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identification of the product of phage 434 cro gene. 1979160335
nucleotide sequence of the cro-cii-oop region of bacteriophage 434 dna.the nucleotide sequence of a 869 bp segment of phage 434 dna including the regulatory genes cro and cii is presented and compared with the corresponding part of the phage lambda dna sequence. the 434 cro protein as deduced from the dna sequence is a highly basic protein of 71 amino acid residues with a calculated molecular weight of 8089. while the cro gene sequences of phage 434 and lambda dna are very different, the nuleotide sequences to the right of the lambda imm434 boundary show difference ...1979375198
primary structure of an ecori fragment of lambda imm434 dna containing regions ci-cro of phage 434 and cii-o of phage lambda.digestion of phage lambda imm434 dna with restriction endonuclease ecori yields 7 fragments. the shortest among them (1287 bp) contains the right part of the phage 434 immunity region and the phage dna portion proximal to it. the complete primary structure of this fragment has been determined using the chemical method of dna sequencing. hypothetical amino-acid sequences of proteins coded by the cro gene of phage 434 and the cii gene of phage lambda, as well as nh2-terminal amino-acid sequences o ...1979478301
cro mutants of phage 434. 19751189298
determination of the nuclear magnetic resonance solution structure of the dna-binding domain (residues 1 to 69) of the 434 repressor and comparison with the x-ray crystal structure.the dna-binding domain of the phage 434 repressor consisting of n-terminal residues 1 to 69 (434 repressor(1-69)), was expressed in escherichia coli with natural isotope abundance, uniform 15n-labeling and biosynthetically directed fractional 13c-labeling in extent of about 10%. with these protein preparations the three-dimensional structure was determined in solution. the techniques used were nuclear magnetic resonance (n.m.r.) spectroscopy for the collection of conformational constraints, calc ...19921311771
complete 15n and 1h nmr assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form.the amino-terminal domain of the phage 434 repressor consisting of residues 1-69 forms a globular structure of five tightly packed helices, with nearly identical molecular architectures in crystals and in solution. upon addition of urea to an aqueous solution of this protein, the nmr spectrum of a second form of the protein appears in addition to the native form, and at a urea concentration of 7 m, this urea-unfolded form is the only species observed. at intermediate urea concentrations, the two ...19921584772
bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.the extent of dna bending induced by 434 repressor, its amino terminal dna binding domain (r1-69), and 434 cro was studied by gel shift assay. the results show that 434 repressor and r1-69 bend dna to the same extent. 434 cro-induced dna bends are similar to those seen with the 434 repressor proteins. on approximately 265 base pair fragments, the cyclic amp receptor protein of escherichia coli (crp) produces larger mobility shifts than does 434 repressor. this indicates that the 434 proteins ben ...19911870967
shared operator recognition specificity between trp repressor and the repressors of bacteriophage 434.trp repressor is the only dna-binding regulatory protein having a helix-turn-helix motif that has been reported to engage its operator target by a mechanism termed indirect readout: the trp repressor-dna interface is replete with hydrogen bonds between amino acid residues and non-esterified oxygen atoms of the sugar-phosphate backbone, and contains numerous specifically positioned water molecules. in escherichia coli mutants deleted for trpr, the immunity repressor of phage 434 led to an eightfo ...19912005612
achilles' heel cleavage: creation of rare restriction sites in lambda phage genomes and evaluation of additional operators, repressors and restriction/modification systems.a novel technique for the creation of rare restriction sites was described by koob et al. [science 241 (1988) 1084-1086]. this technique, achilles' heel cleavage (ac), relies on the use of a bound repressor molecule to protect only one of many identical restriction sites from a modification methyltransferase that inactivates all other restriction sites. the technique was applied to a small plasmid and shown to work efficiently with two repressor/operator systems: lac repressor/laco operator and ...19902165969
dependence of the torsional rigidity of dna on base composition.the escherichia coli phage 434 repressor binds as a dimer to the operator of the dna helix. although the centre of the operator is not in contact with protein, the repressor binding affinity can be reduced at least 50-fold by changing the sequence there: operators with a.t base pairs near their centre bind the repressor more strongly than do operators with g.c base pairs at the same positions. to explain these observations, it has been proposed that the base composition at the centre of the oper ...19902308636
structure of phage 434 cro protein at 2.35 a resolution.the crystal structure of phage 434 cro protein has been determined and refined against 2.35 a data to an r-factor of 19.5%. the protein comprises five alpha-helices and shows the helix-turn-helix motif found in other repressor proteins.19892647998
structure of the amino-terminal domain of phage 434 repressor at 2.0 a resolution.the crystal structure of the amino-terminal domain of phage 434 repressor has been solved using molecular replacement methods and refined to an r-factor of 19.3% against data to 2.0 a resolution. the protein comprises five short alpha-helices. two of these form a helix-turn-helix motif, very similar to those found in related proteins. the protein is remarkably similar to the cro protein from the same phage.19892926803
functional elements of dna upstream from the integrase operon that are conserved in bacteriophages 434 and lambda.a 1488-bp restriction fragment of bacteriophage 434 dna contains the integrase promoter and an adjacent nucleotide sequence (t'i) resembling a rho-independent terminator. to identify and quantitate transcription termination, dna segments were cloned into a plasmid between the galactose promoter and assayable galactokinase gene and tested for termination. whereas the entire fragment effectively terminated transcription, a 331-bp restriction fragment containing the t'i terminator had only weak ter ...19872965063
turning lambda cro into a transcriptional activator.according to our present understanding, lambda repressor bound to dna stimulates transcription by touching rna polymerase bound at an adjacent promoter. the part of repressor required for activation was identified in part by the isolation of mutants specifically impaired in transcriptional activation. the amino acids of repressor altered in these "positive control" mutants lie in an acidic patch on the surface of repressor that is closely apposed to rna polymerase. in this study, we show that th ...19882968842
