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F Rojo

Publications and source records attributed to F Rojo.

At least 73 records · Page 4Linked to original sources
PubMed1994

Requirement for an A-tract structure at the binding site of phage phi 29 transcriptional activator.

The Bacillus subtilis phage phi 29 transcriptional activator, protein p4, binds to the 5'-AACT-TTTT-15 base-pair spacer-AAAATGTT-3' inverted repeat. In this communication, we study the influence in protein p4 binding of the DNA helical structure within the protein p4 recognition sequences, 5'-AAAATAG-3'. Protein p4 could efficiently bind to a modified target in which the A-tracts had been changed into T-tracts (a different sequence with a similar structure). Binding was lost when the structure of the binding site was modified by an interrupting C residue. The results suggest that the DNA helical structure of the A-tracts is critical for p4 binding. Two models are described that would explain how protein p4 recognized its target sequences on the DNA.

Bacillus Phages↗
PubMed1994

Promoters responsive to DNA bending: a common theme in prokaryotic gene expression.

The early notion of DNA as a passive target for regulatory proteins has given way to the realization that higher-order DNA structures and DNA-protein complexes are at the basis of many molecular processes, including control of promoter activity. Protein binding may direct the bending of an otherwise linear DNA, exacerbate the angle of an intrinsic bend, or assist the directional flexibility of certain sequences within prokaryotic promoters. The important, sometimes essential role of intrinsic or protein-induced DNA bending in transcriptional regulation has become evident in virtually every system examined. As discussed throughout this article, not every function of DNA bends is understood, but their presence has been detected in a wide variety of bacterial promoters subjected to positive or negative control. Nonlinear DNA structures facilitate and even determine proximal and distal DNA-protein and protein-protein contacts involved in the various steps leading to transcription initiation.

Bacterial Proteins↗
PubMed1993

Residues of the Bacillus subtilis phage phi 29 transcriptional activator required both to interact with RNA polymerase and to activate transcription.

Regulatory protein p4 from Bacillus subtilis phage phi 29 activates transcription from the viral late promoter, PA3, by stabilizing the binding of RNA polymerase to the DNA as a closed complex. Protein p4-induced DNA bending and direct contacts between p4 and RNA polymerase have been proposed to play a role in P(A3) activation. By site-directed mutagenesis at the carboxyl end of protein p4 we have identified residues that are critical both to interact with RNA polymerase and to activate transcription. Substitution of arginine 120 gives rise to a p4 derivative unable to activate transcription, that can bind to DNA and induce a normal DNA bending, but does not stimulate the binding of RNA polymerase to the promoter and cannot form complexes with RNA polymerase. Modification of the closely located residue leucine 117 had a similar but milder effect. The results obtained suggest that arginine 120 and leucine 117 form part of the activating domain of the protein, and show that direct contacts between protein p4 and RNA polymerase play a critical role in transcription activation. The p4-induced DNA bending is therefore necessary but not sufficient for the activation of the PA3 promoter.

Amino Acid Sequence↗
PubMed1993

Characterization of an RNA-binding domain in the bacteriophage phi 29 connector.

The connector of bacteriophage phi 29 is known to promote the viral prohead assembly, to bind DNA, and to drive DNA packaging into preformed viral shells in an RNA-dependent process. In this report, the phi 29 connector protein, p10, is shown to bind RNA in a sequence-independent fashion, and to possess an RNA recognition motif comprised approximately the region between residues 21 and 94 of the p10 sequence. Substitution mutants in specific amino acids of the RNA-binding domain obtained by site-directed mutagenesis showed that amino acids Phe23, His57, Phe59, and Tyr61 are critical for RNA binding and, subsequently, for DNA packaging into proheads. Proteolytic modified forms of the phi 29 connector have allowed us to conclude that the DNA- and RNA-binding domains are separated within the p10 sequence. It is also shown that RNA is stably associated to DNA-filled proheads during the DNA-packaging process.

