppressor tRNA but no other tRNA in the cell. Here we describe two such aaRSsuppressor tRNA pairs, one for use in the yeast Saccharomyces cerevisiae and another for use in Escherichia coli. The "21st synthetase-tRNA pairs" include E. coli glutaminyl-tRNA synthetase (GlnRS) along with an amber suppressor derived from human initiator tRNA, for use in yeast, and mutants of the yeast tyrosyltRNA synthetase (TyrRS) along with an amber suppressor derived from E. coli initiator tRNA, for use in E. coli. The suppressor tRNAs are aminoacylated in vivo only in the presence of the heterologous aaRSs, and the aminoacylated tRNAs function efficiently in suppression of amber codons. Plasmids carrying the E. coli GlnRS gene can be stably maintained in yeast. However, plasmids carrying the yeast TyrRS gene could not be stably maintained in E. coli. This lack of stability is most likely due to the fact that the wild-type yeast TyrRS misaminoacylates the E. coli proline tRNA. By using errorprone PCR, we have isolated and characterized three mutants of yeast TyrRS, which can be stably expressed in E. coli. These mutants still aminoacylate the suppressor tRNA essentially quantitatively in vivo but show increased discrimination in vitro for the suppressor tRNA over the E. coli proline tRNA by factors of 2.2-to 6.8-fold.

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“…The finding that the hisT1504 regulatory mutation elicits expected constitutive derepression of his enzymes in strains containing hisG deletions further implies that the hisG product is not necessary to mediate effects of the well-established co-regulator of his operon expression-His-tRNAHis (3). Mutations in hisT lead to constitutively derepressed synthesis of his enzymes by inactivating an enzyme that converts uridine to pseudouridine ('I) in the anticodon-loop of tRNAHiS (25). The I modification is necessary for normal regulatory function of tRNAHiS, as well as other tRNA species (26).…”

Section: Discussionmentioning

confidence: 99%

Regulation of histidine operon does not require hisG enzyme.

Scott

Roth²,

Artz

1975

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

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Mutations are described which delete all or part of the first structural gene (hisG) of the histidine operon of Salmonella typhimurium. Physiological regulation of histidine enzymes occurs normally in strains carrying any deletion that has both endpoints within the hisG gene. Constitutive high operon expression is observed in strains carrying any hisG deletion and an unlinked regulatory mutation, hisT1504. These results strongly indicate that the hisG protein is not an essential component of the mechanism for regulating expression of the histidine operon.The biosynthesis of histidine in Salmonella typhimurium requires ten enzymes. The structural genes for these enzymes are located in a cluster of coordinately regulated genes (1, 2) called the histidine operon. Six classes of regulatory mutations have been identified which cause constitutive high level expression of the operon. These are hisR, hisS, hisT, hisU, hisW, and hisO. Characteristics of each of the classes are described in recent review articles (3, 4). None of these classes has been demonstrated to alter any protein that serves directly as a repressor or activator protein. Several lines of evidence have suggested that the hisG enzyme ATP phosphoribosyltransferase [N-1-(5'-phosphoribosyl) ATP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.17] might be the missing regulatory element (5-11). This protein is the first enzyme in the biosynthetic pathway and is encoded by the first structural gene in the operon.In the work reported here, mutants lacking all or part of the hisG gene have been selected as less polar derivatives of hisG frameshift mutations. The general method is similar to that used previously by Jackson and Yanofsky (12). These hisG deletions were mapped at high resolution with known point and deletion mutations. Regulation of the his operon was then studied in strains containing hisG deletions. This paper describes the isolation and mapping of the deletions and the results of studies of their regulatory properties.MATERIALS AND METHODS Bacterial Strains. Genotypes and sources of S. typhimurium strains are listed in Table 1. The strongly polar point mutations hisG6608 and hisG6609 were isolated by selection for temperature resistance of a strain containing the his01242 constitutive regulatory mutation (13). Mutations hisG6608 and hisG6609 were separated from the his01242 mutation by transduction. Strains containing the numerous his mutations used in localizing endpoints of hisG deletions (Fig. 2) were obtained from P. E. Hartman and B. N. Ames.Growth Media. Difco nutrient broth was used as the maximally supplemented liquid medium, with 2 g of agar per 100 ml added for solid medium. The E medium of Abbreviations: HT phage, high-frequency transducing phage; AT, 3-amino-1,2,4-triazole. t Present address:

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Mutant tRNAHis Ineffective in Repression and Lacking Two Pseudouridine Modifications

Cited by 204 publications

References 30 publications

Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines

Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Regulation of histidine operon does not require hisG enzyme.

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Mutant tRNAHis Ineffective in Repression and Lacking Two Pseudouridine Modifications

Cited by 204 publications

References 30 publications

Leucine tRNA1 from HisT mutant of Salmonella typhimurium lacks two pseudouridines

Leucine tRNA1 from HisT mutant of Salmonella typhimurium lacks two pseudouridines

Twenty-first aminoacyl-tRNA synthetase–suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria

Regulation of histidine operon does not require hisG enzyme.

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Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines

Leucine tRNA₁ from HisT mutant of Salmonella typhimurium lacks two pseudouridines