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At least 163 records · Page 9Linked to original sources
PubMed1994

Direct recognition of the trp operator by the trp holorepressor--a review.

Two different X-ray co-crystal structures of the Escherichia coli trp holorepressor complexed with DNA suggest that the TrpR protein recognizes specific DNA sequences primarily with a network of water-mediated H-bonds. However, the more recent nuclear magnetic resonance (NMR) solution structures of the holorepressor-operator complex show no long-lived, ordered water molecules at the protein-DNA interface and place amino acids in intimate contact with nucleotide bases. Both genetic and biochemical studies support a model in which the trp repressor recognizes specific DNA sequences by a direct mechanism, as seen in the NMR solution structures, not by the 'indirect readout' mechanism initially proposed on the basis of X-ray studies.

Bacterial Proteins↗
PubMed2005

Comparative and functional genomic analyses of the pathogenicity of phytopathogen Xanthomonas campestris pv. campestris.

Xanthomonas campestris pathovar campestris (Xcc) is the causative agent of crucifer black rot disease, which causes severe losses in agricultural yield world-wide. This bacterium is a model organism for studying plant-bacteria interactions. We sequenced the complete genome of Xcc 8004 (5,148,708 bp), which is highly conserved relative to that of Xcc ATCC 33913. Comparative genomics analysis indicated that, in addition to a significant genomic-scale rearrangement cross the replication axis between two IS1478 elements, loss and acquisition of blocks of genes, rather than point mutations, constitute the main genetic variation between the two Xcc strains. Screening of a high-density transposon insertional mutant library (16,512 clones) of Xcc 8004 against a host plant (Brassica oleraceae) identified 75 nonredundant, single-copy insertions in protein-coding sequences (CDSs) and intergenic regions. In addition to known virulence factors, full virulence was found to require several additional metabolic pathways and regulatory systems, such as fatty acid degradation, type IV secretion system, cell signaling, and amino acids and nucleotide metabolism. Among the identified pathogenicity-related genes, three of unknown function were found in Xcc 8004-specific chromosomal segments, revealing a direct correlation between genomic dynamics and Xcc virulence. The present combination of comparative and functional genomic analyses provides valuable information about the genetic basis of Xcc pathogenicity, which may offer novel insight toward the development of efficient methods for prevention of this important plant disease.

Bacterial Proteins↗
PubMed1988

Interaction of the Bacillus subtilis phage phi 105 repressor DNA: a genetic analysis.

The Bacillus subtilis phage phi 105 repressor specifically recognizes a 14-bp operator sequence which does not exhibit 2-fold rotational symmetry. To facilitate a genetic analysis of this sequence-dependent DNA binding a B. subtilis strain was constructed in which mutations affecting the phi 105 repressor-operator interaction cause a selectable phenotype, chloramphenicol resistance. After in vivo mutagenesis, we isolated and mapped 22 different mutations in the repressor coding sequence, 15 of which are missense substitutions. These are exclusively located in the N-terminal part (positions 1-43) of the 144 residue long polypeptide. Two nonsense mutants, at positions 70 and 89, respectively, still show partial repressor activity. These data suggest that the phi 105 repressor consists of at least two independently folding structural domains, of which the N-terminal is involved in operator binding. Twelve missense mutations are clustered in a region extending from Gln-18 to Arg-37, which we propose to be the DNA-binding alpha-helix--beta-turn--alpha-helix motif, common to all lambda Cro-like repressors. The second ('recognition') helix shows significant homology with the corresponding sequence in Tn3 resolvase, and there is also a striking similarity between the phi 105 operator and the consensus sequence for a Tn3 res half-site. Based on these observations, and on the previously isolated phi 105 0c mutants, we tentatively assign some specific contacts between base pairs from the first half of a phi 105 operator site and amino acids from the repressor's 'recognition helix'.

Amino Acid Sequence↗
PubMed1997

Molecular genetic characterization of the Escherichia coli gntT gene of GntI, the main system for gluconate metabolism.