dna-bound fos proteins activate transcription in yeast.we constructed genes encoding the dna binding region of the bacterial lexa repressor fused to the v-fos and c-fos oncogene products. the resulting lexa-fos fusion proteins activated transcription in yeast. transcription activation by these proteins was as strong as transcription activation by proteins native to yeast. lexa-fos fusion proteins only activated transcription of genes when they were bound to lexa binding sites inserted upstream of those genes. transcription was activated less strongl ...19883124961
structure of a phage 434 cro/dna complex.comparison of the crystal structure of a complex of the phage 434 cro protein and a synthetic dna operator with the complex of the same operator and the 434 repressor dna-binding domain shows different dna conformations in the two structures. binding of the protein determines the precise conformation of the dna in each case.19883185709
recognition of a dna operator by the repressor of phage 434: a view at high resolution.the repressors of temperate bacteriophages such as 434 and lambda control transcription by binding to a set of dna operator sites. the different affinity of repressor for each of these sites ensures efficient regulation. high-resolution x-ray crystallography was used to study the dna-binding domain of phage 434 repressor in complex with a synthetic dna operator. the structure shows recognition of the operator by direct interactions with base pairs in the major groove, combined with the sequence- ...19883187531
dna twisting and the affinity of bacteriophage 434 operator for bacteriophage 434 repressor.the affinity of the escherichia coli phage 434 operator for phage 434 repressor is affected by changes in the sequence of the noncontacted base pairs near the operator's center. the results presented here show that base composition near the center of the operator affects the operator's affinity for repressor by altering the ease with which the operator can be overtwisted into the proper configuration for complex formation. we show that both dna flexibility and repressor flexibility influence the ...19883387430
activation of transcription by the bacteriophage 434 repressor.bacteriophage 434 encodes a repressor that, like bacteriophage lambda repressor, both activates and represses transcription. as in the lambda chromosome, a region of the 434 chromosome, called the right operator, contains three repressor binding sites (or1, or2, and or3) that mediate these effects on two adjacent promoters. we now show that a part of the 434 repressor, the amino-terminal domain, activates leftward transcription when bound to or2. we show that 434 repressor bound to or2 closely a ...19863467310
structure of the repressor-operator complex of bacteriophage 434.the crystal structure of a specific complex between the dna-binding domain of phage 434 repressor and a synthetic 434 operator dna shows interactions that determine sequence-dependent affinity. the repressor recognizes its operators by its complementarity to a particular dna conformation as well as by direct interaction with base pairs in the major groove.19873553959
effect of non-contacted bases on the affinity of 434 operator for 434 repressor and cro.the repressor of phage 434 binds to six operator sites on the phage chromosome. a comparison of the sequences of these 14-base-pair (bp) operator sites reveals a striking pattern: at five of the six sites, the symmetrically arrayed outer eight base pairs (four in each half-site) are identical and the remaining site differs at only one position (fig. 1b). in contrast, the sequences of the inner four base pairs are highly variable. crystallographic analysis of the repressor-operator complex shows ...19873553960
a new-specificity mutant of 434 repressor that defines an amino acid-base pair contact.the repressor encoded by bacteriophage 434 binds to its operators by inserting a 'recognition' alpha-helix into the major groove of the dna. we have identified an amino acid-base pair contact that determines (in part) the dna-binding specificity of 434 repressor. the identification is based on the properties of a 'new-specificity' mutant, named repressor [ala 28], which bears the substitution of ala for gln at the first residue of its recognition alpha-helix. repressor [ala 28] binds with high a ...19873553961
importance of dna stiffness in protein-dna binding specificity.from the first high-resolution structure of a repressor bound specifically to its dna recognition sequence it has been shown that the phage 434 repressor protein binds as a dimer to the helix. tight, local interactions are made at the ends of the binding site, causing the central four base pairs (bp) to become bent and overtwisted. the centre of the operator is not in contact with protein but repressor binding affinity can be reduced at least 50-fold in response to a sequence change there. this ...19873627268
crystallization and x-ray diffraction studies of a 434 cro-dna complex.crystals have been obtained of the bacteriophage 434 cro protein bound to a synthetic dna operator. an analysis of the packing shows that the complexes stack end-to-end along crystallographic axis, forming long rods with non-crystallographic 11(3) screw symmetry. the average number of dna base-pairs per turn is 10.27, which is somewhat more overwound as compared with the 434 repressor-dna crystals of anderson et al. diffraction extends to 3 a along the rod direction and to 5 a in perpendicular d ...19873681985
a phage repressor-operator complex at 7 a resolution.the crystal structure of a complex between the dna-binding domain of phage 434 repressor and a synthetic 434 operator shows that the protein, very similar in conformation to gamma repressor, binds to b-form dna with the second alpha-helix of a helix-turn-helix motif lying in the major groove.19854033757
mapping of deletions and substitutions in heteroduplex dna molecules of bacteriophage lambda by electron microscopy.electron microscopy of heteroduplex dna molecules, composed of one strand of escherichia coli phage lambda(+) dna annealed to the complementary dna strand of a lambda deletion or substitution mutant, permits visualization, as well as precise measurements and mapping, of the unpaired single-stranded regions of nonhomology in the otherwise double-stranded molecules. in the lambdab2 mutant, the central segment (13 percent) of the lambda(+) dna molecule is shown to be deleted. in the hybrid phages l ...19695765116
homologies between different procaryotic dna-binding regulatory proteins and between their sites of action.comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; cap), laci, galr , lexa, lysr, arac, trpr, and tnpr of escherichia coli, of genes ci, cii and cro of phage lambda, cro of phage 434, and c2 of phage p22, has revealed two regions of homology. the sites of action of these proteins also share common features in their dna sequence. taking into account the models proposed for the lambda repressors, cro and ...19826234163
