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C F Higgins

Publications and source records attributed to C F Higgins.

At least 73 records · Page 4Linked to original sources
PubMed1996

A DEAD-box RNA helicase in the Escherichia coli RNA degradosome.

The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. Both enzymes are important in RNA processing and messenger RNA degradation. Here we report that enolase and RhlB are two other major components of the degradosome. Enolase is a glycolytic enzyme with an unknown role in RNA metabolism. RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes. We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase. Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation. These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase. RhlB is thus an important enzyme in mRNA turnover.

Adenosine Triphosphatases↗
PubMed1996

Localized domains of DNA supercoiling: topological coupling between promoters.

According to the twin-supercoiled domain model, a local domain with a high level of DNA supercoiling can be generated between two divergently transcribed promoters. We have tested this model directly using the rate of DNA photo-cross-linking by 4,5', 8-trimethylpsoralen to measure local regions of unconstrained supercoiling in vivo. We demonstrate that, in plasmids, a localized domain of highly supercoiled DNA is generated upstream of the tetA promoter. The level of supercoiling of this domain depends on the activity of the tetA promoter and on the extent of translation of the tetA gene product. The highest level of supercoiling within this localized domain was observed in a topA strain, although the generation of a localized supercoiling domain could also be detected in a topA+ background, consistent with potential physiological significance. The level of supercoiling in this localized domain correlated with activation of the supercoiling-sensitive leu-500 promoter located upstream of tetA. These data provide a direct demonstration that localized domains of increased negative supercoiling can be generated upstream of an actively transcribed promoter, and that this can result in supercoiling-mediated transcriptional coupling between two promoters.

Antiporters↗
PubMed1996

Na+-induced transcription of nhaA, which encodes an Na+/H+ antiporter in Escherichia coli, is positively regulated by nhaR and affected by hns.

nhaA encodes an Na+/H+ antiporter in Escherichia coli which is essential for adaptation to high salinity and alkaline pH in the presence of Na+. We used Northern (RNA) analysis to measure directly the cellular levels of nhaA mRNA. NhaR belongs to the LysR family of regulatory proteins. Consistent with our previous data with an nhaA'-'lacZ fusion, NhaR was found to be a positive regulator and Na+ was found to be a specific inducer of nhaA transcription. In the nhaA'-'lacZ fusion, maximal induction was observed at alkaline pH. In contrast, in the nhaA+ strain both the level of nhaA expression and the induction ratio were lower at alkaline pH. This difference may be due to the activity of NhaA in the wild-type strain as NhaA efficiently excreted Na+ at alkaline pH and reduced the intracellular concentration of Na+, the signal for induction. We also showed that although the global regulator rpoS was not involved in nhaA regulation, the global regulator hns played a role. Thus, the expression of nhaA'-'lacZ was derepressed in strains bearing hns mutations and transformation with a low-copy-number plasmid carrying hns repressed expression and restored Na+ induction. The derepression in hns strains was nhaR independent. Most interestingly, multicopy nhaR, which in an hns+ background acted only as an Na+-dependent positive regulator, acted as a repressor in an hns strain in the absence of Na+ but was activated in the presence of the ion. Hence, an interplay between nhaR and hns in the regulation of nhaA was suggested.

Adaptation, Biological↗
PubMed1995

The crystal structures of the oligopeptide-binding protein OppA complexed with tripeptide and tetrapeptide ligands.

BACKGROUND: The periplasmic oligopeptide-binding protein OppA has a remarkably broad substrate specificity, binding peptides of two or five amino-acid residues with high affinity, but little regard to sequence. It is therefore an ideal system for studying how different chemical groups can be accommodated in a protein interior. The ability of the protein to bind peptides of different lengths has been studied by co-crystallising it with different ligands. RESULTS: Crystals of OppA from Salmonella typhimurium complexed with the peptides Lys-Lys-Lys (KKK) and Lys-Lys-Lys-Ala (KKKA) have been grown in the presence of uranyl ions which form important crystal contacts. These structures have been refined to 1.4 A and 2.1 A, respectively. The ligands are completely enclosed, their side chains pointing into large hydrated cavities and making few strong interactions with the protein. CONCLUSIONS: Tight peptide binding by OppA arises from strong hydrogen bonding and electrostatic interactions between the protein and the main chain of the ligand. Different basic side chains on the protein form salt bridges with the C terminus of peptide ligands of different lengths.

Amino Acid Sequence↗
PubMed1995

DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium.