The Escherichia coli gntT gene was subcloned from the Kohara library, and its expression was characterized. The cloned gntT gene genetically complemented mutant E. coli strains with defects in gluconate transport and directed the formation of a high-affinity gluconate transporter with a measured apparent Km of 6 microM for gluconate. Primer extension analysis indicated two transcriptional start sites for gntT, which are separated by 66 bp and which give rise to what appears on a Northern blot to be a single, gluconate-inducible, 1.42-kb gntT transcript. Thus, it was concluded that gntT is monocistronic and is regulated by two promoters. Both of the promoters have - 10 and -35 sequence elements typical of sigma70 promoters and catabolite gene activator protein binding sites in appropriate locations to exert glucose catabolite repression. In addition, two putative gnt operator sites were identified in the gntT regulatory region. A search revealed the presence of nearly identical palindromic sequences in the regulatory regions of all known gluconate-inducible genes, and these seven putative gnt operators were used to derive a consensus gnt operator sequence. A gntT::lacZ operon fusion was constructed and used to examine gntT expression. The results indicated that gntT is maximally induced by 500 microM gluconate, modestly induced by very low levels of gluconate (4 microM), and partially catabolite repressed by glucose. The results also showed a pronounced peak of gntT expression very early in the logarithmic phase, a pattern of expression similar to that of the Fis protein. Thus, it is concluded that GntT is important for growth on low concentrations of gluconate, for entry into the logarithmic phase, and for cometabolism of gluconate and glucose.

Bacterial Proteins↗
PubMed1989

Single and double loop formation when deoR repressor binds to its natural operator sites.

Distal effects on the in vivo repression of the deo operon are thought to be mediated by the deoR repressor with DNA loop formation. Such loops are easily observed by electron microscopy when the oligomeric deoR repressor is added to a DNA fragment carrying the three genetically defined operators at their chromosomal distances. Upon binding of deoR to any two operators, single loops are formed, 280, 600, and 880 bp in size. With the deo operon, double loops are also formed, which are the combination of the 280 bp and 600 bp loops and the result of simultaneous binding of the protein to its three sites. The formation of both single and double loops is consistent with the long-range effects observed in vivo and with the cooperative involvement of all three operator sites in the repression.

Binding Sites↗
PubMed2005

Genetic composition of the Bacillus subtilis SOS system.

The SOS response in bacteria includes a global transcriptional response to DNA damage. DNA damage is sensed by the highly conserved recombination protein RecA, which facilitates inactivation of the transcriptional repressor LexA. Inactivation of LexA causes induction (derepression) of genes of the LexA regulon, many of which are involved in DNA repair and survival after DNA damage. To identify potential RecA-LexA-regulated genes in Bacillus subtilis, we searched the genome for putative LexA binding sites within 300 bp upstream of the start codons of all annotated open reading frames. We found 62 genes that could be regulated by putative LexA binding sites. Using mobility shift assays, we found that LexA binds specifically to DNA in the regulatory regions of 54 of these genes, which are organized in 34 putative operons. Using DNA microarray analyses, we found that 33 of the genes with LexA binding sites exhibit RecA-dependent induction by both mitomycin C and UV radiation. Among these 33 SOS genes, there are 22 distinct LexA binding sites preceding 18 putative operons. Alignment of the distinct LexA binding sites reveals an expanded consensus sequence for the B. subtilis operator: 5'-CGAACATATGTTCG-3'. Although the number of genes controlled by RecA and LexA in B. subtilis is similar to that of Escherichia coli, only eight B. subtilis RecA-dependent SOS genes have homologous counterparts in E. coli.

5' Flanking Region↗
PubMed2000

Selection and application of peptide-binding peptides.

