| lambda integrase cleaves dna in cis. | in the int family of site-specific recombinases, dna cleavage is accomplished by nucleophilic attack on the activated scissile phosphodiester bond by a specific tyrosine residue. it has been proposed that this tyrosine is contributed by a protomer bound to a site other than the one being cleaved ('trans' cleavage). to test this hypothesis, the difference in dna binding specificity between closely related integrases (ints) from phages lambda and hk022 was exploited to direct wild type ints and cl ... | 1994 | 7925285 |
| comparative molecular biology of lambdoid phages. | lambdoid phages are natural relatives of phage lambda. as a group, they are highly polymorphic in dna sequence and biological specificity. specificity differences have played a key role in identifying the specific sequences recognized by the n and q antitermination proteins, the initiator o for dna synthesis, the terminase system nv1-a for cutting dna during packaging, and the ci repressor protein. variations that go beyond specificity differences are seen in packaging mechanism (headful in p22, ... | 1994 | 7826005 |
| position and direction of strand exchange in bacteriophage hk022 integration. | the positions of the endonucleolytic cleavages promoted by the integrase protein (int) of coliphage hk022 within its attb site were determined. the protein catalyses a staggered cut, which defines an overlap sequence of 7 bp within the core site. the overlap region is at the center of symmetry of a palindromic sequence which appears in all four putative att core binding sites for int. we confirm that the order of strand exchange is similar to that in phage lambda. | 1994 | 7808413 |
| a zinc-binding region in the beta' subunit of rna polymerase is involved in antitermination of early transcription of phage hk022. | antitermination of early transcription in phage hk022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. it does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. to identify host proteins involved in antitermination, we isolated 14 escherichia coli mutants that are specifically blocked in hk022 growth. the mutations are located in the rpoc gene, which encodes the beta' subunit of rna polymerase. each mutation al ... | 1995 | 7752239 |
| phage hk022 nun protein arrests transcription on phage lambda dna in vitro and competes with the phage lambda n antitermination protein. | phage hk022 nun protein excludes phage lambda by terminating transcription near the lambda nut sites. we have established a purified in vitro system that reproduces the in vivo sequence and factor requirements of nun. nun arrests transcription by e. coli rna polymerase at or near elongation pause sites distal to the nut sites. the boxb sequence of nut is required for optimal nun activity; boxa plays a lesser role. the efficiency of transcription arrest is strongly enhanced by the four e. coli nu ... | 1995 | 7714899 |
| identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases. | bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between dna molecules that carry specific sites. phages lambda and hk022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. to locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these c ... | 1995 | 7674299 |
| temperate coliphage hk022: virions, dna, one-step growth, attachment site, and the prophage genetic map. | | 1981 | 7026732 |
| genetic recombination between phage hk022, lambda, and phi 80. | | 1981 | 6451073 |
| the remarkable specificity of a new transcription termination factor suggests that the mechanisms of termination and antitermination are similar. | e. coli lysogenic for the temperate, lambda-related phage hk022 do not support lambda growth. the exclusion of lambda is caused by the hk022 nun gene product, which blocks the expression of genes located downstream of and in the same transcription unit as the lambda nutl and nutr sequences. transcripts terminating prematurely at or near nutr have been detected after inactivation of lambda repressor in lambda, hk022 dilysogens. nun therefore appears to be a transcription termination factor with a ... | 1987 | 2822258 |
| identification of functional regions of the nun transcription termination protein of phage hk022 and the n antitermination protein of phage lambda using hybrid nun-n genes. | phages lambda and hk022 express proteins n and nun, respectively, each of which acts with a number of escherichia coli host nus factors at lambda nut rna sites, to influence transcription elongation. the lambda nut sites, nearly identical sequences located downstream of the early promoters, pl and pr, were first identified as cis-acting signals required for the action of n in forming termination-resistant transcription complexes. surprisingly, the nun protein, resembling n and expressed by anoth ... | 1996 | 8632463 |
| transcription antitermination: the lambda paradigm updated. | coliphage lambda employs systems of transcription termination and antitermination to regulate gene expression. early gene expression is regulated by the phage-encoded n protein working with a series of escherichia coli proteins, nus, at rna sites, nut, to modify rna polymerase to a termination-resistant form. expression of lambda late genes is regulated by the phage-encoded q antitermination protein. q, which appears to use only one host factor, acts at a dna site, qut, to modify rna polymerase ... | 1995 | 8709839 |
| structure and function of the nun gene and the immunity region of the lambdoid phage hk022. | the immunity region of the lambdoid phage, hk022, has been sequenced. the hk022 repressor gene, its cognate operators and promoters, and several early phage genes can be discerned. the overall design of the immunity region resembles that of other lambdoid phages. the location of the hk022 nun gene, whose product excludes superinfecting lambda by terminating transcription at (or near) the lambda nut sites, is analogous to that of gene n in lambda. nun is preceded by sequences similar to the lambd ... | 1989 | 2760929 |
| determinants of site-specific recombination in the lambdoid coliphage hk022. an evolutionary change in specificity. | the temperate bacteriophage hk022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. however, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. in order to determine the basis for this difference in specificity, we sequenced the hk022 elements that are in ... | 1989 | 2547971 |
| genetic analysis of bacteriophage lambda integrase interactions with arm-type attachment site sequences. | the bacteriophage p22-based challenge phage system was used to study lambda integrase (int) protein binding to its arm-type recognition sequences in the bacteriophage lambda attachment site. challenge phages were constructed that carried inserts containing either the contiguous p'123 arm-type sites or the single p'1 site within the p22 phage promoter, pant, which is required for expression of antirepressor. if int protein binds to these sequences in vivo, it represses transcription from pant. we ... | 1990 | 2155203 |