dna sequence of the att region of coliphage 434.phages lambda and 434 are related phages that insert at the same site on the escherichia coli chromosome. a 5.9-kb sali-bamhi fragment derived from phage 434 was shown to hybridize to a 0.5-kb probe carrying attp-lambda. a 0.8-kb bam hi-taqi fragment subcloned into pbr327 was used for sequencing. the sequence of the 500 bp around the insertion site is given here, comparison of the lambda and 434 sequence shows that the following regions are conserved: the coding sequence for the integrase protei ...19816271639
autoregulation of repressor synthesis in bacteriophage lambda imm434.a 550-bp dna fragment derived from the immunity region of phage lambda imm434 and carrying the wild-type prm promoter has been inserted upstream of the lacz gene, in the beta 2 region of an 'in-vitro'-constructed transducing phage. in strains lysogenized with this phage, the rates of transcription from the 434-prm promoter can be assayed as units of beta-galactosidase in the absence or in the presence of increasing levels of 434 repressor. the results obtained indicate the existence of a mechani ...19826279202
construction of overproducers of the bacteriophage 434 repressor and cro proteins. 19816286819
retroregulation: control of integrase expression by the b2 region of bacteriophages lambda and 434.genetic fusions constructed by a combination of in vivo and in vitro techniques have been used to investigate the cis-regulatory role of a 250-base-pair segment of b2 dna in the expression of int transcripts originating from three different promoters pi, pl, and ptrp. it has been established that (a) the pi and ptrp transcripts, which produce functional int protein, are efficiently terminated by the b2 segment, (b) in the same test system, the pl transcript, which does not result in functional i ...19836297148
the integrase promoter and t'i terminator in bacteriophages lambda and 434.the lambda integrase promoter pi lies immediately downstream from a terminator t'i. the pi promoter is activated by cii protein during lysogenization. the function of t'i is unknown. a deletion (trp-lambda 29), whose fusion point lies within one stem of t'1 retains cii-activable promoter function but shows little if any transcription termination in vivo. the nucleotide sequence of pi is identical in lambda and the related phage 434. however, the t'i sequences of the two phages, though located in ...19836305007
substituting an alpha-helix switches the sequence-specific dna interactions of a repressor.it has been suggested that many dna-binding proteins use an alpha-helix for specific sequence recognition. we have used amino acid sequence homologies to identify the presumptive dna-recognition helices in two related proteins whose structures are unknown--the repressor and cro protein of bacteriophage 434. the 434 repressor and cro protein each bind to three similar sites in the rightward phage 434 operator, or, and they make different contacts in each binding site, as revealed by the chemical ...19846540625
structure of the dna-binding region of lac repressor inferred from its homology with cro repressor.it is shown that the amino acid sequence and the dna gene sequence of the 25 amino-terminal residues of the lac repressor protein of escherichia coli are homologous with the sequences of five dna-binding proteins: the cro repressor proteins from phage lambda and phage 434, the ci and cii proteins from phage lambda, and the repressor protein from salmonella phage p22. the region of homology between lac repressor and the other proteins coincides with the principal dna-binding region of cro repress ...19826951187
rationally designed helix-turn-helix proteins and their conformational changes upon dna binding.circular dichroism and electrophoretic mobility shift studies were performed to confirm that dimerized n-terminal domains of bacterial repressors containing helix-turn-helix motifs are capable of high-affinity and specific dna recognition as opposed to the monomeric n-terminal domains. specific, high-affinity dna binding proteins were designed and produced in which two copies of the n-terminal 1-62 domain of the bacteriophage 434 repressor are connected either in a dyad-symmetric fashion, with a ...19957621832
intermolecular transposition of is10 causes coupled homologous recombination at the transposition site.interplasmid and chromosome to plasmid transposition of is10 were studied by assaying inactivation of the phage 434 ci gene, carried on a low copy number plasmid. this was detected by the activity of the tet gene expressed from the phage 434 pr promoter. each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target dna at the insertion site. cointegrate formation was abolished in delta reca strains, alt ...19957672587
computer modeling of protein folding: conformational and energetic analysis of reduced and detailed protein models.recently we developed methods to generate low-resolution protein tertiary structures using a reduced model of the protein where secondary structure is specified and a simple potential based on a statistical analysis of the protein data bank is employed. here we present the results of an extensive analysis of a large number of detailed, all-atom structures generated from these reduced model structures. following side-chain addition, minimization and simulated annealing simulations are carried out ...19957723045
how 434 repressor discriminates between or1 and or3. the influence of contacted and noncontacted base pairs.the sequence of the bacteriophage 434 or1 (acaaaactttcttgt) differs from its or3 (acagttttcttgt) at positions 4-6. x-ray analysis shows that the side chain of gln33 of the 434 repressor makes van der waals' and h-bond contacts with the t at position 4' in complex with or1, but no specific contact is observed at this position in 434 repressor-or3 complexes. no contacts are made by repressor to the bases at positions 5 or 6 in either binding site. the significance of the sequence differences betwe ...19957836381
the complex between phage 434 repressor dna-binding domain and operator site or3: structural differences between consensus and non-consensus half-sites.the repressor of phage 434 binds to a set of operator sites as a homodimer. its relative affinities for these sites determine the switch from lysogenic to lytic growth. the six 434 operator sites (or1, or2, or3, ol1, ol2 and ol3) have a particularly simple organization; all are 14 base pairs long, with a conserved 5'-acaa sequence symmetrically placed at either end, and a variable central six base pairs. or3 is unique among naturally-occurring 434 operator sites in that it contains a non-consens ...19938081737
differential recognition of or1 and or3 by bacteriophage 434 repressor and cro.the developmental decisions of bacteriophage 434 depend on the ability of 434 repressor and cro to bind or1 and or3 with different relative affinities; repressor binds or1 tighter than or3, whereas cro slightly prefers or3 over or1. studies with operator mutants show that repressor's lower relative affinity for or3 results from a deviation in the sequence of or3 from consensus; an a-->g change at position 4 in one half-site (or1: a-c-a-a-a-c-t-t-t-c-t-t-g-t; or3: a-c-a-g-t-t-t-t-t-c-t-t-g-t). si ...19938226917