Transcription from many bacterial promoters is sensitive to the level of DNA supercoiling. We have investigated the mechanism by which environmentally induced changes in DNA supercoiling might regulate transcription. For the proU promoter of Salmonella typhimurium, osmotically induced changes in DNA topology appear to play a primary regulatory role. Changes in DNA supercoiling (linking number; delta Lk) are partitioned into changes in the winding of the strands of the double helix about themselves (twist; delta Tw) and/or elastic deformations or flexibility of the DNA helix (writhe; delta Wr). Mutations of the proU promoter were isolated in vivo, or generated in vitro, which altered the spacing between the -10 and -35 motifs. Studies on these mutant promoters, both in vivo and in vitro, exclude models in which changes in DNA twist play a regulatory role. Instead, our data suggest that increased DNA flexibility, reflecting the osmotically induced increase in negative supercoiling of DNA, is required for promoter activation.

Amino Acid Transport Systems↗
PubMed1995

Protein kinase C-mediated phosphorylation of the human multidrug resistance P-glycoprotein regulates cell volume-activated chloride channels.

The multidrug resistance P-glycoprotein (P-gp), which transports hydrophobic drugs out of cells, is also associated with volume-activated chloride currents. It is not yet clear whether P-gp is a channel itself, or whether it is a channel regulator. Activation of chloride currents by hypotonicity in cells expressing P-gp was shown to be regulated by protein kinase C (PKC). HeLa cells exhibited volume-activated chloride currents indistinguishable from those obtained in P-gp-expressing cells except that they were insensitive to PKC. HeLa cells did not express detectable P-gp but, following transient transfection with cDNA encoding P-gp, the volume-activated channels acquired PKC regulation. PKC regulation was abolished when serine/threonine residues in the consensus phosphorylation sites of the linker region of P-gp were replaced with alanine. Replacement of these residues with glutamate, in order to mimic the charge of the phosphorylated protein, also mimicked the effects of PKC on channel activation. These data demonstrate that PKC-mediated phosphorylation of P-gp regulates the activity of an endogenous chloride channel and thus indicate that P-gp is a channel regulator.

3T3 Cells↗
PubMed1995

P-glycoprotein and cell volume-activated chloride channels.

The multidrug resistance P-glycoprotein (P-gp) is an active drug transporter which can expel hydrophobic compounds from cells. Expression of P-gp has many effects on cells and tissues and the physiological function, or functions, of P-gp are still unclear. Recently, expression of P-gp has been associated with altered activity of chloride channels which play a role in regulating cell volume of response to osmotic shock or nutrient uptake. The nature and physiological role of this association has been a subject of some debate. In this article, mechanisms by which P-gp might influence cell volume-activated chloride currents is discussed, and the potential physiological role of this regulation considered.

3T3 Cells↗
PubMed1995

Interaction of tamoxifen with the multidrug resistance P-glycoprotein.

Tamoxifen is an anti-oestrogen which is currently being assessed as a prophylactic for women at high risk of breast cancer. Taxoxifen has also been shown to reverse multidrug resistance in P-glycoprotein (P-gp)-expressing cells, although the mechanism of action is unknown. In this study we demonstrate that tamoxifen interacts directly with P-gp. Plasma membranes from P-gp-expressing cells bound [3H]tamoxifen in a specific and saturable fashion. A 180 kDa membrane protein in these membranes, labelled by the affinity analogue tamoxifen aziridine and azidopine, was shown to be P-gp. Tamoxifen reduced the binding of vinblastine and azidopine to P-gp, and tamoxifen increased [3H]vinblastine accumulation in P-gp-expressing cells to levels approaching those in non-P-gp-expressing cells. However, the cellular accumulation of [3H]tamoxifen itself was not influenced by the presence of P-gp. Thus, tamoxifen appears to reverse multidrug resistance by binding to P-gp and inhibiting the transport of cytotoxic drugs, but does not itself appear to be transported by the protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Lens opacification by antioestrogens: tamoxifen vs ICI 182,780.

The antioestrogen, tamoxifen, blocks volume-regulated chloride channels and reduces transparency in bovine lenses maintained in vitro. In contrast to tamoxifen, the steroidal antioestrogen, ICI 182780, did not block volume-regulated chloride currents in three cultured cell lines and required 10 fold higher concentration to induce significant opacification of bovine lenses maintained in vitro. These data suggest that ocular toxic side effects will be minimized by use of the steroidal (ICI 182780) rather than nonsteroidal antioestrogens (tamoxifen).

Animals↗
PubMed1995

Volume-activated chloride currents associated with the multidrug resistance P-glycoprotein.

The ability to regulate volume is an important property of most, if not all cells. In epithelial cells, amongst others, cell volume-activated chloride channels are central to this response. The molecular identities of these channels are not yet known. Expression of the human multidrug resistance P-glycoprotein (P-gp) has been associated with cell volume-regulated chloride currents, although the nature of this association is the subject of debate. Recent data indicate that P-gp acts by regulating the activation of an endogenous channel protein. In this review, evidence associating P-gp with cell volume-activated chloride currents, and the possible mechanisms by which this might be achieved, are discussed.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The structural basis of sequence-independent peptide binding by OppA protein.

