PubMed Health⌕ Search

Biomedical subjects

T R Hoover

Publications and source records attributed to T R Hoover.

At least 19 recordsLinked to original sources
PubMed2001

Transcriptional regulation at a distance in bacteria.

Transcriptional enhancers are cis-acting DNA elements that are binding sites for regulatory proteins and function at large distances from promoter elements to stimulate transcription. Once thought to be unique to eukaryotes, enhancer-like elements have been discovered in a wide variety of bacteria. The regulatory proteins that bind to these bacterial enhancers must contact RNA polymerase to activate transcription. In principle, interactions between bacterial enhancer-binding proteins and RNA polymerase can occur by either DNA looping or tracking of the enhancer-binding protein along the DNA. Paradigms for each of these methods are found in bacterial systems. Activators of sigma(54)-RNA polymerase holoenzyme contact polymerase by DNA looping, while bacteriophage T4 gp45 functions as a sliding clamp that tracks along DNA until it engages RNA polymerase. Significant advances have been made over the last few years towards understanding the mechanisms by which bacterial enhancer-binding proteins activate transcription, but important aspects of these mechanisms are still poorly defined.

Amino Acid Sequence↗
PubMed2000

The amino terminus of Salmonella enterica serovar Typhimurium sigma(54) is required for interactions with an enhancer-binding protein and binding to fork junction DNA.

Transcription initiation by the sigma(54)-RNA polymerase holoenzyme requires an enhancer-binding protein that is thought to contact sigma(54) to activate transcription. To identify potential enhancer-binding protein contact sites in sigma(54), we compared the abilities of wild-type and truncated forms of Salmonella enterica serovar Typhimurium sigma(54) to interact with the enhancer-binding protein DctD in a chemical cross-linking assay. Removal of two regions in the amino-terminal portion of sigma(54), residues 57 to 105 and residues 144 to 179, prevented cross-linking, but removal of either region alone did not. In addition, deletion of 56 amino-terminal residues of sigma(54) (region I) reduced the affinity of the protein for a fork junction DNA probe.

Bacterial Proteins↗
PubMed2000

Transcription initiation-defective forms of sigma(54) that differ in ability To function with a heteroduplex DNA template.

Transcription by sigma(54)-RNA polymerase holoenzyme requires an activator that catalyzes isomerization of the closed promoter complex to an open complex. We examined mutant forms of Salmonella enterica serovar Typhimurium sigma(54) that were defective in transcription initiation but retained core RNA polymerase- and promoter-binding activities. Four of the mutant proteins allowed activator-independent transcription from a heteroduplex DNA template. One of these mutant proteins, L124P V148A, had substitutions in a sequence that had not been shown previously to participate in the prevention of activator-independent transcription. The remaining mutants did not allow efficient activator-independent transcription from the heteroduplex DNA template and had substitutions within a conserved 20-amino-acid segment (Leu-179 to Leu-199), suggesting a role for this sequence in transcription initiation.

Amino Acid Substitution↗
PubMed1999

A rhizobial homolog of IHF stimulates transcription of dctA in Rhizobium leguminosarum but not in Sinorhizobium meliloti.

Sequence inspection identified several potential IHF binding sites adjacent to the Rhizobium leguminosarum dctA promoter. IHF protected the -30 to -76 region from DNase I digestion, but systematic error in quantitative assays suggested that this protein DNA interaction is complex. IHF stimulated DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter both in vivo and in vitro. In contrast to R. leguminosarum dctA, the Sinorhizobium meliloti dctA promoter region was found to have a much weaker match to the consensus IHF binding site and a low affinity for IHF. Moreover, IHF had no effect on transcriptional activation from the S. meliloti dctA promoter in vitro. A base substitution was introduced into the IHF binding site of R. leguminosarum dtA that reduced the affinity of the promoter regulatory region for IHF by approximately 30-fold and resulted in an eight-fold decrease in transcriptional activation in both R. leguminosarum and S. meliloti. These data suggest that both rhizobial species have an IHF homolog that stimulates DctD-mediated transcriptional activation from the R. leguminosarum dctA promoter. Consistent with this hypothesis, a 12.5 kDa protein was identified from R. leguminosarum as a putative homolog of IHF subunit beta by immunoblotting and N-terminal sequence analysis.

Amino Acid Sequence↗
PubMed1999

Mutant forms of Salmonella typhimurium sigma54 defective in transcription initiation but not promoter binding activity.

Transcription initiation with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis. sigma54's binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation. Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium sigma54 that were deficient in transcription initiation but still directed sigma54-holoenzyme to the promoter to form a closed complex. Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for sigma54-holoenzyme to respond to the activator. From the remaining mutants, we identified four residues in region III which when altered affect the function of sigma54 at some point after closed-complex formation. These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

Alleles↗
PubMed1998

Cloning and characterization of Planctomyces limnophilus rpoN: complementation of a Salmonella typhimurium rpoN mutant strain.