the dna-binding properties of an artificial 42-residue polypeptide derived from a natural repressor.bacteriophage 434 repressor recognizes the operator sequences acaag and acaat. as the same or similar sequences occur in the enhancer region of hiv-1, 434 repressor was a potential hiv enhancer-binding protein. we found that the interaction of the dna-binding domain of 434 repressor with a 57-bp hiv enhancer dna was very weak whereas a 42-residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. the results of footprint ...19938416811
de novo design of the hydrophobic cores of proteins.we have developed and experimentally tested a novel computational approach for the de novo design of hydrophobic cores. a pair of computer programs has been written, the first of which creates a "custom" rotamer library for potential hydrophobic residues, based on the backbone structure of the protein of interest. the second program uses a genetic algorithm to globally optimize for a low energy core sequence and structure, using the custom rotamer library as input. success of the programs in pre ...19958535237
design, synthesis, and characterization of hiv-1 enhancer-binding polypeptides derived from bacteriophage 434 repressor.we have designed and synthesized hiv-1 enhancer-binding polypeptides that were derived from bacteriophage 434 repressor. these peptides were 39-54 residues long and contained either the recognition helix or the entire helix-turn-helix motif of the dna-binding domain of 434 repressor. the dissociation constant of the complex formed between the standard peptide (r42) and a synthetic 70-bp hiv enhancer dna was ca. 10(-8) m. the specificity of the interaction of r42 with the two hiv enhancers was de ...19958537188
structure model of a complex between the factor for inversion stimulation (fis) and dna: modeling protein-dna complexes with dyad symmetry and known protein structures.a method is presented to predict overall conformations of protein-dna complexes on the basis of the known three-dimensional structures of the proteins. the method is restricted to proteins with a common twofold symmetry axis, which show only minor conformational changes upon binding to dna. the method uses a numerical finite difference solution of the linearized poisson-boltzmann equation and subsequent energy minimization cycles. structural parameters-the rotation angle of the dna relative to t ...19968865343
cloning and expression of bacillus subtilis phage spp1 in e. coli. ii. expression of lambda/spp1 hybrid phages in e. coli minicells.in the preceding paper (amann et al. 1981) we described the in vitro construction of hybrids between escherichia coli phage lambda nm607 imm434 and b. subtilis phage spp1. these lambda/spp1 hybrids have been used to infect minicells produced by e. coli strain ds410. analysis on polyacrylamide gels of 35s-methionine labeled proteins synthesized in infected minicells revealed the expression of both lambda and spp1 genes. infection of e. coli minicells carrying plasmid pgy101, which encodes and exp ...19816457236
in vitro assembly of bacteriophage lambda heads.the assembly of plaque-forming particles in cell-free extracts of induced lambda lysogens was observed two ways. (i) dna isolated from a lambda-related phage, 434 for example, is added to an extract of an induced lambda lysogen, and plaque-formers with the genotype of the added dna are detected. (ii) one extract from an induced lambda lysogen that carries an amber mutation in one of the head genes (a, b, c, d, or e) is mixed with one carrying an amber mutation in a different head gene; an increa ...19734509659
computer modeling studies of the structure of a repressor.due to advances in molecular biology the dna sequences of structural genes coding for proteins are often known before a protein is characterized or even isolated. the function of a protein whose amino acid sequence has been deduced from a dna sequence may not even be known. this has created greater interest in the development of methods to predict the tertiary structures of proteins. the a priori prediction of a protein's structure from its amino acid sequence is not yet possible. however, since ...19854063476
recognition of dna sequences by the repressor of bacteriophage 434.the structure of a complex between the dna-binding domain of phage 434 repressor and a 14 base-pair synthetic dna operator reveals the molecular interactions important for sequence-specific recognition. a set of contacts with dna backbone, notably involving hydrogen bonds between peptide-nh groups and dna phosphates, position the repressor and fix the dna configuration. direct interactions between amino acid side chains and dna bases involve nonpolar van der waals contacts as well as hydrogen bo ...19883359001
three-dimensional solution structure and stability of phage 434 cro protein.1h nmr resonances of the phage 434 cro protein were assigned using standard 2d nmr methods, and its solution structure determined using 867 distance constraints in distance geometry (diana) calculations ultimately refined by restrained molecular dynamics (gromos). in the 20 best nmr structures, the average pairwise backbone and heavy atom rmsds are 0.63 +/- 0.14 and 1.53 +/- 0.15 a, respectively, for the structurally well-defined residues 4-65. residues 1-3 and 66-71 at the n- and c-termini are ...19979174359
characterization of the lysogeny dna module from the temperate streptococcus thermophilus bacteriophage phi sfi21.phage phi sfi21, the only temperate streptococcus thermophilus phage from our phage collection, showed extensive dna homology with virulent phages from lytic group i. southern blot hybridizations demonstrated that the phi sfi21-specific dna was clustered in an approximately 6.6-kb-long region, the putative lysogeny module. sequence analysis and database research identified an integrase within this module; orf 203 with homology to an anonymous orf 258 from the temperate lactococcal phage bk5-t; o ...19979201223
recognition of dna structure by 434 repressor.in complexes of bacteriophage 434 binding sites with 434 repressor the central 4 bp of the 14 bp site are not contacted by the protein, although changes in these bases alter binding site affinity for the repressor. our previous data suggested that the ability of the non-contacted central bases to be overtwisted in repressor-dna complexes governs affinity of the binding site for 434 repressor. this idea was tested by examining the affinity of two central sequence variant 434 binding sites for 434 ...19989421532
molecular ecology and evolution of streptococcus thermophilus bacteriophages--a review.bacteriophages attacking streptococcus thermophilus, a lactic acid bacterium used in milk fermentation, are a threat to the dairy industry. these small isometric-headed phages possess double-stranded dna genomes of 31 to 45 kb. yoghurt-derived phages exhibit a limited degree of variability, as defined by restriction pattern and host range, while a large diversity of phage types have been isolated from cheese factories. despite this diversity all s. thermophilus phages, virulent and temperate, be ...19989562894