| specificity determinants in the attachment sites of bacteriophages hk022 and lambda. | the int proteins of bacteriophages hk022 and lambda promote recombination between phage and bacterial attachment sites. although the proteins and attachment sites of the two phages are similar, neither protein promotes efficient recombination between the pair of attachment sites used by the other phage. to analyze this difference in specificity, we constructed and characterized chimeric attachment sites in which segments of one site were replaced with corresponding segments of the other. most su ... | 1990 | 2146253 |
| lambda nutr mutations convert hk022 nun protein from a transcription termination factor to a suppressor of termination. | the nun protein of the lambdoid phage hk022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. an hk022 lysogen carrying a fusion of the lambda pr promoter and nutr site to a gal operon that lacks its own promoter is, therefore, gal-. to characterize the target of nun action, spontaneous gal+ revertants of this strain were isolated and characterized. two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within th ... | 1990 | 2139472 |
| escherichia coli nusa is required for efficient rna binding by phage hk022 nun protein. | the nun protein of phage hk022 is an rna binding protein of the arginine-rich motif family. nun binds the phage lambda boxb rna sequence (boxb) on nascent lambda transcripts and arrests transcription elongation. binding to boxb is inhibited by zn2+ and stimulated by the escherichia coli nusa protein. deletion of the nun c-terminal region enhances boxb binding and makes it independent of zn2+ and nusa. the c terminus of nun thus appears to interfere with the n-terminal rna binding motif. nusa rel ... | 1998 | 9465052 |
| escherichia coli mutations that block transcription termination by phage hk022 nun protein. | the nun gene product of the lambdoid coliphage hk022 provokes premature transcription termination at, or near, the phage lambda nut sites. termination by nun and antitermination by lambda n protein both require the nut sites and escherichia coli nusa, nusb and nuse proteins. to characterize further the host requirements for nun termination, we selected host mutations that blocked termination at lambda nutr. in addition to mutations in nusa, nusb and nuse, we obtained mutations in rpoc, encoding ... | 1991 | 1831236 |
| the activity of the ciii regulator of lambdoid bacteriophages resides within a 24-amino acid protein domain. | the ciii protein of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded cii protein, a transcriptional activator of the repressor and integrase genes. we have isolated a set of missense mutations in the ciii gene of phage lambda and of phage hk022 that yield inactive ciii proteins. all the mutations are located in the relatively conserved central region of the protein. a comparative analysis of the ciii protein sequence in lambda, hk022, and the lambdoid bacteriophage p22 ... | 1991 | 1828895 |
| mutations affecting cooperative dna binding of phage hk022 ci repressor. | cooperative protein-dna interactions play critical roles in gene regulation in all organisms. among the best-studied cooperative interactions is that of phage lambda repressor, which binds cooperatively to two adjacent operators. similar cooperative interactions are also shown by several other lambdoid phage repressors, including hk022 ci repressor, which we study here. this protein has a much higher degree of cooperativity than seen with lambda repressor, and previous evidence has suggested tha ... | 1998 | 9636698 |
| genetic analysis of the ciii gene of bacteriophage hk022. | the ciii gene product of lambdoid bacteriophages promotes lysogeny by stabilizing the phage-encoded cii protein, a transcriptional activator of the repressor and integrase genes. previous works showed that the synthesis of the bacteriophage lambda ciii protein has specific translational requirements imposed by the structure of the mrna. to gain insight into the mrna structure and its role in regulating ciii translation, we undertook a mutational analysis of the ciii gene of the related bacteriop ... | 1991 | 1824768 |
| sequence analysis of stx2-converting phage vt2-sa shows a great divergence in early regulation and replication regions. | in enterohemorrhagic escherichia coli, shiga toxin is produced by lysogenic prophages. we have isolated the prophage vt2-sa that is responsible for production of shiga toxin type 2 protein, and determined the complete nucleotide sequence of this phage dna. the entire dna sequence consisted of 60,942 bp, exhibiting marked similarity to the 933w phage genome. however, several differences were observed in the immunity and replication regions, where ci, cii, ciii, n, cro, o, and p genes were present ... | 1999 | 10492170 |
| the early promoters of bacteriophage hk022: contrasts and similarities to other lambdoid phages. | the pl, pr and pm promoters of lambdoid phages direct the transcription of early phage genes and the prophage repressor gene. we have determined the start points of transcription for these three promoters in the lambdoid phage hk022 and have shown that the hk022 repressor represses the early promoters, pl and pr, and activates the repressor promoter, pm. hk022 resembles other phages of the lambda family in these respects, as it does in the functional organization of most of its early genes and s ... | 1991 | 1824767 |
| recombination and modular exchange in the genesis of new lambdoid phages. | comparison of the nucleotide sequence of the integrase genes of lambdoid phages 21 and 434 with the published sequences of phages hk022 and lambda shows that lambda and 434 are very closely related (98% base sequence identity), whereas hk022 and 21 respectively show 73% and 48% identity to lambda. it is likely that several homologous recombination events occurred in the int gene and flanking dna among the progenitors of these phages. sequence divergence to different alternative sequences at a co ... | 1991 | 1715186 |
| requirement for e. coli nusg protein in factor-dependent transcription termination. | the 21 kd nusg protein is essential for e. coli viability. cells depleted for nusg were defective for factor-dependent transcription termination. rho-induced polarity in the gal operon and the rho-dependent lambda tr1 and lambda tl1 terminators were suppressed in nusg-deficient cells. nusg depletion inactivated the phage hk022 nun termination factor. in contrast, the factor-independent lambda tl terminator was fully active in nusg-depleted cells and could be suppressed by phage lambda n function ... | 1992 | 1547498 |