The rpoN gene, which encodes the alternative sigma factor sigma 54, was cloned from the budding, peptidoglycan-less bacterium Planctomyces limnophilus. P. limnophilus rpoN complemented the Ntr- phenotype of a Salmonella typhimurium rpoN mutant strain. The P. limnophilus rpoN gene encoded a predicted polypeptide that was 495 residues in length and shared a significant homology with other members of the sigma 54 family. The protein sequence displayed all of the characteristic motifs found in members of this family, including the C-terminal helix-turn-helix motif and the well-conserved RpoN box. A potential sigma 54-dependent activator was also identified in P. limnophilus. These findings extend the range of phylogenetic groups within the Domain Bacteria that are known to contain sigma 54.

Amino Acid Sequence↗
PubMed1997

A conserved region in the sigma54-dependent activator DctD is involved in both binding to RNA polymerase and coupling ATP hydrolysis to activation.

Rhizobium melioti DctD activates transcription from the dctA promoter by catalysing the isomerization of closed complexes between sigma54-RNA polymerase holoenzyme and the promoter to open complexes. DctD must make productive contact with sigma54-holoenzyme and hydrolyse ATP to catalyse this isomerization. To define further the activation process, we sought to isolate mutants of DctD that had reduced affinities for sigma54-holoenzyme. Mutagenesis was confined to the well-conserved C3 region of the protein, which is required for coupling ATP hydrolysis to open complex formation in sigma54-dependent activators. Mutant forms of DctD that failed to activate transcription and had substitutions in the C-terminal half of the C3 region were efficiently cross-linked to sigma54 and the beta-subunit of RNA polymerase, suggesting that they bound normally to sigma54-holoenzyme. In contrast, some mutant forms of DctD with amino acid substitutions in the N-terminal half of the C3 region had reduced affinities for sigma54 and the beta-subunit in the cross-linking assay. These data suggest that the N-terminal half of the C3 region of DctD contains a site that may contact sigma54-holoenzyme during open complex formation.

Amino Acid Sequence↗
PubMed1997

Alterations within the activation domain of the sigma 54-dependent activator DctD that prevent transcriptional activation.

Rhizobium meliloti DctD (C4-dicarboxylate transport protein D) is a transcriptional activator that catalyzes the ATP-dependent isomerization of closed complexes between sigma 54-RNA polymerase holoenzyme and the dctA promoter to open complexes. Following random mutagenesis of dctD, 55 independent mutant forms of DctD that failed to activate transcription from a dctA'-'lacZ reporter gene in Escherichia coli were selected, and the amino acid substitutions were determined for these mutant proteins. Amino acid substitutions were distributed throughout the central domain of the protein, the domain responsible for transcription activation, but most of the substitutions occurred within three highly conserved regions of the protein. Selected mutant proteins were purified, and their activities were studied in vitro. All of the purified mutant proteins appeared to have normal DNA-binding activity and interacted with sigma 54 and core RNA polymerase, as determined from protein crosslinking assays. Proteins with amino acid substitutions in a region spanning amino acid positions 222 to 225 retained their ATPase activities, whereas proteins with substitutions in other regions had little or no ATPase activity. Taken together, these data suggest that the region that encompasses amino acid residues 222 through 225 probably functions in coupling the energy released from ATP hydrolysis to open complex formation rather than as a major determinant for binding to RNA polymerase.

Adenosine Triphosphate↗
PubMed1997

Genetic analysis of the Rhizobium meliloti nifH promoter, using the P22 challenge phage system.

In several genera of bacteria, the sigma54-RNA polymerase holoenzyme (E sigma54) is a minor form of RNA polymerase that is responsible for transcribing genes whose products are involved in diverse metabolic processes. E sigma54 binds to the promoters of these genes to form a closed promoter complex. An activator protein is required for the transition of this closed promoter complex to an open complex that is transcriptionally competent. In this study, the P22-based challenge phage system was used to investigate interactions between E sigma54 and the Rhizobium meliloti nifH promoter. Challenge phages were constructed in which the R. meliloti nifH promoter replaced the binding site for the Mnt protein, a repressor of the phage P22 ant gene. When a Salmonella typhimurium strain that overexpressed sigma54 was infected with these challenge phages, E sigma54 bound to the nifH promoter and repressed transcription of the ant gene as seen by the increased frequency of lysogeny. Following mutagenesis of challenge phages that carried the R. meliloti nifH promoter, mutant phages that could form plaques on an S. typhimurium strain that overexpressed sigma54 were isolated. These phages had mutations within the nifH promoter that decreased the affinity of the promoter for E sigma54. The mutations were clustered in seven highly conserved residues within the -12 and -24 regions of the nifH promoter.

