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Biomedical subjects

R L Somerville

Publications and source records attributed to R L Somerville.

At least 37 records · Page 2Linked to original sources
PubMed1993

A mutational analysis of the structural basis for transcriptional activation and monomer-monomer interaction in the TyrR system of Escherichia coli K-12.

In response to the binding of tyrosine or phenylalanine, the TyrR protein (513 amino acids) activates certain promoters and represses others. In a previous study (J. Cui and R. L. Somerville, J. Bacteriol. 175:303-306, 1993), it was shown that promoter activation was selectively abolished in mutant proteins lacking amino acid residues 2 to 9. An additional series of constructs that encoded mutant TyrR proteins having deletions or point mutations near the N terminus were analyzed. Residues Arg-2 and Leu-3 were shown to be critical for the activation of the mtr promoter. In confirmation of previous findings, none of the activation-defective mutant TyrR proteins had lost significant repression function. The TyrR protein was shown by chemical cross-linking to be dimeric. The polypeptide segments critical for dimer formation in vivo were identified by evaluating the negative dominance phenotypes of a series of mutant proteins, all defective in DNA binding, lacking progressively greater numbers of amino acid residues from either the N terminus or the C terminus. Amino acid residues 194 to 438 were found to contain all of the essential dimerization determinants.

Amino Acid Sequence↗
PubMed1992

Genetic and biochemical characterization of the trpB8 mutation of Escherichia coli tryptophan synthase. An amino acid switch at the sharp turn of the trypsin-sensitive "hinge" region diminishes substrate binding and alters solubility.

The trpB8 mutation of Escherichia coli tryptophan synthase is unique in that the cells bearing this lesion are not only capable of utilizing indole for growth, but they also accumulate indole, under conditions of tryptophan limitation. The lesion was shown by DNA sequencing to be a G to C transversion at nucleotide 5528 of the trp operon, resulting in a Gly to Arg switch at codon 281. Gly-281, within the trypsin-sensitive "hinge" region, is invariant among all known beta polypeptides. The catalytic activity of the mutant beta 2(B8) protein is dramatically stimulated by alpha subunit, both in vivo and in vitro. In the absence of alpha subunit, ammonium ion effectively stimulated the activity in an apparently cooperative manner. The pH optimum for the mutant subunit was 9.8, which is 2 units higher than that of wild type. In contrast to the wild-type subunit, beta(B8) partially aggregated within cells upon overexpression. At the optimal concentration of ammonium ions (2.25 M), the beta 2(B8) mutant enzyme displayed lower affinity than wild-type enzyme toward indole and L-serine, but the Vmax was almost unchanged. The physicochemical behavior of beta 2(B8) is supported by computer graphic modeling studies. An open versus closed model of conformational change within the beta 2 protein is proposed. A plausible role for the hinge region is discussed.

Amino Acid Sequence↗
PubMed1992

Synergism between the Trp repressor and Tyr repressor in repression of the aroL promoter of Escherichia coli K-12.

Computer analysis identified a potential Trp repressor operator 56 nucleotides downstream of the transcriptional start point of aroL, the gene that encodes shikimate kinase II. Tryptophan-dependent interaction of Trp repressor with this operator was demonstrated in vitro by means of a restriction endonuclease protection assay. Regulation of expression from the aroL promoter was evaluated with several genetically marked Escherichia coli strains by using a single-copy aroL-lacZ transcriptional-translational reporter system. The expression of aroL was repressed 6.9-fold by the Tyr repressor alone and 29-fold when both Tyr and Trp repressors were present. The Trp repressor had no effect on expression from the aroL promoter in the absence of the Tyr repressor. Possible mechanisms for Trp repressor-mediated repression, including cooperative interactions with the Tyr repressor, are discussed.

Bacteriophage lambda↗
PubMed1991

Gene expression from multicopy T7 promoter vectors proceeds at single copy rates in the absence of T7 RNA polymerase.