thymine methyls and dna-protein interactions.evidence is summarized showing that thymine methyls are as important in the recognition of specific sequences by proteins as are the more widely recognized hydrogen bonding sites of bases in the major groove (1). strongest evidence has come from experiments using functional group mutagenesis (2) in which thymines in a specific recognition sequence (e.g., promoters, operators and restriction sites) are replaced by oligonucleotide synthesis with methyl-free uracil or cytosine and 5-methylcytosine. ...19873320959
selective stabilization by the bacteriophage 434 repressor of the plasmid expressing bovine growth hormone in escherichia coli.the maintenance of a plasmid vector-host system that selects for bacteria carrying the plasmid without the need for antibiotics is described. in this system, the bacteriophage 434 repressor gene cloned on the plasmid protects the host from lysis by a lambda imm434 ci- prophage. cells that occasionally lose the plasmid are killed by prophage induction and therefore do not accumulate in the growing culture. the presence of the phage 434 repressor in the cells does not interfere with the process of ...19883291762
prediction of 3-d structure of the cro protein from phage 434.a comparative model building process has been utilized to predict the three-dimensional structure of the bacteriophage 434 cro protein. amino acid sequence similarities between the 434 cro protein and other bacteriophage repressor and cro proteins have been used, in conjunction with secondary structure prediction and the known structures of other base sequence specific dna binding proteins, to derive the model. from this model the interactions between the 434 cro protein and its operator dna hav ...19863271423
cold-sensitive e-lysis systems.the release of recombinant bacteria into the environment is undesirable because of possible risks associated with the genetically modified organisms. the aim of this study was to establish a cold-sensitive killing system with a lethal gene, activated when bacteria encounter lower environmental temperatures. to obtain cold-sensitive lysis vectors, the lambdaci857 repressor/pr promoter expression system was combined with either the laci/laczpo or the phage 434 ci/pr system that control the express ...19989751796
[design and synthesis of peptides capable of specific binding to dna].in the present communication, design, synthesis and dna binding activities of the following two peptides are reported: dns-gly-ala-gln-lys-leu-ala-cly-lys-val-gly-thr-lys-val-lys-val-gl y-thr-lys-thr - val-oh (i) and [(h-ala-lys-leu-ala-thr-lys-ala-gly-val-lys-gln-gln-ser-ile-gln-leu-ile- thr- ala-aca-lys-aca)2lys-aca]2lys-val-oh (ii), where aca = nh(ch2)5co--; dns is a residue of 5-dimethylaminonaphtalene-1-sulfonic acid. peptide i contains a large fraction (ca.30%) of valyl and threonyl residu ...19882851717
carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and dna binding specificities.strong dimerization of the repressor, mediated by the carboxyl (c)-terminal domain, is a prerequisite for forming a specific complex with dna and cooperative dna binding to form tetramers. we have generated a computer model of the c-terminal domain of the 434 repressor based on the crystal structure of the homologous umud' protein. this model predicts that residues in the primary sequence between 93 and 168 contribute to the dimer interface. we changed several amino acid residues located in this ...19989799634
isolation of altered specificity mutants of the single-chain 434 repressor that recognize asymmetric dna sequences containing the ttaa and ttac subsites.a novel single-chain (sc) protein framework containing covalently dimerized dna-binding domains (dbd) of the phage 434 repressor was used to construct combinatorial mutant libraries in order to isolate mutant dbds with altered specificities. the library members contain one wild-type dbd and one mutant domain with randomized amino acids in the dna-contacting region. based on previous studies, the mutant sc derivatives are expected to recognize a general acaa-6 bp-nnnn sequence, where acaa is cont ...199910446235
the phage 434 cro/or1 complex at 2.5 a resolution.the crystal structure of phage 434 cro protein in complex with a 20 base-pair dna fragment has been determined to 2.5 a resolution. the dna fragment contains the sequence of the or1 operator site. the structure shows a bent conformation for the dna, straighter at the center and more bent at the ends. the central base-pairs adopt conformations with significant deviations from coplanarity. the two molecules interact extensively along their common interface, both through hydrogen bonds and van der ...19912038059
modeling helix-turn-helix protein-induced dna bending with knowledge-based distance restraints.a crucial element of many gene functions is protein-induced dna bending. computer-generated models of such bending have generally been derived by using a presumed bending angle for dna. here we describe a knowledge-based docking strategy for modeling the structure of bent dna recognized by a major groove-inserting alpha-helix of proteins with a helix-turn-helix (hth) motif. the method encompasses a series of molecular mechanics and dynamics simulations and incorporates two experimentally derived ...199910465734
dna twisting and the effects of non-contacted bases on affinity of 434 operator for 434 repressor.the bacteriophage 434 repressor regulates gene expression by binding with differing affinities to the six operator sites on the phage chromosome. the symmetrically arrayed outer eight base pairs (four in each half-site) of these 14-base-pair operators are highly conserved but the middle four bases are divergent. although these four base pairs are not in contact with repressor, operators with a.t or t.a base pairs at these positions bind repressor more strongly than those bearing c.g or g.c, sugg ...19921731202
inhibition of hiv-1 enhancer-controlled transcription by artificial enhancer-binding peptides derived from bacteriophage 434 repressor.an artificial hiv-1 enhancer-binding 42-residue peptide (r42) that had been derived from bacteriophage 434 repressor inhibited the cell-free in vitro transcription of hiv-1 enhancer-containing plasmids [hehlgans, t., stolz, m., klauser, s., cui, t., salgam, p., brenz verca, s., widmann, m., leiser, a., städler, k. & gutte, b. (1993) febs lett. 315, 51-55; caderas, g. (1997) phd thesis, university of zürich]. here we show that, after n-terminal extension of r42 with a viral nuclear localization s ...199910561603
control of ci gene expression in bacteriophage lambda imm434, studied in an immunity/trp fusion made in vitro.the trp genes of a lambda imm434 trp-transducing phage have been fused to the immunity region by deletion, in vitro, of the dna between two targets for the restriction enzyme r.ecori. the resulting phage has been used to study the control of expression of the ci gene in vivo. the constitutive rate of expression of the ci gene is between 2 and 5% of the maximally stimulated rate. the products of the cii and ciii genes enhance expression of ci on infection of a sensitive host. the requirement for ...1976785219