Adenosine Triphosphate↗
PubMed1993

Purification of the beta product encoded by the Streptococcus pyogenes plasmid pSM19035. A putative DNA recombinase required to resolve plasmid oligomers.

Genetic evidence suggests that the gene beta product of Streptococcus pyogenes plasmid pSM19035 is required for converting plasmid multimers into monomers. The beta protein was purified from cells overexpressing the cloned gene. N-terminal protein sequence analysis demonstrated that the purified protein had the predicted sequence, except that the N-terminal initiator methionine was not present. Native beta protein consists of a dimer of two identical subunits with a molecular mass of 23.8 kDa (25 kDa in SDS-PAGE). The beta protein (isoelectric point of 9.7) binds specifically to a DNA fragment (312 bp in length) which contains the promoter region of the orf alpha-gene beta operon and two regions (sites I and II) that show dyad axes of symmetry. It is proposed that protein beta binds to sites I and II to mediate resolution of plasmid oligomers.

Bacterial Proteins↗
PubMed1993

The main early and late promoters of Bacillus subtilis phage phi 29 form unstable open complexes with sigma A-RNA polymerase that are stabilized by DNA supercoiling.

Most Escherichia coli promoters studied so far form stable open complexes with sigma 70-RNA polymerase which have relatively long half-lives and, therefore, are resistant to a competitor challenge. A few exceptions are nevertheless known. The analysis of a number of promoters in Bacillus subtilis has suggested that the instability of open complexes formed by the vegetative sigma A-RNA polymerase may be a more general phenomenon than in Escherichia coli. We show that the main early and late promoters from the Bacillus subtilis phage phi 29 form unstable open complexes that are stabilized either by the formation of the first phosphodiester bond between the initiating nucleoside triphosphates or by DNA supercoiling. The functional characteristics of these two strong promoters suggest that they are not optimized for a tight and stable RNA polymerase binding. Their high activity is probably the consequence of the efficiency of further steps leading to the formation of an elongation complex.

Bacillus Phages↗
PubMed1992

Phage phi 29 regulatory protein p4 stabilizes the binding of the RNA polymerase to the late promoter in a process involving direct protein-protein contacts.

Transcription from the late promoter, PA3, of Bacillus subtilis phage phi 29 is activated by the viral regulatory protein p4. A kinetic analysis of the activation process has revealed that the role of protein p4 is to stabilize the binding of RNA polymerase to the promoter as a closed complex without significantly affecting further steps of the initiation process. Electrophoretic band-shift assays performed with a DNA fragment spanning only the protein p4 binding site showed that RNA polymerase could efficiently retard the complex formed by protein p4 bound to the DNA. Similarly, when a DNA fragment containing only the RNA polymerase-binding region of PA3 was used, p4 greatly stimulated the binding of RNA polymerase to the DNA. These results strongly suggest that p4 and RNA polymerase contact each other at the PA3 promoter. In the light of current knowledge of the p4 activation mechanism, we propose that direct contacts between the two proteins participate in the activation process.

Bacillus Phages↗
PubMed1992

Role of the amino-terminal domain of bacteriophage phi 29 connector in DNA binding and packaging.

The connector of bacteriophage phi 29 is required for prohead assembly, binds DNA, and drives DNA packaging into viral proheads. Limited proteolysis of the connector protein with endoproteinase Glu-C from Staphylococcus aureus V8 and chymotrypsin showed that a domain of the NH2-terminal region is involved in DNA binding and in the subsequent packaging into preformed proheads, but not in prohead assembly. Mutants in specific amino acids of the NH2-terminal domain, obtained by directed mutagenesis techniques, showed that the Ala1-Arg2-Lys3-Arg4 region of the connector is absolutely necessary for DNA packaging into the proheads as well as for efficient DNA binding.

Amino Acid Sequence↗
PubMed1992

von Willebrand factor antigen in hypertrophic osteoarthropathy.