Peptide-binding ligands would be useful for directing reagents to particular epitopes in a protein, the detection of peptide hormones, and many other applications. Here we show that peptides of modest size isolated from a library using a simple genetic assay can act as specific receptors for other peptides. The equilibrium dissociation constants of these peptide-peptide complexes are higher than those of typical monoclonal antibody-epitope complexes. Nonetheless, as shown here, these peptide-binding peptides can be used to detect or purify proteins containing the partner peptide.

Amino Acid Sequence↗
PubMed1989

Cooperative tandem binding of met repressor of Escherichia coli.

We present biochemical and genetic data to support the hypothesis that the Escherichia coli met repressor, MetJ, binds to synthetic and natural operator sequences in tandem arrays such that repression depends not only on the affinity of the DNA-protein interaction, but also on protein-protein contacts along the tandem array. This represents a novel form of regulatory switch. Furthermore, there seems to be homology between the organization of the met and trp operators.

Apoproteins↗
PubMed1989

Genetic analysis of the promoter region of the Bacillus subtilis alpha-amylase gene.

The amyR2 allele of the Bacillus subtilis alpha-amylase cis-regulatory region enhances production of amylase and transcription of amyE, the structural gene, by two- to threefold over amyR1. The amylase gene bearing each of these alleles was cloned on plasmids of about 10 to 15 copies per chromosome. Transcription of the cloned amylase gene by each amyR allele was activated at the end of exponential growth and was subject to catabolite repression by glucose. The amount of amylase produced was roughly proportional to the copy number of the plasmid, and cells containing the amyR2-bearing plasmid, pAR2, produced two- to threefold more amylase than cells with the amyR1 plasmid, pAMY10. Deletion of DNA 5' to the alpha-amylase promoter, including deletion of the A + T-rich inverted repeat found in amyR1 and amyR2, had no effect on expression or transcription of alpha-amylase. Deletion of DNA 3' to the amyR1 promoter did not impair temporal activation of chloramphenicol acetyltransferase in amyR1-cat-86 transcriptional fusions, but catabolite repression was abolished. When an 8-base-pair linker was inserted in pAMY10 at the same site from which the 3' deletion was made, amylase expression doubled and was repressed less by glucose. Both the deletion and the insertion disrupted four bases at the 3' end of the putative amylase operator region. Site-directed mutagenesis was used to change bases in the promoter-operator region of amyR1 to their amyR2 counterparts. Either change alone increased amylase production twofold, but only the change at +7, next to the linker insertion of 3' deletion site, yielded the increased amylase activity in the presence of glucose that is characteristic of the amyR2 strain. The double mutant behaved most like strains carrying the amyR2 allele.

Bacillus subtilis↗
PubMed2001

Study of adaptive mutations in Salmonella typhimurium by using a super-repressing mutant of a trans regulatory gene purR.

Salmonella typhimurium purR encodes a transcriptional repressor regulating gene expression of de novo purine nucleotide biosynthesis. It represses purD gene transcription by binding to the 16-base pair purD operator (PUR box). A S. typhimurium strain carrying a super-repressing mutant of purR, purR(s), has been used as an experimental system to study adaptive mutation. Escherichia coli lac genes were genetically engineered into S. typhimurium chromosome and repressed by purR(s) so that they could be used as an indicator of adaptive mutations in purR(s) or in the purD operator. Mutations in purR(s) or in the purD operator accumulated when the mutant strain was placed on a minimal lactose plate supplemented with 10 microg/ml of adenine during prolonged incubation. These specific mutations reverted the mutant strain from lac(-) to lac(+) phenotype. The lac(+) strains were categorized into the early- and late-arising mutants according to the time for colony appearance. Our genetic studies indicate that (i) Poisson distributed mutations accumulated in the chromosomal regulatory gene purR or the purD operator in very slowly dividing cells under selection; (ii) after about 8 days of selection, the frequency of mutations in purD operator reached the high value of about two mutations per 10(8) cells; (iii) the mutational spectrum in the purD operator during growth was not significantly different from that during selection; (iv) defects in mutL or mutS appeared to have a stronger effect on growth-dependent mutations than on adaptive mutations.

