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W A Walter

Publications and source records attributed to W A Walter.

18 recordsLinked to original sources
PubMed1996

Involvement of the DnaK-DnaJ-GrpE chaperone team in protein secretion in Escherichia coli.

We used depletion studies designed to further investigate the role of the DnaK, DnaJ, and GrpE heat shock proteins in the SecB-dependent and SecB-independent secretion pathways. Our previous finding that SecB-deficient strains containing the grpE280 mutation were still secretion proficient raised the possibility that GrpE was not involved in this secretory pathway. Using depletion studies, we now demonstrate a requirement for GrpE in this pathway. In addition, depletion studies demonstrate that while DnaK, DnaJ, and GrpE are involved in the secretion of the SecB-independent proteins (alkaline phosphatase, ribose-binding protein, and beta-lactamase), they are not the primary chaperones in this process.

Alkaline Phosphatase↗
PubMed1993

Genetic evidence for the interaction between cluster I and cluster III rifampicin resistant mutations.

Rifampicin-resistant (Rifr) mutations of Escherichia coli map to the central portion of the rpoB gene, which encodes the beta subunit of RNA polymerase. These mutations are located in three distinct clusters, designated I, II and III. Three intragenic suppressors of the cluster III Rifr mutation, rpoB3406(RH687), restore the ability of the mutant strain to grow at low and high temperatures and map to a single locus in cluster I. These suppressors are identical to two previously characterized Rifr alleles, rpoB3401(RC529) and rpoB3402(RS529). None of the other 14 previously identified Rifr mutations that we have characterized confers this phenotype. We suggest that this allele-specific suppression results from interaction between Cluster I and Cluster III of the beta subunit.

Alleles↗
PubMed1993

Amino-terminal amino acids modulate sigma-factor DNA-binding activity.

Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase. Genetic studies have indicated that sigma itself interacts with DNA at the -10 and -35 promoter consensus sequences. Binding of Escherichia coli sigma 70 to DNA in vitro, however, can only be observed for truncated polypeptides lacking the amino-terminal amino acids. We have investigated the role of the amino terminus of E. coli sigma 70 in controlling DNA-binding ability. Deletion analysis indicates that amino acids within amino-terminal region 1.1 of sigma 70 inhibit DNA binding by the carboxy-terminal DNA-binding domains. Furthermore, inhibition of binding by the amino-terminal inhibitory domain of sigma 70 can be observed in trans. Likewise, the amino-terminal extensions of two alternative sigma-factors, E. coli sigma 32 and Bacillus subtilis sigma K, negatively affect the DNA binding activity of their carboxy-terminal domains. We propose that initiation of transcription is subject to modulation as a result of the composition and/or structure of the amino terminus of the sigma-subunit and that the sigma family of proteins belong to a larger class of intramolecularly regulated transcriptional effectors.

Amino Acids↗
PubMed1993

Accumulation of secretory protein precursors in Escherichia coli induces the heat shock response.

The accumulation of secretory protein precursors, caused either by mutations in secB or secA or by the overproduction of export-defective proteins, results in a two- to fivefold increase in the synthesis of heat shock proteins. In such strains, sigma 32, the alternative sigma factor responsible for transcription of the heat shock genes, is stabilized. The resultant increase in the level of sigma 32 leads to increased transcription of heat shock genes and increased synthesis of heat shock proteins. We have also found that although a secB null mutant does not grow on rich medium at a temperature range of 30 to 42 degrees C, it does grow at 44 degrees C. In addition, we found that a secB null mutant exhibits greater thermotolerance than the wild-type parental strain. Elevated levels of heat shock proteins, as well as some other non-heat shock proteins, may account for the partial heat resistance of a SecB-lacking strain.

Bacterial Proteins↗
PubMed1993

The role of the sigma subunit in promoter recognition by RNA polymerase.

Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase holoenzyme. Genetic and physiological studies have indicated that sigma interacts with promoter DNA sequences but biochemical analysis did not demonstrate DNA binding by the sigma subunit itself. We have investigated both the DNA binding properties and the regulation of DNA binding for several sigma factors using partial polypeptides. In this report we demonstrate that partial sigmas can bind to promoter DNA in the absence of the core subunits of RNA polymerase and the binding is regulated by an N-terminal inhibitory domain.

Amino Acid Sequence↗
PubMed1992

Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA.