| mutations of the phage lambda nutl region that prevent the action of nun, a site-specific transcription termination factor. | phage hk022 encodes a protein, nun, that promotes transcription termination within the pl and pr operons of its relative, phage lambda. the lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. to further specify the nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutl region and measured nun-de ... | 1992 | 1532174 |
| the escherichia coli rpob60 mutation blocks antitermination by coliphage hk022 q-function. | the lambdoid bacteriophage regulate gene expression by suppressing transcription terminators. although similar in sequence to lambda, hk022 lacks an analogue to the lambda n antitermination gene and a distinct nutr sequence. to define the hk022 antitermination system, we plated the phage on escherichia coli nus mutants that inhibit lambda n function. only rpob60 (also called nusc60) blocked hk022 lytic growth. analyses of hk022-lambda hybrid phage suggested that a hk022 function analogous to lam ... | 1992 | 1522593 |
| temperate coliphage hk022. clear plaque mutants and preliminary vegetative map. | wild type phage hk022 was mutagenized by n-methyl-n'-nitro-n-nitrosoguanidine to induce clear plaque mutants. a total of 225 clear plaque mutants were isolated and 198 of these were assignable to one or the other of two complementation groups of the corresponding cistrons which have been designated as ci and cii, respectively. approximately 25% of the c mutants were found to be temperature-sensitive (cts); producing turbid plaques at 32 c and clear plaques at 38 c and above. from complementation ... | 1976 | 994348 |
| core-binding specificity of bacteriophage integrases. | the site-specific recombination systems of bacteriophages lambda and hk022 share the same mechanism and their integrase proteins show strong homology. nevertheless the integrase protein of each phage can only catalyze recombination between its own att sites. previous work has shown that the specificity determinants in the att sites are located within the sequences that bind the integrase to the core of att. dna fragments that carry attl and attr sites of each phage were challenged with each of t ... | 2000 | 10852483 |
| studies on bacteriophage distribution: virulent and temperate bacteriophage content of mammalian feces. | freshly voided samples of the feces of cows, pigs, and humans were analyzed for the enumeration of cell-free plaque-forming units (pfu) of coliphages and salmonella phages. coliphage pfu counts per gram (wet weight) of feces were found to range from less than 10(1) to greater than 10(7). salmonella phages were found in three out of five porcine samples, but none were found in the four bovine samples analyzed. virulent coliphages related to the phix174/s13 serological group showed some "habitat p ... | 1976 | 987749 |
| a segment of the phage hk022 chromosome is a mosaic of other lambdoid chromosomes. | we report the sequence of a region of the pr operon of lambdoid phage hk022 and an analysis of the proteins it encodes. this region has dna sequence elements and open reading frames that resemble those found in phages lambda, p22, and phi 80. the open reading frames encode homologs of the lambda cii transcription activator, the p22 dna replication proteins, and a fourth protein of unknown function. | 1994 | 8127672 |
| use of a gene encoding a suppressor trna as a reporter of transcription: analyzing the action of the nun protein of bacteriophage hk022. | the nun protein of phage hk022 blocks the expression of genes that lie downstream of the nut sites of phage lambda. nun is believed to act by promoting premature termination of transcription at or near these sites. to test this hypothesis and to facilitate mapping the sites of termination, we inserted a gene encoding a suppressor trna immediately downstream of the lambda nutl site and determined the effect of nun on trna level. we found that nun severely reduced the accumulation of mature, biolo ... | 1993 | 8234323 |
| a novel antivirulence element in the temperate bacteriophage hk022. | lysogens of the temperate lambdoid phage hk022 are immune to superinfection by hk022. superinfection immunity is conferred in part by the action of the hk022 ci repressor at the o.r operators. in this work, we have identified an additional regulatory element involved in immunity. this site, termed ofr (operator far right), is located just downstream of the cro gene, more than 250 nucleotides distant from or. the behavior of phage containing a mutation in ofr suggests that the wild-type site func ... | 1993 | 8244923 |
| cooperative dna-protein interactions. effects of changing the spacing between adjacent binding sites. | cooperative binding of specific dna-binding proteins plays crucial roles in gene regulatory circuitry, and is a model system for interactions between proteins bound to dna. we have studied coliphage hk022 repressor, which binds to two adjacent operators with a cooperativity parameter of approximately 2000. we examined the effect of changing the spacing between these two operators on cooperativity and on the conformation of the complex. maximum cooperativity was seen with the wild-type spacing; c ... | 1994 | 8289280 |
| the effect of mutations in the xis-binding sites on site-specific recombination in coliphage hk022. | excisionase (xis) is an accessory protein that is required for the excision of the related prophages lambda and hk022. xis binds to two tandemly arranged binding sites (x1 and x2) on the p arm of the recombination sites attp and attr. gel-retardation analyses and site-specific recombination assays were conducted on derivatives bearing site-directed mutations in the x1 and x2 sites of phage hk022. the results confirm the cooperative binding of xis to its sites, showing that binding to x1 stimulat ... | 2001 | 11810229 |
| xis and fis proteins prevent site-specific dna inversion in lysogens of phage hk022. | hk022, a temperate coliphage related to lambda, forms lysogens by inserting its dna into the bacterial chromosome through site-specific recombination. the escherichia coli fis and phage xis proteins promote excision of hk022 dna from the bacterial chromosome. these two proteins also act during lysogenization to prevent a prophage rearrangement: lysogens formed in the absence of either fis or xis frequently carried a prophage that had suffered a site-specific internal dna inversion. the inversion ... | 1993 | 8423145 |
| phage hk022-based integrative vectors for the insertion of genes in the chromosome of multiply marked escherichia coli strains. | we constructed a series of plasmids that allow the insertion of cloned dna in the escherichia coli chromosome by site-specific integration into the bacteriophage hk022 bacterial attachment site. these plasmids make use of a cole1 origin of replication, the phage hk022 attachment site attp, antibiotic resistance genes for selection and unique restriction sites. circularisation of non-replicative fragments containing the hk022 attachment site attp is performed in vitro and site-specific integratio ... | 2002 | 12127487 |
| identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases. | the integrases of bacteriophages lambda and hk022 promote recombination between dna molecules that carry attachment sites. the two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. to identify the amino acids that determine this difference in specificity, we selected mutants of lambda integrase with increased ability to recombine hk022 sites. this selection yielded eleven different amino acid substitutions at eight diff ... | 1995 | 7674300 |
| antitermination of early transcription in phage hk022. absence of a phage-encoded antitermination factor. | in phage lambda and its relatives most early phage genes are located downstream from transcription termination sites, and full gene expression requires suppression of termination (or antitermination). phage hk022, a lambda relative, also antiterminates early transcription, but, unlike its relatives, does so in the absence of any active phage gene product. we found no functional equivalent of the lambda n antitermination protein in hk022. in addition, nus mutations, which alter host proteins requ ... | 1993 | 8429552 |
| highly cooperative dna binding by the coliphage hk022 repressor. | the ci repressor protein from the temperate lambdoid phage hk022 was purified to near homogeneity and used in dnase i footprinting analyses to identify six binding sites in this phage. all these sites contained homologous 15 bp inverted repeats. three of these 15 bp inverted repeats were located between the ci and cro (or1 to or3), and the other three were 3' to the ci gene (ol1 to ol3). two of these sites were identified as operator sites for the repressor by dna sequence analyses of virulent p ... | 1993 | 8487297 |
| a spring-loaded state of nusg in its functional cycle is suggested by x-ray crystallography and supported by site-directed mutants. | transcription factor nusg is present in all prokaryotes, and orthologous proteins have also been identified in yeast and humans. nusg contains a 27-residue kow motif, found in ribosomal protein l24 where it interacts with rrna. nusg in escherichia coli (ecnusg) is an essential protein and functions as a regulator of rho-dependent transcription termination, phage lambda n and rrna transcription antitermination, and phage hk022 nun termination. relative to ecnusg, aquifex aeolicus nusg (aanusg) an ... | 2003 | 12600194 |
| interaction between the phage hk022 nun protein and the nut rna of phage lambda. | the nun gene product of prophage hk022 excludes phage lambda infection by blocking the expression of genes downstream from the lambda nut sequence. the nun protein functions both by competing with lambda n transcription-antitermination protein and by actively inducing transcription termination on the lambda chromosome. we demonstrate that nun binds directly to a stem-loop structure within nut rna, boxb, which is also the target for the n antiterminator. the two proteins show comparable affinitie ... | 1995 | 8618858 |
| phage hk022 roi protein inhibits phage lytic growth in escherichia coli integration host factor mutants. | temperate coliphage hk022 requires integration host factor (ihf) for lytic growth. the determinant responsible for this requirement was identified as a new gene (roi) located between genes p and q. this gene encodes a dna-binding protein (roi) containing a helix-turn-helix motif. we have shown that roi binds a site within its own gene that is closely linked to an ihf binding site. by gel retardation experiments, we have found that ihf binding stabilizes the interaction of roi with its gene. we h ... | 1996 | 8763934 |
| transcripts that increase the processivity and elongation rate of rna polymerase. | transcripts encoded by the cis-acting antitermination sites (put sites) of lambdoid phage hk022 promote readthrough of downstream transcription terminators. proper conformation of the transcripts is essential for activity, since put mutations that prevent the formation of predicted rna stems prevented antitermination, and suppressor mutations that restore the stems restored antitermination. antitermination does not appear to require proteins other than rna polymerase, since put-dependent readthr ... | 1996 | 8945516 |
| a second site-specific recombination event in the lambdoid bacteriophage hk022. | an in vitro site-specific recombination reaction of the lambdoid phage hk022 has revealed two supercoiled products that proved to be holliday intermediates. one of them is the holliday intermediate which has resulted from an attp x attb reaction. the other is an intermediate which has resulted from a recombination reaction between attp and the attl site of the product from the first reaction. the preferential attl x attp over attr x attp reaction was confirmed in vitro and in vivo by challenging ... | 1996 | 9003323 |
| the nun protein of bacteriophage hk022 inhibits translocation of escherichia coli rna polymerase without abolishing its catalytic activities. | bacteriophage hk022 nun protein blocks transcription elongation by escherichia coli rna polymerase in vitro without dissociating the transcription complex. nun is active on complexes located at any template site tested. ultimately, only the 3'-oh terminal nucleotide of the nascent transcript in an arrested complex can turn over; it is removed by pyrophosphate and restored with ntps. this suggests that nun inhibits the translocation of rna polymerase without abolishing its catalytic activities. u ... | 1997 | 9334329 |
| the antiterminator rna of phage hk022. | the put sites of phage hk022 increase the processivity of transcription and thereby promote the expression of viral genes that are located downstream of transcription terminators. rna polymerase molecules that have traversed a put site are converted to a terminator-resistant form by the put transcript. we analyzed the structure and function of put transcripts by determining the effects of put mutations on terminator read-through, and by probing wild-type and mutant put rnas with structure-specif ... | 1997 | 9368650 |
| recognition of core binding sites by bacteriophage integrases. | bacteriophage integrases promote recombination between dna molecules that carry attachment sites. they are members of a large and widely distributed family of site-specific recombinases with diverse biological roles. the integrases of phages lambda and hk022 are closely related members of this family, but neither protein efficiently recombines the attachment sites of the other phage. the nucleotides responsible for this specificity difference are located close to the points of recombinational st ... | 1998 | 9571022 |