Adaptation, Physiological↗
PubMed1994

Iron, DtxR, and the regulation of diphtheria toxin expression.

In recent years considerable advances have been made in the understanding of the molecular basis of iron-mediated regulation of diphtheria toxin expression. The tox gene has been shown to be regulated by the heavy metal ion-activated regulatory element DtxR. In the presence of divalent heavy metal ions, DtxR becomes activated and binds to a 9 bp interrupted palindromic sequence. The consensus-binding site has been determined by both the sequence analysis of DtxR-responsive operators cloned from genomic libraries of Corynebacterium diphtheriae as well as by in vitro genetic methods using cyclic amplification of selected targets (CASTing). It is now clear that DtxR functions as a global iron-sensitive regulatory element in the control of gene expression in C. diphtheriae. In addition, the metal ion-activation domain of DtxR is being characterized by both mutational analysis and determination of the X-ray structure at 3.0 A resolution.

Bacterial Proteins↗
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↗
PubMed1997

MS2 coat protein mutants which bind Qbeta RNA.

The coat proteins of the RNA phages MS2 and Qbetaare structurally homologous, yet they specifically bind different RNA structures. In an effort to identify the basis of RNA binding specificity we sought to isolate mutants that convert MS2 coat protein to the RNA binding specificity of Qbeta. A library of mutations was created which selectively substitutes amino acids within the RNA binding site. Genetic selection for the ability to repress translation from the Qbetatranslational operator led to the isolation of several MS2 mutants that acquired binding activity for QbetaRNA. Some of these also had reduced abilities to repress translation from the MS2 translational operator. These changes in RNA binding specificity were the results of substitutions of amino acid residues 87 and 89. Additional codon- directed mutagenesis experiments confirmed earlier results showing that the identity of Asn87 is important for specific binding of MS2 RNA. Glu89, on the other hand, is not required for recognition of MS2 RNA, but prevents binding of QbetaRNA.

Allolevivirus↗
PubMed1994

DNA recognition by beta-sheets in the Arc repressor-operator crystal structure.

Transcription of the ant gene during lytic growth of bacteriophage P22 (ref. 1) is regulated by the cooperative binding of two Arc repressor dimers to a 21-base-pair operator site. Here we report the co-crystal structure of this Arc tetramer-operator complex at 2.6 A resolution. As expected from genetic and structural studies and from the co-crystal structure of the homologous Escherichia coli MetJ repressor, each Arc dimer uses an antiparallel beta-sheet to recognize bases in the major groove. However, the Arc and MetJ complexes differ in several important ways: the beta-sheet-DNA interactions of Arc are far less symmetrical; DNA binding by Arc is accompanied by important conformational changes in the beta-sheet; and Arc uses a different part of its protein surface for dimer-dimer interactions.

Base Sequence↗
PubMed2002

Lactococcus lactis lytic bacteriophages of the P335 group are inhibited by overexpression of a truncated CI repressor.

Phages of the P335 group have recently emerged as important taxa among lactococcal phages that disrupt dairy fermentations. DNA sequencing has revealed extensive homologies between the lytic and temperate phages of this group. The P335 lytic phage phi31 encodes a genetic switch region of cI and cro homologs but lacks the phage attachment site and integrase necessary to establish lysogeny. When the putative cI repressor gene of phage phi31 was subcloned into the medium-copy-number vector pAK80, no superinfection immunity was conferred to the host, Lactococcus lactis subsp. lactis NCK203, indicating that the wild-type CI repressor was dysfunctional. Attempts to clone the full-length cI gene in Lactococcus in the high-copy-number shuttle vector pTRKH2 were unsuccessful. The single clone that was recovered harbored an ochre mutation in the cI gene after the first 128 amino acids of the predicted 180-amino-acid protein. In the presence of the truncated CI construct, pTRKH2::CI-per1, phage phi31 was inhibited to an efficiency of plaquing (EOP) of 10(-6) in NCK203. A pTRKH2 subclone which lacked the DNA downstream of the ochre mutation, pTRKH2::CI-per2, confirmed the phenotype and further reduced the phi31 EOP to <10(-7). Phage phi31 mutants, partially resistant to CI-per, were isolated and showed changes in two of three putative operator sites for CI and Cro binding. Both the wild-type and truncated CI proteins bound the two wild-type operators in gel mobility shift experiments, but the mutated operators were not bound by the truncated CI. Twelve of 16 lytic P335 group phages failed to form plaques on L. lactis harboring pTRKH2::CI-per2, while 4 phages formed plaques at normal efficiencies. Comparisons of amino acid and DNA level homologies with other lactococcal temperate phage repressors suggest that evolutionary events may have led to inactivation of the phi31 CI repressor. This study demonstrated that a number of different P335 phages, lytic for L. lactis NCK203, have a common operator region which can be targeted by a truncated derivative of a dysfunctional CI repressor.