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Bacterial Proteins↗
PubMed1992

A mutant sigma 32 with a small deletion in conserved region 3 of sigma has reduced affinity for core RNA polymerase.

sigma 70, encoded by rpoD, is the major sigma factor in Escherichia coli. rpoD285 (rpoD800) is a small deletion mutation in rpoD that confers a temperature-sensitive growth phenotype because the mutant sigma 70 is rapidly degraded at high temperature. Extragenic mutations which reduce the rate of degradation of RpoD285 sigma 70 permit growth at high temperature. One class of such suppressors is located in rpoH, the gene encoding sigma 32, an alternative sigma factor required for transcription of the heat shock genes. One of these, rpoH113, is incompatible with rpoD+. We determined the mechanism of incompatibility. Although RpoH113 sigma 32 continues to be made when wild-type sigma 70 is present, cells show reduced ability to express heat shock genes and to transcribe from heat shock promoters. Glycerol gradient fractionation of sigma 32 into the holoenzyme and free sigma suggests that RpoH113 sigma 32 has a lower binding affinity for core RNA polymerase than does wild-type sigma 32. The presence of wild-type sigma 70 exacerbates this defect. We suggest that the reduced ability of RpoH113 sigma 32 to compete with wild-type sigma 70 for core RNA polymerase explains the incompatibility between rpoH113 and rpoD+. The rpoH113 cells would have reduced amounts of sigma 32 holoenzyme and thus be unable to express sufficient amounts of the essential heat shock proteins to maintain viability.

Chromosome Deletion↗
PubMed1991

Physiological effects of the fructose-1,6-diphosphate aldolase ts8 mutation on stable RNA synthesis in Escherichia coli.

The conditional lethal mutations ts8 and h8 are located in fda, the gene encoding aldolase, and they inhibit RNA synthesis upon shift to the nonpermissive temperature. We demonstrate that both mutations preferentially inhibit stable RNA synthesis and that this inhibition occurs at the level of transcription initiation. The susceptibility of a promoter to the inhibitory effects of ts8 is correlated with the ability of the promoter to be growth rate regulated. This effect is independent of relA and spoT function. Inhibition is dependent upon glucose metabolism past the generation of glucose-6-phosphate; however, the mechanism of this effect is unknown.

Escherichia coli↗
PubMed1989

Altered promoter recognition by mutant forms of the sigma 70 subunit of Escherichia coli RNA polymerase.

We have systematically assayed the in vivo promoter recognition properties of 13 mutations in rpoD, the gene that encodes the sigma 70 subunit of Escherichia coli RNA polymerase holoenzyme, using transcriptional fusions to 37 mutant and wild-type promoters. We found three classes of rpoD mutations: (1) mutations that suggest contacts between amino acid side-chains of sigma 70 and specific bases in the promoter; (2) mutations that appear to affect either sequence independent contacts to promoter DNA or isomerization of the polymerase; and (3) mutations that have little or no effect on promoter recognition. Our results lead us to suggest that a sequence near the C terminus of sigma 70, which is similar to the helix-turn-helix DNA binding motif of phage and bacterial DNA binding proteins, is responsible for recognition of the -35 region, and that a sequence internal to sigma 70, in a region which is highly conserved among sigma factors, recognizes the -10 region of the promoter. rpoD mutations that lie in the recognition helix of the proposed helix-turn-helix motif affect interactions with specific bases in the -35 region, while mutations in the upstream helix, which is thought to contact the phosphate backbone, have sequence-independent effect on promoter recognition.

Base Sequence↗
PubMed1989

The activity of sigma 32 is reduced under conditions of excess heat shock protein production in Escherichia coli.

The expression of heat shock genes in Escherichia coli is controlled by the action of an alternate sigma-factor of RNA polymerase, sigma 32, which directs core RNA polymerase to recognize the promoters for heat shock genes. After a shift from 30 degrees C to 42 degrees C, both the level of sigma 32 and transcription initiation at heat shock promoters transiently increase, indicating that heat shock gene expression is regulated by changes in the concentration of sigma 32. Here, we report that heat shock gene expression is regulated by changes in the activity of sigma 32 under some conditions. Our results show that the transient repression of heat shock protein synthesis, which follows a shift down from 42 degrees C to 30 degrees, occurs as a result of decreased transcription initiation at heat shock promoters, but this repression is accompanied by only a small decrease in the level of sigma 32. In addition, the induction of heat shock proteins following overproduction of sigma 32 from a multicopy plasmid is only transient, despite the fact that the level of sigma 32 remains elevated. Constitutive overproduction of sigma 32 also fails to cause a proportionate increase in heat shock gene transcription. These three examples suggest that the activity of sigma 32 is reduced under conditions of excess heat shock gene expression.

Chloramphenicol↗
PubMed1988

Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli.

Rifampicin resistant (Rifr mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have used our collection of 17 sequenced Rifr mutations to investigate the involvement of E. coli RNA polymerase in the antitermination systems enhancing expression of delayed early lambda genes or stable RNA. We have found that Rifr mutations affect both lambda N-mediated antitermination and the cellular antitermination system involved in synthesis of stable RNA. Because NusA is involved in antitermination and termination, we also investigated the interaction of NusA and RNA polymerase by determining whether Rifr mutations alter NusA-dependent termination or antitermination in cells with defective nusA alleles. We have shown that Rifr mutations can either enhance or suppress the phenotypes of defective nusA alleles. Most Rifr mutations alter the temperature range over which the nusA1 allele supports lambda N-mediated antitermination. In addition, a number of Rifr alleles restore termination to the nusA10(Cs) and the nusA11(Ts) mutants defective in this process. Our results indicate that the region of the rpoB gene defined by the Rifr mutations is involved in the antitermination process and affects the activity of the NusA protein directly or indirectly.