Three different genes (trpR+, tyrR+ and phi (trpR-lacZ)) were inserted into pET3a, a multicopy transcription-translation vector designed by Rosenberg et al. (1) for the T7 RNA polymerase-driven overexpression of proteins in Escherichia coli. Gene orientation was in the anticlockwise ("silent") direction. Gene expression in the absence of T7 RNA polymerase was evaluated either directly using lacZ reporter systems or indirectly by observing the susceptibility of plasmid-bearing tester strains to inhibition by an aromatic amino acid analog. The production of repressor proteins and of a Trp repressor-LacZ chimera was readily detected, at levels comparable to those of haploid trpR+ or tyrR+ E. coli strains. Such T7 vector constructs thus have two especially useful properties: first, they provide a means for the high-level production of various proteins in E. coli; second, they offer a technically advantageous point of departure for structure-function studies of genes whose overexpression from multicopy plasmids would normally be cytotoxic.

Bacterial Proteins↗
PubMed1991

Shared operator recognition specificity between Trp repressor and the repressors of bacteriophage 434.

Trp repressor is the only DNA-binding regulatory protein having a helix-turn-helix motif that has been reported to engage its operator target by a mechanism termed indirect readout: the Trp repressor-DNA interface is replete with hydrogen bonds between amino acid residues and non-esterified oxygen atoms of the sugar-phosphate backbone, and contains numerous specifically positioned water molecules. In Escherichia coli mutants deleted for trpR, the immunity repressor of phage 434 led to an eightfold reduction in trp promoter utilization. The Cro434 repressor also inhibited transcription from the trp promoter. The 434 repressors, considered to interact directly with operator targets, carry recognition helices positioned near the N terminus of each protein. The DNA-recognizing elements of Trp repressor lie toward the C terminus. The trp operator thus appears to possess significant plasticity in terms of its ability to assume conformational states that allow complex formation with more than one class of regulatory protein.

Amino Acid Sequence↗
PubMed1991

Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12.

The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG. Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter.

Amino Acid Sequence↗
PubMed1991

The tryptophan-specific permease gene, mtr, is differentially regulated by the tryptophan and tyrosine repressors in Escherichia coli K-12.

The regulation of transcription of the gene for the tryptophan-specific permease, mtr, was evaluated in several genetically marked Escherichia coli strains through the use of a single-copy lacZ reporter system. The expression of mtr was repressed 97-fold by tryptophan via the Trp repressor and induced 10-fold by phenylalanine or tyrosine via the Tyr repressor. By primer extension analysis two distinct mtr transcripts and their corresponding promoters were identified. One transcript was induced by the Tyr repressor. The tryptophan-dependent interaction of Trp repressor with an operator target within the mtr promoter was demonstrated by means of a restriction endonuclease protection assay.

Amino Acid Transport Systems↗
PubMed1991

The tyrosine repressor negatively regulates aroH expression in Escherichia coli.

The levels of the tryptophan-sensitive isoenzyme of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli, encoded by the aroH gene, were elevated in tyrR and/or trpR mutants. The effect of tyrR and trpR lesions on aroH expression was confirmed by using a lacZ reporter system. The mutational elimination of either repressor led to a threefold increase in beta-galactosidase.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1991

The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes.

The tdh promoter of Escherichia coli is induced seven- to eightfold when cells are grown in the presence of exogenous leucine. A scheme was devised to select mutants that exhibited high constitutive expression of the tdh promoter. The mutations in these strains were shown to lie within a previously identified gene (lrp) that encodes Lrp (leucine-responsive regulatory protein). By deletion analysis, the site of action of Lrp was localized to a 25-bp region between coordinates -69 and -44 of the tdh promoter. Disruption of a 12-bp presumptive target sequence found in this region of tdh resulted in constitutively derepressed expression from the tdh promoter. Similar DNA segments (consensus, TTTATTCtNaAT) were also identified in a number of other promoters, including each of the Lrp-regulated promoters whose nucleotide sequence is known. The sequence of the promoter region of serA, an Lrp-regulated gene, was determined. No Lrp consensus target sequence was present upstream of serA, suggesting that Lrp acts indirectly on the serA promoter. A previously described mutation in a leucine-responsive trans-acting factor, LivR (J. J. Anderson, S. C. Quay, and D. L. Oxender, J. Bacteriol. 126:80-90, 1976), resulted in constitutively repressed expression from the tdh promoter and constitutively induced expression from the serA promoter. The possibility that LivR and Lrp are allelic is discussed.

Alcohol Oxidoreductases↗
PubMed1990

A human hepatocellular carcinoma 3.0-kilobase DNA sequence transforms both rat liver cells and NIH3T3 fibroblasts and encodes a 52-kilodalton protein.

