The nucleotide sequence in the promoter region of the gene for an Escherichia coli tyrosine transfer ribonucleic acid.

Journal of Biological Chemistry (Impact Factor: 4.6). 09/1976; 251(15):4481-9.
Source: PubMed

ABSTRACT The sequence of the first 29 nucleotides in the promoter region of a tyrosine tRNA gene has previously been determined (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). This work has now been extended to give the sequence of a total of 59 nucleotides; the sequence is as follows: (see article). The general approach used in the determination of the sequence involved the DNA polymerase I-catalyzed elongation of synthetic deoxyribopolynucleotide primers hydridized to the l-strand of phi80psu+III DNA at the appropriate site. Sequencing of the newly added nucleotides was facilitated by the use of a number of techniques including (a) elongation of the primer with the use of all of the four nucleoside 5'-triphosphates but limiting the concentration of one of the triphosphates, (b) insertion of ribonucleotide units at appropriate sites so as to permit subsequent specific cleavages by pancreatic RNase, and (c) two-dimensional fingerprinting of the oligonucleotides in conjunction with partial exonucleolytic degradation, comprehensive nearest neighbor analyses, and the determination of pyrimidine tracts.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have cloned the Escherichia coli tyrosine-inserting amber suppressor tRNA gene into the recombinant single-strand phage M12mp3. By using the M13mp3SuIII+ recombinant phage DNA as template and an oligonucleotide bearing a mismatch as primer, we have synthesized in vitro an M13mp3SuIII heteroduplex DNA that has a single mismatch at a predetermined site in the tRNA gene. Transformation of E. coli with the heteroduplex DNA yielded M13 recombinant phages carrying a mutant suppressor tRNA gene in which the sequence G-T-T-C, corresponding to the universal G-T-pseudouracil-C sequence in E. coli tRNAs, is changed to G-A-T-C. The mutant DNA has been characterized by restriction mapping and by sequence analysis. In contrast to results with the wild-type suppressor tRNA gene, cells transformed with recombinant plasmids carrying the mutant tRNA gene are phenotypically Su-. Thus, the single nucleotide change introduced has inactivated the function of the tRNA gene. By using E. coli minicells for studying the expression in vivo of cloned tRNA genes, we have found that cells transformed with recombinant plasmids carrying the mutant tRNA gene contain very little, if any, mature mutant suppressor tRNA. In contrast, the predominant low molecular weight RNA in cells transformed with recombinant plasmids carrying the wild-type suppressor tRNA gene is the mature tyrosine suppressor tRNA. Thus, while our results imply an important role for the G-T-pseudouracil-C sequence common to all E. coli tRNAs, whether this sequence is essential for tRNA biosynthesis, tRNA stability in vivo, or tRNA function remains to be determined. The procedures used to generate the mutant should be of general application toward site-specific mutagenesis on cloned DNAs, including regions that possess high degrees of secondary structure. In addition, the frequency of mutants among the progeny is high enough to enable one to identify and isolate site-specific mutants on any cloned DNA without requiring phenotypic selection.
    Proceedings of the National Academy of Sciences 08/1981; 78(8):4753-7. DOI:10.1073/pnas.78.8.4753 · 9.81 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: virR is the central regulatory locus required for coordinate temperature-regulated virulence gene expression in the human enteric pathogens of Shigella species. Detailed characterization of VirR+ clones revealed that virR consisted of a 411 bp open reading frame (ORF) that mapped to a chromosomally located 1.8kb EcoRI-Accl DNA fragment from Shigella flexneri. Insertional inactivation of the virR ORF at a unique Hpal restriction site resulted in a loss of VirR+ activity. The vir R ORF nucleotide sequence was virtually identical to the Escherichia coli hns gene, which encodes the histone-like protein, H-NS. Based on the predicted amino acid sequence of E. coli H-NS, only a single conservative base-pair change was identified in the virR gene. An additional clone, designated VirRP, which only partially complemented the virR mutation, was also characterized and determined by Southern hybridization and nucleotide sequence analysis to be unique from virR. Subclone mapping of this clone indicated that the VirRP phenotype was a result of the multiple copy expression of the S. flexneri gene for tRNATyr. These data constitute the first direct genetic evidence that virR is an analogue of the E. coli hns gene, and suggest a model for temperature regulation of Shigella species virulence via the bacterial translational machinery.
    Molecular Microbiology 08/1992; 6(15):2113-2124. DOI:10.1111/j.1365-2958.1992.tb01385.x · 5.03 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The DNA sequences for nine independent promoter mutants of a tyrosine tRNA gene, tyrT of Escherichia coli, are reported. The nine mutations involve six transitions, two transversions, and one deletion. They are located at four different sites in the first 30 base pairs preceding the start point of transcription. The changes found are: a T.A to A.T transversion at position -8 (two mutants); a T.A to C.G transition at position -8 (three mutants); a T.A to C.G transition at position -13 (two mutants); a T.A to C.G transition at position -16 (one mutant); and a deletion of a G.C base pair at position -26/27 (one mutant). Four of the five different mutant tyrT promoters have alterations at positions that might have been expected from DNA sequence studies with other prokaryote promoters. One of these mutants (a G.C deletion at position -26/27) occurs in a stretch of eight consecutive G.C base pairs which may be characteristic of stable RNA promoters.
    Proceedings of the National Academy of Sciences 10/1979; 76(9):4303-7. DOI:10.1073/pnas.76.9.4303 · 9.81 Impact Factor


Available from