Recent evidence suggests that a key feature in the pathogenesis of hypertrophic osteoarthropathy (HOA) is an enhanced local platelet vessel wall interaction in the affected extremities. In our investigation we measured plasma levels of von Willebrand Factor Antigen (vWF:Ag; ELISA assay), a marker of platelet and/or endothelial activation, in 5 patients with the primary form of HOA and in 6 patients with the secondary form (due to cyanotic heart disease). Seven subjects matched for sex and age were used as controls. Patients showed statistically significantly higher levels of vWF:Ag in the primary (163.8 +/- 13.9%) and secondary form (152.6 +/- 9.6%, p less than 0.001 for both) when compared to controls (100.4 +/- 6.3%). Our results support the notion that platelet endothelial cell interaction may play a key role in the development of HOA.

Adolescent↗
PubMed1991

Identification of the sequences recognized by phage phi 29 transcriptional activator: possible interaction between the activator and the RNA polymerase.

Expression of Bacillus subtilis phage phi 29 late genes requires the transcriptional activator protein p4. This activator binds to a region of the late A3 promoter spanning nucleotides -56 to -102 relative to the transcription start site, generating a strong bending Tin the DNA. In this work the target sequences recognized by protein p4 in the phage phi 29 late A3 promoter have been characterized. The binding of protein p4 to derivatives of the late A3 promoter harbouring deletions in the protein p4 binding site has been studied. When protein p4 recognition sequences were altered, the activator could only bind to the promoter in the presence of RNA polymerase. This strong cooperativity in the binding of protein p4 and RNA polymerase to the promoter suggests the presence of direct protein-protein contacts between them.

Bacillus subtilis↗
PubMed1991

A DNA curvature can substitute phage phi 29 regulatory protein p4 when acting as a transcriptional repressor.

Binding of phage phi 29 regulatory protein p4 to its target sequences produces a strong bend in the DNA that is important for activation of the late A3 promoter (PA3). Protein p4 binding site in PA3 overlaps with the divergently transcribed main early promoter. PA2b, which suggested that p4 could also act as a repressor. We show that protein p4 both excludes Bacillus subtilis sigma A-RNA polymerase from PA2b and directs it to the divergently transcribed A3 promoter. Although steric hindrance is likely to be involved in the repression process, we have also analysed the effect on PA2b activity of a sequence-dependent curvature that simulates that induced by protein p4. A progressive increase in the DNA curvature of protein p4 binding region, performed by site-directed mutagenesis, has indicated that a static DNA curvature by itself can inhibit transcription from PA2b, both by impairing the binding of sigma A-RNA polymerase to the promoter and by reducing its ability to form transcriptionally active open complexes. These results indicate that bending promoter sequences in a direction unfavourable for RNA polymerase binding can repress transcription. Protein p4-induced DNA bending could therefore participate in PA2b repression by producing a DNA structure not recognized as a promoter by sigma A-RNA polymerase.

Bacteriophages↗
PubMed1990

Short N-terminal deletions in the phage phi 29 transcriptional activator protein impair its DNA-binding ability.

The expression of Bacillus subtilis phage phi 29 late genes from the A3 promoter requires the viral protein p4. This protein is a transcriptional activator which binds to a region of the A3 promoter located between nucleotides -56 to -102, relative to the transcription start point. Mutants at the N terminus of protein p4 have been constructed and their function investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation and DNase I footprinting assays. The results indicate that the N terminus of protein p4 could be involved in its binding to the A3 promoter, suggesting that it may not be a typical Cro-like helix-turn-helix DNA-binding protein.

Amino Acid Sequence↗
PubMed1990

A family of positive regulators related to the Pseudomonas putida TOL plasmid XylS and the Escherichia coli AraC activators.