Neoplastic transformation of rat liver cells in vitro by DNA-mediated gene transfer with an oncogene, hhcM, derived from human (Mahlavu) hepatocellular carcinoma, is described and compared with that of NIH3T3 cells. hhcM was cloned in a neomycin-resistant simian virus 40 promoter vector (pNeor/S) and was designated pNrpM-1. BRL-1 or NIH3T3 cells, transfected with pNrpM-1 DNA, showed significant morphological changes, loss of contact inhibition, and anchorage-independent growth. They became highly tumorigenic in nude rats and nu/nu mice. Control BRL-1 and NIH3T3 cells, whether transfected with pNeor/S DNA or not, remained contact inhibited and nontumorigenic. Both the transformants and the tumor cells contained integrated hhcM DNA as shown by Southern blot hybridization. The complete nucleotide sequence of the hhcM 3.0-kilobase DNA was also determined, and it consisted of a possible open reading frame for a protein of 52 kilodaltons (467 amino acids). The high-level production of a slightly modified form of this 52-kilodalton protein in a bacterial expression system has been successfully achieved. The bacteria-produced protein was similar in electrophoretic behavior to the 52- to 53-kilodalton protein synthesized in a cell-free translation system using rabbit reticulocyte lysate programmed with hybrid-selected hhcM-specific mRNA from Mahlavu hepatocellular carcinoma cells.

Animals↗
PubMed1990

Fragment tagging.

A tactic known as fragment tagging, which has proven to be exceptionally useful in expediting DNA cloning and plasmid construction schemes, is described. The advantage of fragment tagging is that it facilitates the isolation of specific plasmid DNA molecules present in small amounts within mixed pools of DNA. Four examples that illustrate several variations of the fragment tagging concept are presented.

Cloning, Molecular↗
PubMed1989

The primary structure of Escherichia coli L-threonine dehydrogenase.

The complete primary structures of Escherichia coli L-threonine dehydrogenase has been deduced by sequencing the cloned tdh gene. The primary structure so determined agrees with results obtained independently for the amino acid composition, the N-terminal amino acid sequence (20 residues), and a short sequence at the end of an internal peptide of the purified enzyme. The presence of a predicted Asp-Pro bond at residues 148 and 149 was confirmed by treatment of purified threonine dehydrogenase with dilute acid and subsequent analysis of the resulting cleavage products. The primary structure of L-threonine dehydrogenase from E. coli has been examined for possible homology to other NAD+-dependent dehydrogenases; indications are that this enzyme is a member of the zinc-containing long-chain alcohol/polyol dehydrogenase family.

Acids↗
PubMed1989

Activation of a cryptic pathway for threonine metabolism via specific IS3-mediated alteration of promoter structure in Escherichia coli.

The tdh operon of Escherichia coli consists of two genes whose products catalyze sequential steps in the formation of glycine and acetyl coenzyme A from threonine. The operation of the tdh pathway can potentially confer at least two capabilities on the cell: the first is to provide a biosynthetic source of glycine, serine, or both that is an alternative to the conventional (triose phosphate) pathway; the second is to enable cells to utilize threonine as the sole carbon source. The latter capability is referred to as the Tuc+ phenotype. In wild-type E. coli, the tdh operon is expressed at levels that are too low to bestow the Tuc+ phenotype, even in leucine-supplemented media, where the operon is induced eightfold. In eight Tuc+ mutants, the expression of the tdh operon was elevated 100-fold relative to the uninduced wild-type operon. The physical state of the DNA at the tdh locus in these Tuc+ strains was analyzed by Southern blotting and by DNA sequencing. In eight independent isolates the mobile genetic element IS3 was found to lie within the tdh promoter region in identical orientations. In six cases that were examined by DNA sequencing, IS3 occupied identical sites between the -10 and -35 elements of the tdh promoter. The transcription start points for the wild-type tdh promoter and one IS3-activated tdh promoter were identical. In effect, the repeatedly observed transposition event juxtaposed an IS3-borne -35 region and the tdh-specific -10 region, generating a hybrid promoter whose utilization led to elevated, constitutive expression of the tdh operon. This is the first case of promoter activation by IS3 where the site of transcription initiation is unaltered.

Base Sequence↗