operators and promoters in the or region of phage 434.the or operator region of phage 434 contains three 14 bp blocks with sequence acaaga-a--ttgt which are presumed to be the 434 repressor recognition sites. operator constitutive mutations are located in two of these blocks, while a mutation affecting repressor levels in the lysogenic state is located in the third. two transcripts obtained in vitro, one leftwards and one rightwards, are tentatively identified as the prm and pr transcription starts. the arrangement of the 434 operator region appear ...1979450705
the site controlling the specificity of n action is outside the promoter-operator region: a triple hybrid phage lambda n21 imm434nin5.a short interval of homology between imm lambda, imm434 and imm21 dnas was identified near the leftward promoter-operator region. this homology, denoted hs, was revealed by electron microscopic examination of lambda imm lambda/lambda imm21 and lambda imm434/lambda imm21 heteroduplexes, and permitted us to construct a special lambda hybrid (lambda hyb) which contains the n region of phage 21 and the adjacent imm region from phage 434. this triple hybrid, labmda n21 imm434nin5, was analysed by gen ...1979381108
major outer membrane proteins of e. coli k12 serve as receptors for the phages t2 (protein ia) and 434 (protein ib).mutants of e. coli resistant to bacteriophage t2 have lowered amounts of protein ia in their outer membrane. bacteriophage t2 was inactivated by a mixture of protein ia-lipopolysaccharide. protein ia or lipopolysaccharide alone had no neutralizing activity. however, only protein ia was required to inactivate a t2 host range mutant. in the presence of polymyxin b t2 receptor activity of protein ia--lipopolysaccharide mixtures could not be restored. e. coli strains missing protein ib were resistan ...1978360042
inhibition of spontaneous induction of lambdoid prophages in escherichia coli cultures: simple procedures with possible biotechnological applications.infections of bacterial cultures by bacteriophages are serious problems in biotechnological laboratories. apart from such infections, prophage induction in the host cells may also be dangerous. escherichia coli is a commonly used host in biotechnological production, and many laboratory strains of this bacterium harbour lambdoid prophages. these prophages may be induced under certain conditions leading to phage lytic development. this is fatal for further cultivations as relatively low, though st ...200111316465
ab initio prediction of helical segments in polypeptides.an ab initio method has been developed to predict helix formation for polypeptides. the approach relies on the systematic analysis of overlapping oligopeptides to determine the helical propensity for individual residues. detailed atomistic level modeling, including entropic contributions, and solvation/ionization energies calculated through the solution of the poisson-boltzmann equation, is utilized. the calculation of probabilities for helix formation is based on the generation of ensembles of ...200211924737
expression of a 434:vp16 chimeric activator leads to high-level activation of gene expression in stable transformants of arabidopsis.the performance of an expression system based on a fusion of the bacteriophage 434-repressor to the vp16 activation domain of herpes simplex virus type 1 (434:vp16) was tested after stable integration into arabidopsis. a special feature of this system was the use of the monocot maize ubiquitin1 and rice actin1 promoters to drive the expression of the 434:vp16 activator and 434-repressor, respectively. our results demonstrate that the maize ubiquitin1 and the rice actin1 promoters, each of which ...200212054349
purification and some properties of presumptive tof gene product of coli phage 434.the presumptive tof gene product of coli phage 434 has been purified from cells carrying lambdaimm434cidv plasmid known to contain only some of the "early" genes of phage 434 and lambda. it was detected and tentatively identified as tof protein primarily by its ability to specifically bind to phage 434 dna. the protein has a molecular weight of about 11,000 and requires mg2+ for specific dna binding, unlike 434 ci-repressor.1977340902
a comparative study of dynamic structures between phage 434 cro and repressor proteins by normal mode analysis.two dna binding proteins, cro and the amino-terminal domain of the repressor of bacteriophage 434 (434 cro and 434 repressor) that regulate gene expression and contain a helix-turn-helix (hth) motif responsible for their site-specific dna recognition adopt very similar three-dimensional structures when compared to each other. to reveal structural differences between these two similar proteins, their dynamic structures, as examined by normal mode analysis, are compared in this paper. two kinds of ...19968880931
operator recognition by the phage 434 ci repressor: md simulations of free and bound 50-bp dna reveal important differences between the or1 and or2 sites.using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp dna sequence containing the 434 bacteriophage operators or1 and or2 separated by an 8-bp spacer. two simulations of 1 ns each were carried out, with dna alone and with dna complexed to dimers of the r1-69 dna binding domain of the phage 434 ci repressor protein at the or1 and or2 sites. strong correlations among average structural parameters are observed between our simulations and available experim ...200312548627
dna-stimulated assembly of oligomeric bacteriophage 434 repressor: evidence for cooperative binding by recruitment.typical of many transcriptional regulatory proteins, the lambdoid bacteriophage repressors bind cooperatively to multiple sites on dna. this cooperative binding is essential for establishment and maintenance of phage lysogeny. in the phage, two repressor homodimers, one bound at each of the adjacent operator sites, interact to form the tetramer that is necessary for the cooperative binding of the repressor. bacteriophage 434 repressor does not form tetramers in the absence of dna, and the mechan ...200312680780
purification and properties of a deoxyribonucleic acid exonuclease associated with the formation of phage 434. 196414257619
the preferred substrate for reca-mediated cleavage of bacteriophage 434 repressor is the dna-bound dimer.induction of a lysogen of a lambdoid bacteriophage usually involves reca-stimulated autoproteolysis of the bacteriophage repressor protein. previous work on the phage repressors showed that the monomeric form of the protein is the target of reca. our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo. hence, if the repressor in a lysogen is present as a dimer, how can reca-stimulated autoproteolysis play a role in bacter ...200414679217
