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R A Weisberg

Publications and source records attributed to R A Weisberg.

At least 19 recordsLinked to original sources
PubMed1992

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-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N.

Bacteriophage lambda↗
PubMed1991

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 sites. One exception is nun, the first gene of the HK022 pL operon, which is expressed in the presence of prophage repressor and thus differs from its lambda counterpart, gene N. We show that transcription of nun in a lysogen does not initiate at pL but instead starts upstream at the pM promoter. This difference in transcription fits the different roles of Nun and N proteins in the physiology of the two phages: Nun protects HK022 lysogens against superinfection with certain other lambdoid phages, while N promotes the transcription of early lambda genes.

Bacteriophage lambda↗
PubMed1990

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 the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

Bacteriophage lambda↗
PubMed1990

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 such chimeras recombined with appropriate partner sites in vivo and in vitro, and their differential responses to the Int proteins of the two phages allowed us to locate determinants of the specificity difference in the bacterial attachment sites and a central segment of the phage attachment sites. The location of these determinants encompasses three of the four core-type binding sites for lambda Int: C, B, and most importantly, B'. The regions corresponding to the C' core binding site and the arm-type binding sites of lambda Int play no role in the specificity difference and, indeed, are well conserved in the two phages. We found, unexpectedly, that the effect of replacement of an Int-binding region on the recombinational potency of one chimeric site was reversed by a change of partner. This novel context effect suggests that postsynaptic interactions affect the specificity of recognition of attachment sites by Int.

Bacteriophage lambda↗
PubMed1989

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 lambda nut sites and the lambda pL promoter and is followed by several transcription termination signals. Despite these similarities, Nun is required neither for the lytic nor the lysogenic pathway of phage development. Again, unlike N, Nun is expressed in a prophage, perhaps from a promoter other than pL. We suggest that Nun and N have diverged in evolution and now perform different functions for their respective phages. Although Nun and N compete at the lambda nut sites and interact with the same host Nus proteins, they are only distantly related in predicted amino acid sequence. The presence of transcription terminators in the pL operon suggests that the expression of the HK022 early functions, like those of lambda, entails an antitermination mechanism. However, Nun does not appear to be an essential component of this mechanism. Our most economic model is that the HK022 nutL sequence suppresses pL operon terminators in the absence of a phage-encoded antitermination protein. Striking homologies between the HK022 nutL sequence and related sequences in the Escherichia coli rrn operons support this notion. Alternatively, a phage antitermination gene may be located outside the pL operon.

Bacteriophages↗
PubMed1989

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 involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

Amino Acid Sequence↗
PubMed1989

Mutations of the phage lambda attachment site alter the directionality of resolution of Holliday structures.

Integrative recombination of bacteriophage lambda occurs by two sequential, reciprocal strand exchanges at specific positions within the attachment sites. Both exchanges are promoted by the lambda Int protein; the first forms a Holliday structure, and the second resolves it to recombinant products. Recombination requires sequence homology within the 7 bp 'overlap' region that separates the two points of strand exchange. To see if homology promotes the second strand exchange, we constructed attachment site Holliday structures by annealing DNA strands and then assayed Int-promoted resolution. Holliday structures corresponding to strand exchange between sites with homologous overlap regions were efficiently resolved to give mixtures of recombinants and parents. Holliday structures corresponding to exchanges between heterologous sites fell into two classes. Members of the first class, in which heterology limited but did not completely prevent migration of the branchpoint within the overlap region, were resolved efficiently and preferentially to parental molecules. We propose that resolution to recombinants occurs only if homology allows branch migration from the first to the second exchange site. Members of the second class, in which heterology constrained the branchpoint within an Int binding site, were resolved poorly. We suggest that Holliday structures that have a branchpoint within an Int binding site are poor substrates for Int.

Attachment Sites, Microbiological↗
PubMed1989

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Attachment Sites, Microbiological↗
PubMed1987

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 remarkable specificity; it converts the lambda nut sequences, which normally interact with lambda N protein to suppress transcription termination, into terminators. These and other similarities between Nun-promoted termination and N-promoted antitermination argue strongly that the mechanisms of the two reactions have steps in common.

Bacteriophage lambda↗
PubMed1987

Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA.

Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates. Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches. Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them. Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome. The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration. The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA. Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.

Attachment Sites, Microbiological↗
PubMed1987

Overproduction of Escherichia coli integration host factor, a protein with nonidentical subunits.

Integration host factor (IHF) is a small, basic protein that is needed for efficient recombination of bacteriophage lambda, as well as for other host and viral functions. We have constructed strains in which the two subunits of IHF, encoded by the himA and hip genes of Escherichia coli, are expressed under the control of the lambda rho L promoter. Separate overexpression of himA and hip led to the production of unstable and insoluble peptides, respectively. In contrast, the overexpression of both genes conjointly led to the accumulation of large amounts of active IHF. Extracts of such cells provided the starting material for a rapid purification procedure that results in milligram quantities of apparently homogeneous IHF.

Bacterial Proteins↗
PubMed1986

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.

Amino Acid Sequence↗
PubMed1986

Bacteriophage lambda int protein may recognize structural features of the attachment sites.

The bacteriophage lambda int protein binds to and promotes polynucleotide strand exchange within specific DNA segments called attachment sites. Previous work strongly suggests that the specificity of int protein action is based, at least in part, on its ability to recognize nucleotide sequences in the attachment sites. We suggest that int protein also recognizes structural features of the attachment sites such as the twist and roll angles between adjacent base pairs. This proposal is based on statistical analysis of the predicted twist and roll angles of a large collection of secondary attachment sites. The analysis shows that the oscillation patterns of these parameters are conserved in regions where int proteins binds.

Attachment Sites, Microbiological↗
PubMed1985

Primary structure of the hip gene of Escherichia coli and of its product, the beta subunit of integration host factor.

We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the beta subunit of integration host factor (IHF beta). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip+ and which hip- by complementation, and then sequenced the ends of the critical deletions. The 5' end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3' end of the neighboring gene, rpsA, and we constructed an IHF beta overproducer by fusing the coding sequences to the lambda pL promoter. A survey of known protein sequences revealed a close relationship between IHF beta and the type II prokaryotic DNA binding proteins (the "histone-like" proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHF alpha. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.

Amino Acid Sequence↗
PubMed1985

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda.

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.

Bacterial Proteins↗
PubMed1984

An integration-proficient int mutant of bacteriophage lambda.

We have isolated and characterized a novel int mutant of phage lambda. This mutant promotes efficient recombination between the phage and bacterial attachment sites, but, unlike wild type, does not promote efficient recombination of any other pair of attachment sites tested in most conditions. In particular, recombination between two phage or two prophage attachment sites is poor relative to the wild type frequency. We attribute this unusual phenotype to differences in the distribution of int protein binding sites among different attachment sites (Ross and Landy 1982, 1983). We suggest that int protein molecules bound to one of two recombining DNAs interact with empty sites or with bound proteins on the other, and that the mutant protein acts efficiently only if the distribution of protein binding sites within the two attachment sites is that of the attP-attB pair. Similar discrimination among attachment site pairs by wild type int protein may also modulate recombination frequencies.

Bacteriophage lambda↗
PubMed1983

Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda.

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.

Attachment Sites, Microbiological↗