| the spacing between binding sites controls the mode of cooperative dna-protein interactions: implications for evolution of regulatory circuitry. | the ci repressors of lambdoid phages bind cooperatively to adjacent binding sites. although these binding sites are found at complex operators containing three binding sites, cooperative binding occurs only between dimers bound at two of the sites, a mode of binding termed pairwise cooperativity. at the level of regulation, pairwise cooperativity allows the proper operation of the genetic switch. at the mechanistic level, it has been proposed to result from steric distortion of the complex, such ... | 1998 | 9571055 |
| bacteriophage hk022 nun protein: a specific transcription termination factor that excludes bacteriophage lambda. | | 2003 | 14712713 |
| escherichia coli nusg mutations that block transcription termination by coliphage hk022 nun protein. | the escherichia coli nusg gene product is required for transcription termination by phage hk022 nun protein at the lambda nutr site in vivo. we show that it is also essential for nun termination at lambda nutl. three recessive mis-sense nusg mutations have been isolated that inhibit termination by nun at lambda nutr. the mutations are ineffective in a lambda pl nutl fusion, even when lambda nutr replaces lambda nutl. the mutant strains support lambda growth, indicating that lambda n antiterminat ... | 1999 | 10209750 |
| site-specific recombination in mammalian cells expressing the int recombinase of bacteriophage hk022. | the int gene of bacteriophage hk022, coding for the integrase protein, was cloned in a mammalian expression vector downstream of the human cytomegalovirus (cmv) promoter. green monkey kidney cells (cos-1) and mouse embryo fibroblast cells (nih3t3) transiently transfected with the recombinant plasmid express the integrase protein. co-transfection of this plasmid with reporter plasmids for site-specific recombination and pcr analyses show that the integrase promotes site-specific integration as we ... | 1999 | 10532317 |
| binding of transcription termination protein nun to nascent rna and template dna. | the amino-terminal arginine-rich motif of coliphage hk022 nun binds phage lambda nascent transcript, whereas the carboxyl-terminal domain interacts with rna polymerase (rnap) and blocks transcription elongation. rna binding is inhibited by zinc (zn2+) and stimulated by escherichia coli nusa. to study these interactions, the nun carboxyl terminus was extended by a cysteine residue conjugated to a photochemical cross-linker. the carboxyl terminus contacted nusa and made zn2+-dependent intramolecul ... | 1999 | 10600743 |
| constitutive expression of a transcription termination factor by a repressed prophage: promoters for transcribing the phage hk022 nun gene. | lysogens of phage hk022 are resistant to infection by phage lambda. lambda resistance is caused by the action of the hk022 nun protein, which prematurely terminates early lambda transcripts. we report here that transcription of the nun gene initiates at a constitutive prophage promoter, p(nun), located just upstream of the protein coding sequence. the 5' end of the transcript was determined by primer extension analysis of rna isolated from hk022 lysogens or rna made in vitro by transcribing a te ... | 2000 | 10629193 |
| family values in the age of genomics: comparative analyses of temperate bacteriophage hk022. | hk022 is a temperate coliphage related to phage lambda. its chromosome has been completely sequenced, and several aspects of its life cycle have been intensively studied. in the overall arrangement, expression, and function of most of its genes, hk022 broadly resembles lambda and other members of the lambda family. upon closer view, significant differences emerge. the differences reveal alternative strategies used by related phages to cope with similar problems and illuminate previously unknown ... | 1999 | 10690418 |
| the carboxyl terminus of phage hk022 nun includes a novel zinc-binding motif and a tryptophan required for transcription termination. | the amino-terminal arginine-rich motif of the phage hk022 nun protein binds phage lambda nascent mrna transcripts while the carboxy-terminal domain binds rna polymerase and arrests transcription. the role of specific residues in the carboxy-terminal domain in transcription termination were investigated by mutagenesis, in vitro and in vivo functional assays, and nmr spectroscopy. coordination of zinc to three histidine residues in the carboxy-terminus inhibited rna binding by the amino-terminal d ... | 2000 | 10733532 |
| specificity determinants for bacteriophage hong kong 022 integrase: analysis of mutants with relaxed core-binding specificities. | the integrase (int) proteins encoded by bacteriophages hk022 and lambda catalyse similar site-specific integration and excision reactions between specific dna regions known as attachment (att) sites. however, the int proteins of hk022 and lambda are unable to catalyse recombination between non-cognate att sites. the att sites of both phages contain weak binding sites for int, known as 'core-type' sites. negatively acting nucleotide determinants associated with specific core sites (lambda b', hk0 ... | 2000 | 10792728 |
| protection of antiterminator rna by the transcript elongation complex. | nascent transcripts encoded by the putl and putr sites of phage hk022 bind the transcript elongation complex and suppress termination at downstream transcription terminators. we report here that the chemical stability of putl rna is considerably greater than that of the typical escherichia coli message because the elongation complex protects this rna from degradation. when binding to the elongation complex was prevented by mutation of either putl or rna polymerase, rna stability decreased more t ... | 2007 | 17238921 |
| a comparative study of the immunity region of lambdoid phages including shiga-toxin-converting phages: molecular basis for cross immunity. | comparison of eight lambdoid phages, including three shiga-toxin converting phages, has been carried out with respect to the immunity region, especially the recognition helices of their repressor and cro proteins on the one hand, and operator sequences on the other. some as yet unassigned components of the regulatory circuits have been inferred by computer search. the cross immunity phenomenon shown by phages vt2-sa and lambda is explained on the basis of similarity in their sequences. in additi ... | 2000 | 11245215 |
| the structure of the coliphage hk022 nun protein-lambda-phage boxb rna complex. implications for the mechanism of transcription termination. | nun protein from coliphage hk022 binds to phage boxb rna and functions, in contrast to phage lambda n protein, as a transcriptional terminator. the basic nun-(10-44) peptide contains the boxb rna binding arginine rich motif, arm. the peptide binds boxb rna and competes with the phage lambda arm peptide n-(1-36) as indicated by nuclear magnetic resonance (nmr) spectroscopy titrations. in two-dimensional nuclear overhauser enhancement spectroscopy experiments boxb rna in complex with nun-(20-44) e ... | 2001 | 11356847 |