Bacteriophages↗
PubMed1995

Protein crosslinking studies suggest that Rhizobium meliloti C4-dicarboxylic acid transport protein D, a sigma 54-dependent transcriptional activator, interacts with sigma 54 and the beta subunit of RNA polymerase.

Rhizobium meliloti C4-dicarboxylic acid transport protein D (DCTD) activates transcription by a form of RNA polymerase holoenzyme that has sigma 54 as its sigma factor (referred to as E sigma 54). DCTD catalyzes the ATP-dependent isomerization of closed complexes between E sigma 54 and the dctA promoter to transcriptionally productive open complexes. Transcriptional activation probably involves specific protein-protein interactions between DCTD and E sigma 54. Interactions between sigma 54-dependent activators and E sigma 54 are transient, and there has been no report of a biochemical assay for contact between E sigma 54 and any activator to date. Heterobifunctional crosslinking reagents were used to examine protein-protein interactions between the various subunits of E sigma 54 and DCTD. DCTD was crosslinked to Salmonella typhimurium sigma 54 with the crosslinking reagents succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-hydroxysulfosuccinimidyl-4-azidobenzoate. Cys-307 of sigma 54 was identified by site-directed mutagenesis as the residue that was crosslinked to DCTD. DCTD was also crosslinked to the beta subunit of Escherichia coli core RNA polymerase with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, but not with N-hydroxysulfosuccinimidyl-4-azidobenzoate. These data suggest that interactions of DCTD with sigma 54 and the beta subunit may be important for transcriptional activation and offer evidence for interactions between a sigma 54-dependent activator and sigma 54, as well as the beta subunit of RNA polymerase.

Azides↗
PubMed1994

Constitutive ATP hydrolysis and transcription activation by a stable, truncated form of Rhizobium meliloti DCTD, a sigma 54-dependent transcriptional activator.

The dctD gene product (DCTD) activates transcription from dctA by the sigma 54-holoenzyme form of RNA polymerase in Rhizobium meliloti. We have purified a constitutively active form of R. meliloti DCTD that lacks 142 amino acid residues from the N terminus (designated DCTDL143). Purified DCTDL143 recognized the DCTD-binding sites at the dctA promoter region and catalyzed the isomerization of closed complexes between sigma 54-holoenzyme and the dctA promoter to open complexes. Like the related sigma 54-dependent activators NTRC and NIFA, a purine nucleoside triphosphate with a hydrolyzable beta-gamma bond was required prior to transcription initiation for this isomerization. DCTDL143 hydrolyzed purine nucleoside triphosphates but not pyrimidine nucleoside triphosphates. As observed with NTRC-phosphate, the specific activity for the ATPase of DCTDL143 was strongly dependent on the enzyme concentration and was stimulated by DNA fragments bearing the binding sites for the protein. These DNA fragments increased the Vmax for MgATP hydrolysis but did not significantly lower the apparent Km for MgATP. These data are consistent with the idea proposed for related activators that DCTDL143 must assemble into an active, oligomeric form before it can hydrolyze MgATP and presumably activate transcription.

Adenosine Triphosphate↗
PubMed1994

Rhizobium meliloti DctD, a sigma 54-dependent transcriptional activator, may be negatively controlled by a subdomain in the C-terminal end of its two-component receiver module.

Rhizobium meliloti DctD is believed to have three functional domains: an N-terminal, two-component receiver domain; and like other sigma 54-dependent activators, C-terminal and central domains for DNA binding and transcription activation. We have characterized a progressive series of N-terminal deletions of R. meliloti DctD. The N-terminal domain was not needed for binding the dctA upstream activation sequence. Only 25% of the C-terminal end of the receive domain was needed to significantly inhibit the central domain, and proteins lacking up to 60% of the N-terminal end of the receiver domain were 'inducible' in R. meliloti cells. We hypothesize that the N-terminal two-thirds of the DctD receiver domain augments and controls an adjacent subdomain for inhibiting the central domain.

Bacterial Proteins↗
PubMed1992

Role of integration host factor in stimulating transcription from the sigma 54-dependent nifH promoter.