Specific protein-ligand interactions are critical for cellular function, and most proteins select their partners with sharp discrimination. However, the oligopeptide-binding protein of Salmonella typhimurium (OppA) binds peptides of two to five amino acid residues without regard to sequence. The crystal structure of OppA reveals a three-domain organization, unlike other periplasmic binding proteins. In OppA-peptide complexes, the ligands are completely enclosed in the protein interior, a mode of binding that normally imposes tight specificity. The protein fulfills the hydrogen bonding and electrostatic potential of the ligand main chain and accommodates the peptide side chains in voluminous hydrated cavities.

Amino Acid Sequence↗
PubMed1994

P-glycoprotein. To flip or not to flip?

The phenotype of mice homozygous for mutations of the mdr2 gene suggests that the mdr2 protein, which is closely related to the multidrug resistance P-glycoprotein, has a role in phospholipid transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

The chromatin-associated protein H-NS alters DNA topology in vitro.

H-NS is one of the two most abundant proteins in the bacterial nucleoid and influences the expression of a number of genes. We have studied the interaction of H-NS with DNA; purified H-NS was demonstrated to constrain negative DNA supercoils in vitro. This provides support for the hypothesis that H-NS influences transcription via changes in DNA topology, and is evidence of a structural role for H-NS in bacterial chromatin. The effects of H-NS on topology were only observed at sub-saturating concentrations of the protein. In addition, a preferred binding site on DNA was identified by DNase I footprinting at sub-saturating H-NS concentrations. This site corresponded to a curved sequence element which we previously showed, by in vivo studies, to be a site at which H-NS influences transcription of the proU operon. When present in saturating concentrations, H-NS did not constrain supercoils and bound to DNA in a sequence-independent fashion, covering all DNA molecules from end to end, suggesting that H-NS may form distinct complexes with DNA at different H-NS:DNA ratios. The data presented here provide direct support for the hypothesis that H-NS acts at specific sites to influence DNA topology and, hence, transcription.

Bacterial Proteins↗
PubMed1994

The chromatin-associated protein H-NS.

H-NS is a major component of chromatin in enteric bacteria. H-NS plays a structural role in organising the chromosome, and influences DNA rearrangements as well as the expression of many genes. The biochemical and functional characteristics of H-NS are distinct from those of 'typical' DNA-binding proteins and much remains to be learned about the mechanism(s) by which H-NS acts. In this article we review our current understanding of the role of H-NS, and describe possible models by which H-NS might influence DNA structure and gene expression.

Amino Acid Sequence↗
PubMed1994

Role of hns in the virulence phenotype of pathogenic salmonellae.

A TnphoA-generated mutant C5060, attenuated for virulence, was derived from the mouse-virulent Salmonella typhimurium strain C5. This mutation, designated hns-112::TnphoA, harbours the transposon in the 3' end of hns, with the alkaline phosphatase open reading frame in the opposite orientation to that of hns. Bacterial strains harbouring hns-112::TnphoA were mucoid and had altered levels of DNA supercoiling, as monitored using pUC18 as a reporter plasmid. Transduction of hns-112::TnphoA into mouse virulent strains, including S. typhimurium SL1344 and Salmonella enteritidis Se795, resulted in attenuation. When an independent hns mutation, harbouring a kanamycin-resistance cassette inserted into the Kpnl site at base pair 237 of the hns gene, was introduced into S. typhimurium C5, the isolates were also attenuated. S. typhimurium C5 isolates harbouring the multicopy plasmid pGB651, which encodes the Escherichia coli hns gene, were partially attenuated in mice. Transductional analysis, using Tn10 insertions located close to the hns gene, showed that virulence could be restored in genetic crosses that eliminated the resident hns mutations. However, some hns+ transductants were still attenuated, suggesting that secondary attenuating lesions can accumulate in hns-deficient strains. These studies show that the hns locus plays a role in Salmonella virulence.

Animals↗
PubMed1994

A protein complex mediating mRNA degradation in Escherichia coli.

mRNA degradation in Escherichia coli is mediated by a combination of exo- and endoribonucleases. We present evidence for a multiprotein complex which includes at least two enzymes that play important roles in mRNA degradation: the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E. An activity which impedes the processive activity of PNPase at stem-loop structures also appears to be associated with the complex. This complex is estimated to have a molecular mass of about 500 kDa and includes several additional polypeptides whose functions are unknown. The identification of a complex which includes several activities associated with mRNA degradation has implications for the mechanisms and co-ordinated control of mRNA degradation.

Amino Acid Sequence↗