| modification of the properties of elongating rna polymerase by persistent association with nascent antiterminator rna. | nascent rna encoded by putl, a cis-acting antitermination site of bacteriophage hk022, increases readthrough of terminators by directly modifying the transcript elongation complex. to characterize the interaction between the antiterminator rna and rna polymerase, we stalled the elongation complex downstream of putl and determined the sensitivity of the transcript to ribonuclease cleavage. part of putl rna was protected from cleavage by wild-type polymerase, but not by a mutant with a defect in p ... | 2001 | 11389846 |
| gamma integrase complementation at the level of dna binding and complex formation. | site-specific recombinases of the gamma int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type dna sites. each protomer may cleave its own site as a monomer in cis (as for cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for flp recombinase). the crystal structure of the gamma int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamm ... | 2002 | 11844768 |
| sequence-specific interaction of nascent antiterminator rna with the zinc-finger motif of escherichia coli rna polymerase. | the n-terminal zn-finger motif of the beta' subunit of rna polymerase contains two pairs of invariant cysteines flanking a moderately well-conserved segment of 13 amino acids that is rich in basic residues. previous work showed that replacement of certain zn-finger residues prevented transcription antitermination in response to phage hk022 put sites. nascent put rna binds to and modifies transcribing polymerase, so that it becomes resistant to termination. to characterize the zn finger further, ... | 2002 | 12366844 |
| specificity in dna recognition by phage integrases. | the lambda-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. analysis of phages lambda and hk022 by weisberg and collaborators previously showed that changes in five noncontiguous am ... | 2002 | 12468081 |
| determinants that target the integrase of phage hk022 into the mammalian nucleus. | the integrase (int) protein of coliphage hk022 catalyzes the site-specific integration and excision of the phage into and from its escherichia coli host chromosome. int expressed from a plasmid in cos1 monkey cells is localized in the nucleus, as is a fusion protein between int and the green fluorescent protein (gfp). mutation analysis of the gfp-int fusion has revealed in int two regions of positively charged amino acid residues that cooperate in the nuclear localization. one region harbors res ... | 2003 | 12507468 |
| phage hk022 nun protein represses translation of phage lambda n (transcription termination/translation repression). | the n-terminal arginine-rich motif of phage hk022 nun protein binds to nut sequences in phage lambda nascent transcripts and induces transcription termination. interactions between the nun c terminus and rna polymerase as well as the dna template are required for termination. we have isolated nun c-terminal point and deletion mutants that are unable to block transcription. the mutants bind nut rna and inhibit translation of the lambda n gene. thus hk022 excludes lambda both by terminating transc ... | 2003 | 12684530 |
| role of e.coli transcription-repair coupling factor mfd in nun-mediated transcription termination. | phage hk022 nun protein excludes phage lambda by binding nascent lambda-nut rna and inducing termination and transcript release. in contrast, in a purified in vitro system, nun arrests transcription on lambdadna templates without dissociation of the transcription elongation complex (tec). our evidence indicates that transcription-repair coupling factor (mfd) frees nun-arrested rna polymerase. the activity of nun is enhanced in an mfd-null mutant, consistent with prolonged association of nun with ... | 2003 | 12787667 |
| activity of coliphage hk022 excisionase (xis) in the absence of dna binding. | a mutated excisionase (xis) protein of coliphage hk022 whose single cys residue was replaced by ser does not bind to its two tandem binding sites (x1, x2) on the p arm of attr. despite its dna-binding inability the protein showed 30% excision activity of the wild type xis both in vitro and in vivo. this partial activity is attributed to the interaction of xis with integrase that is retained in the mutant protein. this protein-protein interaction occurs in the absence of dna binding. | 2003 | 12804763 |
| site-specific recombination in human cells catalyzed by the wild-type integrase protein of coliphage hk022. | the activity of the integrase (int) protein encoded by coliphage hk022 was tested in a human cell culture. plasmids were constructed as substrates that carry the sites of the integration reaction (attp and attb) or the sites of excision (attl and attr). the site-specific recombination reactions were monitored in cis and in trans configurations by the expression of the green fluorescent protein (gfp) as a reporter. cells cotransfected with the substrate plasmid(s) and with a plasmid that expresse ... | 2003 | 12910543 |
| the n-terminus is unstructured, but not dynamically disordered, in the complex between hk022 nun protein and lambda-phage boxb rna hairpin. | the nun protein of lambdoid phage hk022 excludes lambda-phage superinfection by blocking expression of genes downstream from the lambda nut sequences. heteronuclear nmr studies have been performed on a nun peptide comprising residues 1-49 bound to the nutr boxb rna. the pattern of (13)c chemical shifts indicates that the arginine-rich motif of nun forms an induced alpha-helix, consisting of residues 23-43, when bound to boxb rna, consistent with the structure of a shorter (residues 22-44) nun pe ... | 2003 | 14550553 |
| bacteriophage st64b, a genetic mosaic of genes from diverse sources isolated from salmonella enterica serovar typhimurium dt 64. | the complete sequence of the double-stranded dna (dsdna) genome of the salmonella enterica serovar typhimurium st64b bacteriophage was determined. the 40,149-bp genomic sequence of st64b has an overall g+c content of 51.3% and is distinct from that of p22. the genome architecture is similar to that of the lambdoid phages, particularly that of coliphage lambda. most of the putative tail genes showed sequence similarity to tail genes of mu, a nonlambdoid phage. in addition, it is likely that these ... | 2003 | 14563886 |