a permutational approach toward protein-dna recognition.the ci repressor of bacteriophage 434, known as 434 repressor, binds to 14-bp operator sequences by means of a helix-turn-helix motif. to probe the requirements for selective dna recognition by this class of dna binding proteins, as well as to generate new proteins with altered specificities, a library of approximately 3 x 10(6) mutants was generated that contains all permutations of five residues in the recognition helix (helix 3) of the repressor. these mutants were then selected in vivo for t ...19948171021
structure based prediction of protein folding intermediates.the complete unfolding of a protein involves the disruption of non-covalent intramolecular interactions within the protein and the subsequent hydration of the backbone and amino acid side-chains. the magnitude of the thermodynamic parameters associated with this process is known accurately for a growing number of globular proteins for which high-resolution structures are also available. the existence of this database of structural and thermodynamic information has facilitated the development of ...19948078072
nmr structures of salt-refolded forms of the 434-repressor dna-binding domain in 6 m urea.the n-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in h(2)o solution, and is unfolded in 6 m urea at ph 7.5. in this study, 434(1-63) has been refolded by adding either 1.7 m nacl or 0.47 m natfa to the solution in 6 m urea, and the nmr structures of both refolded forms have been determined. the two refolded forms have similar free energies of unfolding and are approximately 16 kj/mol less stable than the protein in h(2)o solution. 434(1-63 ...200415518542
the bacteriophage 434 repressor dimer preferentially undergoes autoproteolysis by an intramolecular mechanism.inactivation of the lambdoid phage repressor protein is necessary to induce lytic growth of a lambdoid prophage. activated reca, the mediator of the host sos response to dna damage, causes inactivation of the repressor by stimulating the repressor's nascent autocleavage activity. the repressor of bacteriophage lambda and its homolog, lexa, preferentially undergo reca-stimulated autocleavage as free monomers, which requires that each monomer mediates its own (intramolecular) cleavage. the ci repr ...200516077107
a quantitative methodology for the de novo design of proteins.we have developed a general quantitative methodology for designing proteins de novo, which automatically produces sequences for any given plausible protein structure. the method incorporates statistical information, a theoretical description of protein structure, and motifs described in the literature. a model system embodying a portion of the quantitative methodology has been used to design many protein sequences for the phage 434 cro and fibronectin type iii domain folds, as well as several ot ...19947849602
indirect readout: detection of optimized subsequences and calculation of relative binding affinities using different dna elastic potentials.essential biological processes require that proteins bind to a set of specific dna sites with tuned relative affinities. we focus on the indirect readout mechanism and discuss its theoretical description in relation to the present understanding of dna elasticity on the rigid base pair level. combining existing parametrizations of elastic potentials for dna, we derive elastic free energies directly related to competitive binding experiments, and propose a computationally inexpensive local marker ...200617038333
the bacteriophage 434 operator/repressor system in yeast.the ability of the bacteriophage 434 operator/repressor system to function in a eukaryotic cell has been explored. an idealized 434 operator was placed at various positions in the pgk promoter of saccharomyces cerevisiae: within the upstream activator sequence, close to the tata box, and downstream of the transcription-initiation site. expression of the 434 ci gene from a 2 microns-based plasmid resulted in significant repression of gene expression from constructs containing the altered promoter ...19957496531
cocrystals of the dna-binding domain of phage 434 repressor and a synthetic phage 434 operator.the amino-terminal domain of the phage 434 repressor forms cocrystals with a synthetic phage 434 operator. the cocrystals diffract to at least 4 a, and x-ray crystallographic analysis of them is in progress. an analysis of the packing in the cocrystals shows that complexes consisting of dimers of amino-terminal domain bound specifically to operators are stacked end to end in longer protein-dna rods parallel to the unit cell body diagonals. the dna in the complexes has 10.5 base pairs per turn an ...19846369323
the construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the ci gene of coliphage 434.a new plasmid vector, pns1, is described that allows positive selection for bacterial transformants carrying recombinant plasmids. it is a derivative of pbr327, and it includes a regulatory region from the lambdoid phage 434. the expression of the tcr gene of pns1 is under the control of the orpr operator-promoter of phage 434, which is regulated by the repressor gene ci. the cloning sites of pns1 (stui, ndei, hpai, hindiii, asuii and ecori) are situated within ci; hence insertion of foreign dna ...19846096221
single-chain repressors containing engineered dna-binding domains of the phage 434 repressor recognize symmetric or asymmetric dna operators.single-chain (sc) dna-binding proteins containing covalently dimerized n-terminal domains of the bacteriophage 434 repressor ci have been constructed. the dna-binding domains (amino acid residues 1 to 69) were connected in a head-to-tail arrangement with a part of the natural linker sequence that connects the n and c-terminal domains of the intact repressor. compared to the isolated n-terminal dna-binding domain, the sc molecule showed at least 100-fold higher binding affinity in vitro and a sli ...19979096211
the bacteriophage 434 right operator. roles of o(r)1, o(r)2 and o(r)3.lysogenic induction of bacteriophage lambda is controlled by the action of the phage repressor and cro proteins at the phage right operator (o(r)). this study examines the roles of the repressor and cro proteins of the related phage 434. the start sites of transcription of the divergently oriented promoters in the 434 o(r) region, pr and prm, were mapped, and the effects of 434 repressor and cro on promoter activity were assessed using promoter fusions to lacz. the effects of repressor or cro bo ...19938450541
monovalent cations regulate dna sequence recognition by 434 repressor.the bacteriophage 434 repressor distinguishes between its six naturally occurring binding sites using indirect readout. in indirect readout, sequence-dependent differences in the structure and flexibility of non-contacted bases in a protein's dna-binding site modulate the affinity of dna for protein. the conformation and flexibility of a dna sequence can be influenced by the interaction of the dna bases or backbone with solution components. we examined the effect of changing the cation-type pres ...200415210346
