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G W Hatfield

Publications and source records attributed to G W Hatfield.

At least 19 recordsLinked to original sources
PubMed1992

DNA topology-mediated regulation of transcription initiation from the tandem promoters of the ilvGMEDA operon of Escherichia coli.

It is becoming increasingly clear that the intrinsic and protein-induced topological properties of the DNA helix influence transcriptional efficiency. In this report we describe the properties of two upstream activating regions that influence transcription from the non-overlapping tandem promoters of the ilvGMEDA operon of Escherichia coli. One 20 base-pair region between the promoter sites contains an intrinsic DNA bend that activates transcription from the downstream promoter. The other region contains an integration host factor (IHF) binding site that overlaps the upstream promoter site. IHF binding at this site represses transcription from the upstream promoter and enhances transcription from the downstream promoter. IHF also induces a severe bend in the DNA at its target binding site in the upstream promoter region. The activating property of the 20 base-pair DNA sequence located between the promoters is dependent upon the helical phasing of the sequence-directed DNA bend that it encodes. However, the IHF-mediated activation of transcription is not dependent upon the helical phasing (spatial orientation) of the upstream IHF and downstream promoter sites. The IHF-mediated activation of transcription is also uninfluenced by the presence or absence of the intrinsic DNA bend between its binding site and the downstream promoter site. These results suggest the interesting possibility that IHF activates transcription from the nearby downstream promoter simply by bending the DNA helix in the absence of specific IHF-RNA polymerase or upstream DNA-RNA polymerase interactions.

Bacterial Proteins↗
PubMed1991

Integration host factor-mediated expression of the ilvGMEDA operon of Escherichia coli.

The structural genes of the ilvGMEDA operon of Escherichia coli are preceded by two promoters, ilvPG1 and ilvPG2, and a leader-attenuator region. Alkylation protection and hydroxyl radical footprinting techniques have been used to demonstrate that integration host factor (IHF) interacts with the nucleotides in a consensus-like DNA sequence located immediately downstream of the RNA polymerase transcriptional pause site in the leader-attenuator region. In the presence of purified IHF protein, in vitro transcriptional pausing of RNA polymerase at the leader-attenuator pause site is increased 2-fold and, concomitantly, a 2-fold increase in transcriptional termination at the attenuator is observed. Strains containing chromosomal transcriptional fusions of various segments of the ilvGMEDA promoter-attenuator region to the galK gene were used to show that IHF also decreases the in vivo basal level of transcriptional readthrough at the attenuator 2-fold. The binding of IHF to another target site in the ilvPG1 promoter region represses transcription from this promoter and causes a 4-fold stimulation of transcription initiation from the downstream ilvPG2 promoter 4-fold. This IHF-mediated control of transcription initiation from the upstream promoter region is independent of the regulation of transcription termination effected by IHF interaction at the attenuator site. Thus, IHF is capable of regulating the expression of the ilvGMEDA operon in opposing manners; it can activate transcription initiation of this operon from the ilvPG2 promoter 4-fold and increase the termination of this transcription at the downstream attenuator 2-fold.

Amino Acids, Branched-Chain↗
PubMed1979

The nucleotide sequence preceding and including the beginning of the ilvE gene of the ilvGEDA operon of Escherichia coli K12.

The DNA sequence of the 570 base pairs that precedes and includes the beginning of the ilvE gene shows no evidence of a leader-attenuator region. Instead, the sequence shows that the ilvE gene is preceded by another structural gene, presumably the normally cryptic ilvG gene. In vitro transcription of the two plasmids (pLC26-3 and pRL5) containing the regulatory region of the ilvEDA operon results in the formation of two "leader" RNAs. These results are consistent with the suggestion that the ilvEDA operon is regulated by an attenuator mechanism and that the attenuator region lies before the ilvG gene.

Base Composition↗
PubMed1978

A site of action for tRNA mediated regulation of the ilvOEDA operon of Escherichia coli K12.

Transfer RNA (tRNA), rho factor threonine deaminase and the ilvO locus are molecular participants in the regulation of isoleucine-valine (ilv) biosynthesis. Isogenic strains have been constructed with the hisT76 mutation in pairwise combination with ilvO mutations, the rho221 mutation and the ilvDAC115 deletion mutation. The role of the altered tRNA of the hisT76 mutation was found to be independent of the sites of action of the ilvO- mutation, rho factor, and threonine deaminase. The expression of the ilvOEDA operon is stimulated 2-fold when the hisT76 mutation is present in strains containing either ilvO- or rho221 mutations. The expression of the ilvOEDA operon remains nonrepressed in a hisT76 strain deleted for threonine deaminase. These results indicate that the hisT76 undermodified tRNAs are influencing the initiation of transcription of the ilvOEDA operon.