| suppression of factor-dependent transcription termination by antiterminator rna. | nascent transcripts of the phage hk022 put sites modify the transcription elongation complex so that it terminates less efficiently at intrinsic transcription terminators and accelerates through pause sites. we show here that the modification also suppresses termination in vivo at two factor-dependent terminators, one that depends on the bacterial rho protein and a second that depends on the hk022-encoded nun protein. suppression was efficient when the termination factors were present at physiol ... | 2003 | 14645267 |
| solution structure and stability of the full-length excisionase from bacteriophage hk022. | heteronuclear high-resolution nmr spectroscopy was employed to determine the solution structure of the excisionase protein (xis) from the lambda-like bacteriophage hk022 and to study its sequence-specific dna interaction. as wild-type xis was previously characterized as a generally unstable protein, a biologically active hk022 xis mutant with a single amino acid substitution cys28-->ser was used in this work. this substitution has been shown to diminish the irreversibility of xis denaturation an ... | 2003 | 14653811 |
| transcription termination by phage hk022 nun is facilitated by cooh-terminal lysine residues. | the 109-amino acid nun protein of prophage hk022 excludes superinfecting bacteriophage lambda by blocking transcription elongation on the lambda chromosome. multiple interactions between nun and the transcription elongation complex are involved in this reaction. the nun nh(2)-terminal arginine-rich motif binds boxb sequence in nascent lambda transcripts, whereas the cooh terminus binds rna polymerase and contacts dna template. nun trp(108) is required for interaction with dna and transcription a ... | 2004 | 14742436 |
| a conserved zinc binding domain in the largest subunit of dna-dependent rna polymerase modulates intrinsic transcription termination and antitermination but does not stabilize the elongation complex. | an evolutionarily conserved zinc-binding motif is found close to the amino terminus of the largest subunits of dna-dependent rna polymerases from bacteria, archaea, and eukaryotes. in bacterial rna polymerase, this motif, the zinc binding domain, has been implicated in protein-dna interactions that stabilize the transcription elongation complex and that occur downstream of the catalytic center. here, we show that this view is incorrect, and instead, the zinc binding domain interacts with product ... | 2004 | 15351641 |
| an orthologue of the cor gene is involved in the exclusion of temperate lambdoid phages. evidence that cor inactivates fhua receptor functions. | a new set of lambdoid phages (mep) classified into different immunity groups was previously described. phages mep213, mep237, and mep410 were unable to grow in mep167 lysogenic cells, presumably due to an exclusion mechanism expressed constitutively by the mep167 repressed prophage. in this work, to analyze the exclusion phenomenon, we constructed a genomic library from mep167 phage in a pproex derivative plasmid. a dna fragment containing an open reading frame for a 77 amino acid polypeptide wa ... | 2004 | 15518820 |
| protein-protein interaction between monomers of coliphage hk022 excisionase. | excisionase (xis) is an accessory protein that is required for the site-specific excision reaction of the coliphages hk022 and lambda. xis binds in a strong cooperative manner to two tandem binding sites (x1 and x2) located on the p arm of the attachment (att) sites on the phage genome. as a result of crosslinking experiments in vivo and in vitro of xis-overexpressing cells, by gel filtration of purified xis and by fret analyses we show that xis monomers of hk022 interact and form dimers that ar ... | 2004 | 15527755 |
| site-specific recombination in arabidopsis plants promoted by the integrase protein of coliphage hk022. | the gene encoding the wild type integrase protein of coliphage hk022 was integrated chromosomally and expressed in arabidopsis thaliana plants. double-transgenic plants cloned with the int gene as well as with a t-dna fragment carrying the proper att sites in a tandem orientation showed that int catalyzed a site-specific integration reaction (attp x attb) as well as a site-specific excision reaction (attl x attr). the reactions took place without the need to provide any of the accessory proteins ... | 2005 | 15830132 |
| role of an rnase iii binding site in transcription termination at lambda nutl by hk022 nun protein. | the phage hk022 nun protein excludes phage lambda by binding nascent lambda pl and pr transcripts at nutl and nutr, respectively, and inducing transcription termination just downstream of these sites. termination is more efficient at nutl than at nutr. one difference between nutl and nutr is the presence of rnase iii processing sites (riii) located immediately promoter distal to lambda nutl. we found that deletion of riii dramatically reduced nun transcription arrest in vitro but had little effe ... | 2006 | 16980485 |
| genomic sequences of bacteriophages hk97 and hk022: pervasive genetic mosaicism in the lambdoid bacteriophages. | we report the complete genome dna sequences of hk97 (39,732 bp) and hk022 (40,751 bp), double-stranded dna bacteriophages of escherichia coli and members of the lambdoid or lambda-like group of phages. we provide a comparative analysis of these sequences with each other and with two previously determined lambdoid family genome sequences, those of e. coli phage lambda and salmonella typhimurium phage p22. the comparisons confirm that these phages are genetic mosaics, with mosaic segments separate ... | 2000 | 10860721 |
| sequence of the genome of the temperate, serotype-converting, pseudomonas aeruginosa bacteriophage d3. | temperate bacteriophage d3, a member of the virus family siphoviridae, is responsible for serotype conversion in its host, pseudomonas aeruginosa. the complete sequence of the double-stranded dna genome has been determined. the 56,426 bp contains 90 putative open reading frames (orfs) and four genes specifying trnas. the latter are specific for methionine (aug), glycine (gga), asparagine (aac), and threonine (aca). the trnas may function in the translation of certain highly expressed proteins fr ... | 2000 | 11029426 |
| phosphorylation of the integrase protein of coliphage hk022. | the integrase (int) proteins of coliphages hk022 and lambda, are phosphorylated in one or more of their tyrosine residues. in int of hk022 the phosphorylated residue(s) belong to its core-binding/catalytic domains. wzc, a protein tyrosine kinase of escherichia coli, is not required for int phosphorylation in vivo, however, it can transphosphorylate the conserved tyr(342) catalytic residue of int in vitro. int purified from cells that overexpress wzc has a reduced activity in vitro. in vivo, the ... | 2008 | 18353423 |