dna-mediated assembly of weakly interacting dna-binding protein subunits: in vitro recruitment of phage 434 repressor and yeast gcn4 dna-binding domains.the specificity of dna-mediated protein assembly was studied in two in vitro systems, based on (i) the dna-binding domain of bacteriophage 434 repressor ci (amino acid residues 1-69), or (ii) the dna-binding domain of the yeast transcription factor gcn4, (amino acids 1-34) and their respective oligonucleotide cognates. in vivo, both of these peptides are part of larger protein molecules that also contain dimerization domains, and the resulting dimers recognize cognate palindromic dna sequences t ...200415388801
recognition of dna by single-chain derivatives of the phage 434 repressor: high affinity binding depends on both the contacted and non-contacted base pairs.single-chain derivatives of the phage 434 repressor, termed single-chain repressors, contain covalently dimerized dna-binding domains (dbd) which are connected with a peptide linker in a head-to-tail arrangement. the prototype rr69 contains two wild-type dbds, while rr*69 contains a wild-type and an engineered dbd. in this latter domain, the dna- contacting amino acids of thealpha3 helix of the 434 repressor are replaced by the corresponding residues of the related p22 repressor. we have used bi ...19979153301
uv light induces is10 transposition in escherichia coli.a new mutagenesis assay system based on the phage 434 ci gene carried on a low-copy number plasmid was used to investigate the effect of uv light on intermolecular transposition of is10. inactivation of the target gene by is10 insertion was detected by the expression of the tet gene from the phage 434 pr promoter, followed by southern blot analysis of plasmids isolated from tetr colonies. uv irradiation of cells harboring the target plasmid and a donor plasmid carrying an is10 element led to an ...19989649512
folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 cro protein.the phage 434 cro protein, the n-terminal domain of its repressor (r1-69) and that of phage lambda (lambda6-85) constitute a group of small, monomeric, single-domain folding units consisting of five helices with striking structural similarity. the intrinsic helix stabilities in lambda6-85 have been correlated to its rapid folding behavior, and a residual hydrophobic cluster found in r1-69 in 7 m urea has been proposed as a folding initiation site. to understand the early events in the folding of ...199910452612
molecular dynamics simulation in solvent of the bacteriophage 434 ci repressor protein dna binding domain amino acids (r1-69) in complex with its cognate operator (or1) dna sequence.we investigated protein/dna interactions, using molecular dynamics simulations computed between a 10 angstom water layer model of the 434 ci repressor protein dna binding domain (dbd) amino acids (r1-69) and dna of operator (or1) and its flanks consisting of 28 nucleotide base pairs. hydrogen bonding interactions were monitored. in addition, van der waals and electrostatic interaction energies were calculated. amino acids of the 434 ci repressor dna recognition helix 3 formed both direct and wat ...199910496417
thermodynamic analysis of the structural stability of phage 434 cro protein.thermodynamic parameters describing the phage 434 cro protein have been determined by calorimetry and, independently, by far-uv circular dichroism (cd) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. these equilibrium unfolding transitions are adequately described by the two-state model. the far-uv cd denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) m(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) k ...199910569937
dna-induced conformational changes in bacteriophage 434 repressor.although bacteriophage 434 repressor binds to its specific dna sites only as a dimer, formation of the dimers in solution occurs at concentrations three orders of magnitude higher than those needed to bind the 434 operator dna. our results suggest that both specific and non-specific dna induce conformational changes in repressor that lead to formation of repressor dimers. the repressor conformational changes induced by dna occur at concentrations much lower than those needed for binding of repre ...199910588892
mutually exclusive utilization of p(r) and p(rm) promoters in bacteriophage 434 o(r).establishment and maintenance of a lysogen of the lambdoid bacteriophage 434 require that the 434 repressor both activate transcription from the p(rm) promoter and repress transcription from the divergent p(r) promoter. several lines of evidence indicate that the 434 repressor activates initiation of p(rm) transcription by occupying a binding site adjacent to the p(rm) promoter and directly contacting rna polymerase. the overlapping architecture of the p(rm) and p(r) promoters suggests that an r ...200010809696
design of protein cores by screening combinatorial sequence library.we have developed a new method, heterogeneous self-consistent ensemble optimization (hetero-sceo), to select appropriate hydrophobic cores of proteins. it has been tested with five kinds of proteins: lambda-repressor, phage 434 cro protein, interleukin-4, thioredoxin and ubiquitin. the results show that the method can be used for the de novo desigh of the hydrophobic cores of proteins.199812168026
combinatorial redesign of the dna binding specificity of a prokaryotic helix-turn-helix repressor.redesign of the bacteriophage 434 cro repressor was accomplished by using an in vivo genetic screening system to identify new variants that specifically bound previously unrecognized dna sequences. site-directed, combinatorial mutagenesis of the 434 cro helix-turn-helix (hth) motif generated libraries of new variants which were screened for binding to new target sequences. multiple mutations of 434 cro that functionally converted wild-type (wt) 434 cro dna binding-sequence specificity to that of ...200312511493
on the importance of van der waals interaction in the groove binding of dna with ligands: restrained molecular dynamics study.comparison of interaction energy between an oligonucleotide and a dna-binding ligand in the minor and major groove modes was made by use of restrained molecular dynamics. distortion in dna was found for the major groove mode whereas less significant changes for both ligand and dna were detected for the minor groove binding after molecular dynamics simulation. the conformation of the ligand obtained from the major groove modes resembles that computed with the ligand soaked in water. the van der w ...19969024904
mechanisms that determine the differential stability of stx⁺ and stx(-) lysogens.phages 933w, baa2326, 434, and λ are evolutionarily-related temperate lambdoid phages that infect escherichia coli. although these are highly-similar phages, baa2326 and 933w naturally encode shiga toxin 2 (stx⁺), but phage 434 and λ do not (stx(-)). previous reports suggest that the 933w stx⁺ prophage forms less stable lysogens in e. coli than does the stx(-) prophages λ, p22, and 434. the higher spontaneous induction frequency of the stx⁺ prophage may be correlated with both virulence and disp ...201627043626
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