In a wide variety of nitrogen-fixing organisms among the Purple Bacteria (large division of Gram-negative bacteria) the nitrogen fixation (nif) operons are transcribed by an alternative holoenzyme form of RNA polymerase, sigma 54-holoenzyme. Transcription depends on the activator protein NIFA (nitrogen fixation protein A), which catalyzes isomerization of closed complexes between this polymerase and a promoter to transcriptionally productive open complexes. NIFA-mediated activation of transcription from the nifH promoter of Klebsiella pneumoniae is greatly stimulated by the integration host factor IHF, which binds to a site between the upstream binding site for NIFA and the promoter, and bends the DNA. IHF fails to stimulate activation of transcription from this promoter by another activator of sigma 54-holoenzyme, NTRC (nitrogen regulatory protein C), which lacks a specific binding site in the nifH promoter region. As predicted, if the IHF-induced bend facilitates interaction between NIFA and sigma 54-holoenzyme, substitution of an NTRC-binding site for the NIFA-binding site allowed IHF to stimulate NTRC-mediated activation of transcription from the nifH promoter. The stimulation was of the same order of magnitude as that for NIFA in the native configuration of the promoter-regulatory region (up to 20-fold). With purified NTRC and the substitution construct we could demonstrate that stimulation by IHF in a purified transcription system was comparable to that in a crude coupled transcription-translation system, indicating that the stimulation in the crude system could be accounted for by IHF. The IHF stimulation was observed on linear as well as supercoiled templates, indicating that the geometric requirements are relatively simple. We have attempted to visualize the arrangement of proteins on DNA fragments carrying the nifH promoter-regulatory region of K. pneumoniae by electron microscopy. IHF stimulated NIFA-mediated activation of transcription from the nifH and nifD promoters of Bradyrhizobium japonicum and less so from the nifH promoters of Rhizobium meliloti and Thiobacillus ferrooxidans, consistent with previous observations that stimulation is greatest at promoters that are weak binding sites for sigma 54-holoenzyme in closed complexes.

Bacterial Proteins↗
PubMed1992

Entomophthoramycosis conidiobolae in a llama.

A 9-year-old female llama was evaluated for chronic dermatosis involving the external nares. The condition had been diagnosed as a fungal infection. Examination of punch biopsy specimens confirmed the diagnosis of Conidiobolus coronatus. Because of the chronicity of the disease and history of ineffective drug treatment, excision of infected tissue was attempted. Permanent nasal openings were created by suturing the nasal mucosa to the skin after debulking of infected tissue. Excision of most of the infected tissue enabled the llama to breathe easily for 3 years. Because C coronatus does not usually induce deep infections, it may continue to remain dormant in this llama.

Animals↗
PubMed1990

The integration host factor stimulates interaction of RNA polymerase with NIFA, the transcriptional activator for nitrogen fixation operons.

The regulatory protein NIFA activates transcription of nitrogen fixation (nif) operons by the sigma 54 holoenzyme form of RNA polymerase. NIFA from Klebsiella pneumoniae activates transcription from the nifH promoter in vitro; in addition, the integration host factor, IHF, binds between the nifH promoter and an upstream binding site for NIFA. We demonstrate here that IHF greatly stimulates NIFA-mediated activation of nifH transcription in vitro and thus that the two factors are functionally synergistic. Electron micrographs indicate that IHF bends the DNA in the nifH promoter regulatory region. Although IHF binds close to the nifH promoter, it does not directly stimulate binding of sigma 54 holoenzyme. Rather, the IHF-induced bend may facilitate productive contacts between NIFA and sigma 54 holoenzyme that lead to the formation of open complexes. IHF binds to nif promoter regulatory regions from a variety of organisms within the phylum "purple bacteria," suggesting a general ability to stimulate NIFA-mediated activation of nif transcription.

Bacterial Proteins↗
PubMed1990

Effectiveness of two commercial infectious bovine keratoconjunctivitis vaccines.

Two commercially available infectious bovine keratoconjunctivitis (IBK) vaccines were evaluated for their effectiveness in protecting cattle from disease caused by experimental challenge exposure and natural transmission of Moraxella bovis infections. The study was conducted as 2 experiments, using a total of 81 cattle that were culture-negative for M bovis prior to vaccination. In each experiment, young adult cattle were randomly allotted to 4 groups. Each calf in groups 1 and 2 was vaccinated according to the vaccine manufacturer's directions. Groups 3 and 4 were unvaccinated controls. Three weeks after the last vaccination, each calf in groups 1 and 3 was experimentally challenge exposed by dropping a suspension of viable cells of a virulent strain of M bovis directly onto the corneal surface of each eye. Calves in all 4 groups were then commingled in open pastures so that calves in groups 2 and 4 could be naturally exposed to the calves with experimentally induced infections. Each calf was examined for signs of ocular disease on a regular basis by 2 experienced clinicians who scored each eye for severity of disease on the basis of a prearranged scale. Neither clinician was aware of the vaccination or exposure status of the calf nor to which experimental group they belonged. Lacrimal secretions were collected regularly to determine the number of eyes in which the virulent organism became established. Moraxella bovis with bacterial cultural characteristics similar to those of the virulent strain placed in the eyes of groups 1 and 3 was cultured from greater than or equal to 83% of the eyes of calves in all groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