| the y39a mutation of hk022 nun disrupts a boxb interaction but preserves termination activity. | coliphage hk022 nun protein targets phage lambda nut boxb rna and acts as a transcriptional terminator, counteracting the phage lambda n protein, a suppressor of transcription termination. both nun and n protein interact directly with rna polymerase, and nun competes with n protein for boxb binding and prevents superinfection of escherichia coli hk022 lysogens by lambda. interaction of trp18 of lambda n and a7 of boxb rna in the n- boxb complex is essential for efficient antitermination. we foun ... | 2008 | 18563916 |
| overexpression of phage hk022 nun protein is toxic for escherichia coli. | the nun protein of coliphage hk022 excludes superinfecting lambda phage. nun recognizes and binds to the n utilization (nut) sites on phage lambda nascent rna and induces transcription termination. overexpression of nun from a high-copy plasmid is toxic for escherichia coli, despite the fact that nut sites are not encoded in the e. coli genome. cells expressing nun cannot exit stationary phase. toxicity is related to transcription termination, since host and nun mutations that block termination ... | 2008 | 18571198 |
| inhibition of a transcriptional pause by rna anchoring to rna polymerase. | we describe a mechanism by which nascent rna inhibits transcriptional pausing. putl rna of bacteriophage hk022 suppresses transcription termination at downstream terminators and pausing within a nearby u-rich sequence. in vitro transcription and footprinting assays reveal that this pausing results from backtracking of rna polymerase and that binding of nascent putl rna to polymerase limits backtracking by restricting re-entry of the transcript into the rna exit channel. the restriction is local ... | 2008 | 18775328 |
| molecular analysis of recombinase-mediated cassette exchange reactions catalyzed by integrase of coliphage hk022. | the integrase (int) protein of coliphage hk022 can catalyze in escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. atomic force microscopy images have revealed that in the protein-dna complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. this observation, together with the elucidation of intermediate co-integrates between the two circular plasmids ... | 2008 | 18848986 |
| optimization of coliphage hk022 integrase activity in human cells. | the integrase (int) site-specific recombinase of coliphage hk022 catalyzes integrative and excisive dna recombination between two attachment (att) sites in human cells without the need to supply the accessory proteins integration host factor (ihf) and excisionase (xis). previous work has shown that under these conditions, reactions in cis, i.e. both att sites are located on the same chromosome, can be detected without selection. however, recombination in trans, i.e. one att site positioned on a ... | 2009 | 19268511 |
| site-specific recombination in the cyanobacterium anabaena sp. strain pcc 7120 catalyzed by the integrase of coliphage hk022. | the integrase (int) of the lambda-like coliphage hk022 catalyzes the site-specific integration and excision of the phage dna into and from the chromosome of its host, escherichia coli. int recognizes two different pairs of recombining sites attp x attb and attl x attr for integration and excision, respectively. this system was adapted to the cyanobacterium anabaena sp. strain pcc 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. two plasmids were consecutively ... | 2009 | 19429625 |
| two structurally independent domains of e. coli nusg create regulatory plasticity via distinct interactions with rna polymerase and regulators. | nusg is a conserved regulatory protein that interacts with elongation complexes (ecs) of rna polymerase, dna, and rna to modulate transcription in multiple and sometimes opposite ways. in escherichia coli, nusg suppresses pausing and increases elongation rate, enhances termination by e. coli rho and phage hk022 nun protein, and promotes antitermination by lambdan and in ribosomal rna operons. we report nmr studies that suggest that e. coli nusg consists of two largely independent n- and c-termin ... | 2009 | 19500594 |
| high efficiency of a sequential recombinase-mediated cassette exchange reaction in escherichia coli. | a comparison between the efficiency of recombinase-mediated cassette exchange (rmce) reactions catalyzed in escherichia coli by the site-specific recombinases flp of yeast and int of coliphage hk022 has revealed that an flp-catalyzed rmce reaction is more efficient than an int-hk022 catalyzed reaction. in contrast, an rmce reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. however, the same reaction catalyzed by each reco ... | 2010 | 20924197 |
| arm site independence of coliphage hk022 integrase in human cells. | the integrase encoded by the lambdoid phage hk022 (int-hk022) resembles its coliphage ? counterpart (int-?) in the roles of the cognate dna arm binding sites and in controlling the direction of the reaction. we show here that within mammalian cells, int-hk022 does not exhibit such a control. rather, int-hk022 recombined between all ten possible pairwise att site combinations, including attb × attb that was more effective than the conventional integrative attp × attb reaction. we further show tha ... | 2011 | 21442327 |
| newly discovered antiterminator rnas in bacteriophage. | antiterminator rna directly modifies the transcription elongation complex so that it terminates less efficiently at intrinsic and factor-dependent terminators. these unusual rnas were first discovered in bacteriophage hk022 where the nascent transcripts of the phage put sites promote full expression of phage genes during lytic infection. the activity of antiterminator rna depends on specific structural elements that form as the transcript exits rna polymerase. to further our understanding of the ... | 2011 | 21840976 |
| role of e.coli nusa in phage hk022 nun-mediated transcription termination. | the 109 amino acid residue nun protein expressed from prophage hk022 excludes superinfecting phage lambda by arresting transcription on the lambda chromosome near the lambdanut sites. in vitro, the nun n terminus binds to nascent lambdanutrna, whereas the c terminus interacts with rna polymerase and dna template. escherichia coli host factors, nusa, nusb, nuse (s10), and nusg, stimulate nun-arrest. nusa binds the nun c terminus and enhances formation of the nun-nutrna complex. because of these i ... | 2006 | 16631197 |
| human genomic site-specific recombination catalyzed by coliphage hk022 integrase. | it has been previously demonstrated that the wild type integrase (int) protein of coliphage hk022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. in the present report it is shown that int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. these include integrative (attp x attb) as well as excisive (attl x attr) reactions each in two configurations. in the c ... | 2008 | 18282626 |