Alleles↗
PubMed1988

Characterization of the termination phenotypes of rifampicin-resistant mutants.

Rifampicin-resistant (Rifr) mutations map in the rpoB gene encoding the beta subunit of Escherichia coli RNA polymerase. We have examined the effect of each of the 17 sequenced Rifr mutations in our collection on transcription termination. The effect of each Rifr mutation was measured at three types of terminators: simple terminators requiring only RNA polymerase to terminate in vitro, and complex terminators requiring either Rho or Tau for in-vitro termination. Almost every Rifr allele examined (14/17) affected readthrough at one or more of these terminators. We found that mutations with similar termination phenotypes were clustered suggesting functional specialization within the region of rpoB defined by the Rifr mutations. The interaction of the Rifr mutations with the defective rho15 allele was also investigated. Only two Rifr mutations suppress the termination defect of rho15 strains. We discuss models to explain how this region of the beta polypeptide might be involved in the process of transcription termination.

Drug Resistance, Microbial↗
PubMed1988

Escherichia coli heat shock gene mutants are defective in proteolysis.

Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown. The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides. We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase. Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis. Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay. The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation. These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides.

Adenosine Triphosphate↗
PubMed1987

Sigma 32 synthesis can regulate the synthesis of heat shock proteins in Escherichia coli.

The Escherichia coli rpoH (htpR) gene product, sigma 32, is required for the normal expression of heat shock genes and for the heat shock response. We present experiments indicating a direct role for sigma 32 in controlling the heat shock response. Both the induction and decline in the synthesis of heat shock proteins can be controlled by changes in the rate of synthesis of sigma 32. Specifically, we show that: (1) sigma 32 is an unstable protein, degraded with a half-life of approximately 4 min; (2) increasing the rate of synthesis of sigma 32, by inducing expression from a Plac or Ptac-rpoH fusion, is sufficient to increase the rate of synthesis of heat shock proteins; (3) during the shut-off phase of the heat shock response synthesis of sigma 32 is repressed post-transcriptionally, and the dnaK756 mutation, which causes a defect in the shut-off phase, prevents the post-transcriptional repression of synthesis of sigma 32. These results serve as a basis for understanding the role of DnaK in the heat shock response, the regulation of sigma 32 synthesis, and the role of sigma 32 in controlling transcription of heat shock genes.

Escherichia coli↗
PubMed1987

Regulation of the promoters and transcripts of rpoH, the Escherichia coli heat shock regulatory gene.

In Escherichia coli the product of the rpoH (htpR) gene, sigma 32, directs RNA polymerase to initiate transcription from heat shock promoters at all temperatures. Transcription of the heat shock genes is increased when cells are exposed to high temperatures because of increased transcription initiation by sigma 32-RNA polymerase. As a step toward understanding the regulation of the heat shock response we have examined the transcription of the rpoH gene. Using S1 mapping, promoter cloning, and in vitro transcription, we have identified the promoters and the terminator for the rpoH transcription unit. The rpoH transcripts are monocistronic and originate from at least three promoters. None of the promoters is recognized by sigma 32-RNA polymerase. Two are recognized by sigma 70-RNA polymerase and are active at both low and high growth temperatures. We do not know what form of RNA polymerase recognizes the third promoter. Transcripts from this promoter are abundant only at high temperature and are present after shift to the lethal temperature of 50 degrees C, even at times when there are no detectable transcripts from the other rpoH promoters. The amount of rpoH mRNA increases fivefold by 8 min after shift from 30 to 43.5 degrees C but rpoH mRNA synthesis increases by less than twofold, indicating that there is post-transcriptional control of the level of rpoH mRNA and presumably of sigma 32.

Base Sequence↗
PubMedDate not supplied

The heat shock response of E. coli is regulated by changes in the concentration of sigma 32.

Cells subjected to a heat shock, or a variety of other stresses increase the synthesis of a set of proteins, known as heat shock proteins. This response is apparently universal, occurring in the entire range from bacterial to mammalian cells. In Escherichia coli heat shock protein synthesis transiently increases following a shift from 30 degrees C to 42 degrees C as a result of changes in transcription initiation at heat shock promoters. Heat shock promoters are recognized by RNA polymerase containing a sigma factor of relative molecular mass (Mr) 32,000 (32K) E sigma 32 and not E sigma 70, the major form of RNA polymerase holoenzyme. To determine whether changes in the concentration of sigma 32 regulate this response, we measured the amount of sigma 32 before and after shift to high temperature and found that it increased transiently during heat shock as a result of changes in sigma 32 synthesis and stability. Our results indicate that sigma 32 is directly responsible for regulation of the heat shock response.

Bacterial Proteins↗