The XylS family consists of a least 8 different transcriptional regulators. Six of these proteins are positive regulators for the catabolism of carbon sources (benzoate and sugars) in Escherichia coli, Pseudomonas putida and Erwinia carotovora, and two of them are involved in pathogenesis in Escherichia coli and Yersinia enterocolitica. Based on protein alignments, the members of this family exhibit a long stretch of homology at the C-terminal end. The regulators involved in the catabolism of carbon sources stimulate transcription from their respectively regulated promoters only in the presence of effectors. In two of the regulators, mutations at the non-homologous N-terminus alter affinity and specificity for effectors while mutations at the conserved C-terminus part decrease activation of transcription from their corresponding regulated promoters. It is thus probable that the variable N-terminus end in this family of regulators contains the motif involved in effector recognition, while the C-terminal end is involved in DNA-binding. These proteins seem to be related by common ancestry and may act through similar mechanisms of positive regulation effected through similar folding patterns.

Amino Acid Sequence↗
PubMed1990

Bend induced by the phage phi 29 transcriptional activator in the viral late promoter is required for activation.

Transcription initiation from the Bacillus subtilis phage phi 29 late A3 promoter requires the viral protein p4, a transcriptional activator. Protein p4 binds to a region of the A3 promoter, located between nucleotides -50 and -100 relative to the transcription start site, that presents a sequence-directed curvature. This curvature is enhanced when protein p4 binds to the promoter. A number of deletion mutants at the carboxyl end of protein p4 have been constructed and their behavior as transcriptional activators of the late A3 promoter has been investigated. The binding of these deletion mutants to the late A3 promoter has been analyzed by gel retardation, DNase I footprinting, methylation interference and circular permutation assays. The results suggest that the last 12 amino acid residues of protein p4, six of which are positively charged, although not involved in the specific recognition of the promoter are responsible for part of the bend induced by protein p4 in its binding site. Evidence is presented which suggests that full induction of this curvature is needed for the transcription activation process. A model is proposed for protein p4 interaction with the A3 promoter, in which the bend is induced in two steps: first, two monomers of protein p4 bind to the inverted recognition sequences, subsequent interaction between them generating a bend between these sequences; second, the highly basic carboxyl terminus of protein p4 establishes non-specific electrostatic interactions with the DNA backbone inducing a bend at both ends of the protein p4 binding region.

Amino Acid Sequence↗
PubMed1990

Signal-regulator interactions. Genetic analysis of the effector binding site of xylS, the benzoate-activated positive regulator of Pseudomonas TOL plasmid meta-cleavage pathway operon.

This study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. Transcription of the TOL plasmid meta-cleavage pathway operon is specifically stimulated by the XylS protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. One xylS mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding altered XylS proteins that recognize as effectors benzoate analogues that are non-effectors for the XylS wild-type protein. The changes in two mutant regulators are located at the N-terminal end of the protein, within a putative beta-pleated domain. These mutant proteins exhibit a markedly increased affinity for normal benzoate effectors, with K's values fivefold to 60-fold lower than those of the wild-type XylS protein. They are additionally activated by new effectors having certain substituents at position 2, 3 and 4 of the aromatic ring. Two other mutant proteins recognize new effectors having substituents at position 4 and 5 of the aromatic ring, and contain mutations at their C-terminal end within a putative alpha-helix-rich domain. Three other mutations, one of which leads to constitutive expression from Pm, each result in an amino acid change in the central region of the regulator. These findings suggest but do not prove that the effector binding pocket of the XylS protein may be composed of two or more non-contiguous segments of its primary structure. The XylS protein exhibits homology with the AraC protein of Escherichia coli, a protein that stimulates transcription from ara promoters when it is activated by arabinose or benzoate. Mutations influencing effector activation of the XylS protein characterized in this study are all located in regions exhibiting a high degree of homology with the corresponding aligned sequence of AraC protein.

Amino Acid Sequence↗
PubMed1990

Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.

Ampicillin↗