Escherichia coli↗
PubMed1978

Biochemical characterization of a mutant asparaginyl-tRNA synthetase from Chinese hamster ovary cells.

The biochemical and physical properties of asparaginyl-tRNA synthetase from wild type Chinese hamster ovary cells and a temperature sensitive mutant strain (lys 65a) are compared. The asparaginyl-tRNA synthetase in the mutant strain exhibits a greater temperature lability in vitro, a higher temperature-independent Km for asparagine, and a lower temperature-dependent catalytic capacity than the enzyme from the wild type strain. The mutant enzyme shows no differences in its molecular weight, its Km for tRNAAsn, or its ability to aminoacylate tRNAAsn isoacceptor species compared to the wild type enzyme. These observations, as well as the growth properties of the mutant cells as a function of temperature and exogenous asparagine concentrations, are consistent with their decreased ability to aminoacylate tRNAAsn in vivo.

Amino Acyl-tRNA Synthetases↗
PubMed1978

Branched-chain amino acid transport regulation in mutants blocked in tRNA maturation and transcriptional termination.

The regulation of branched-chain amino acid transport and binding protein biosynthesis was studied in Escherichia coli strains containing hisT (the structural gene for pseudouridine synthetase) and rho (the structural gene for the mRNA transcriptional termination factor rho) mutations. The results indicate that the hisT strain cannot be fully derepressed for transport and that the hisT rho double mutant is partially derepressed under excess leucine conditions, but cannot be further derepressed by leucine deprivation. These data are consistent with a model in which fully mature tRNALeu is required for derepression and in which rho interacts with tRNALeu in regulating transport by terminating transcription, especially in excess-leucine growth conditions.

Amino Acids↗
PubMed1977

Differential utilization of leucyl-tRNAs by Escherichia coli.

The utilization of the isoaccepting species of leucyl-tRNA in protein synthesis has been examined in E. coli. Two minor leucyl-tRNA species, isoacceptors tRNA3Leu and tRNA4Leu, are the predominant species found bound to ribosomes during exponential growth in minimal medium. In rich medium, an increased proportion of tRNA1Leu is found bound to ribosomes. One species, tRNA5Leu, is always absent from ribosomes. In a mutant strain in which normal tRNA3Leu and tRNA5Leu are reduced or absent, tRNA1Leu is a major ribosome-bound species in minimal medium. Protein synthesis in vitro with RNA from E. coli further suggests that tRNA5Leu is rarely used for total protein synthesis; however, this species is active with RNA from MS2 phage. We propose that tRNA1Leu can substitute for tRNA3Leu under rapid growth conditions, and that tRNA5Leu is used minimally in total protein synthesis.

Bacterial Proteins↗
PubMed1977

A role for asparaginyl-tRNA in the regulation of asparagine synthetase in a mammalian cell line.

The expression of asparagine synthetase activity [L-aspartate:ammonia ligase (AMP-forming), EC 6.3.1.1] in cultured Chinese hamster ovary (CHO) cells is regulated by asparagine. After transfer of CHO cells from an asparagine-supplemented medium to a medium lacking asparagine, activity increases 1.5- to 2-fold. If asparagine is added back to the medium, activity returns to control levels. To test the possible involvement of Asn-tRNAAsn in regulating the levels of asparagine synthetase, we have examined the levels of asparagine synthetase in a mutant of CHO cells containing a temperature-sensitive asparaginyl-tRNA synthetase [L-asparagine:tRNA ligase (AMP-forming), EC 6.1.1.22]. Under conditions of limited asparaginyl-tRNA synthetase activity in the mutant, there is a 2- to 3-fold increase in the level of asparagine synthetase activity. Under identical conditions, there is no change in asparagine synthetase activity in the wild type. This correlation between asparaginyl-tRNA synthetase activity and asparagine synthetase levels may be a consequence of a direct role of tRNAAsn in the regulation of the in vivo expression of the asparagine synthetase structural gene.

Asparagine↗
PubMed1975

Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.

Chromatography, Ion Exchange↗