Amino Acid Sequence↗
PubMed2000

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Amino Acid Sequence↗
PubMed1996

Mutants in position 69 of the Trp repressor of Escherichia coli K12 with altered DNA-binding specificity.

Structural analysis by X-ray crystallography has indicated that direct contact occurs between Arg69, the second residue of the first helix of the helix-turnhelix (HTH) motif of the Trp repressor, and guanine in position 9 of the alpha-centred consensus trp operator. We therefore replaced residue 69 of the Trp repressor with Gly, Ile, Leu or Gln and tested the resultant repressor mutants for their binding to synthetic symmetrical alpha- or beta-centred trp operator variants, in vivo and in vitro. We present genetic and biochemical evidence that Ile in position 69 of the Trp repressor interacts specifically with thymine in position 9 of the alpha-centred trp operator. There are also interactions with other bases in positions 8 and 9 of the alpha-centred trp operator. In vitro, the Trp repressor of mutant RI69 binds to the consensus alpha-centred trp operator and a similar trp operator variant that carries a T in position 9. In vivo analysis of the interactions of Trp repressor mutant RI69 with symmetrical variants of the beta-centred trp operator shows a change in the specificity of binding to a beta-centred symmetrical trp operator variant with a gua-nine to thymine substitution in position 5, which corresponds to position 9 of the alpha-centred trp operator.

Amino Acids↗
PubMed1993

Structure of the complex of lac repressor headpiece and an 11 base-pair half-operator determined by nuclear magnetic resonance spectroscopy and restrained molecular dynamics.

The structure of the complex of lac repressor headpiece and an 11 base-pair lac half-operator has been determined by NMR spectroscopy and restrained Molecular Dynamics calculations. In total 508 distances were derived from two-dimensional nuclear Overhauser enhancement measurements, 260 of which are within the headpiece, 212 within the operator and 36 between operator and headpiece. An equilibrium restrained Molecular Dynamics calculation of the complex in aqueous solution, spanning 85 picoseconds, has been used to analyze the structure. Configuration sampling by an annealing procedure has been undertaken as well in order to estimate the precision of the structure determination. Our data confirm the results of previous two-dimensional NMR studies that the orientation of the recognition helix of lac repressor in the major groove of DNA with respect to the operator dyad axis is opposite to the orientation found in complexes of other DNA binding proteins of the helix-turn-helix class. We find a number of tight contacts between the protein and the operator that are in agreement with the available genetic and biochemical data. The anchoring of lac headpiece on the operator is similar to that of other repressors. Other features are unique for lac headpiece: relative few direct hydrogen bonds between side-chains and bases; extensive apolar contacts; many direct and water-bridged contacts to phosphates from residues in or close to the recognition helix. Overall, an interconnected set of interactions is observed, involving base-specific contacts, phosphate contacts, intra-protein and water-bridged hydrogen bonds. Several of these interactions appear to be dynamic, i.e. fluctuating in time, rather than static.

Amino Acid Sequence↗