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M Feiss

Publications and source records attributed to M Feiss.

At least 19 recordsLinked to original sources
PubMed1992

Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda.

cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli. cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1. A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1. A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation. lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF. Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB. The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site. In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF. A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host. The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.

Bacteriophage lambda↗
PubMed1992

Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site.

Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules. Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged. Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity. cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein. In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations. One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1. Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4. The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1. The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site. The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix. To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered. The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.

Amino Acid Sequence↗
PubMed1992

Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase.

Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging. lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding. Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding. Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.

Bacteriophage lambda↗
PubMed1991

Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase.

A collection of mutations affecting the site (cosN) at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces nicks to generate mature lambda chromosomes has been studied. A good correlation was found for mutational effects on burst size, accumulation of unused proheads, packaging of DNA into heads and cos cutting by terminase in vitro, indicating that defective cosN cleavage by terminase is the molecular explanation for the phenotypic effects of the mutations. Although the base-pairs of cosN display partial twofold rotational symmetry, cosN was found to be asymmetric functionally. Certain mutations to the left side of the center of rotational symmetry have more pronounced phenotypic effects than rotationally symmetric mutations to the right. The cosN11G mutation has no phenotypic effects when present as a single mutation, but does affect DNA packaging and cosN cutting in the presence of the symmetrically disposed cosN2C mutation. Mutations that decrease cosN cleavage result in the accumulation of unexpanded proheads, indicating that prohead expansion depends on cosN cutting.

Bacteriophage lambda↗
PubMed1991

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.

Bacteriophage lambda↗
PubMed1991

Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to the 3' end of the 16S rRNA from GGA to GGAG. The sti30 mutation causes a approximately 50-fold increase in the level of expression of a Nul-lacZ reporter gene, indicating that the sti30 mutation overcomes the gp1 inhibition by increasing the level of expression of gpNul. Although the Nul and A genes of lambda overlap, the sti30 mutation has little effect on the level of gpA expression, indicating that translational coupling does not occur.

Bacteriophage lambda↗
PubMed1990

Structure of the bacteriophage lambda cohesive end site: bent DNA on both sides of the site, cosN, at which terminase introduces nicks during chromosome maturation.

Packaging of lambda DNA is mediated by the phage-encoded enzyme, terminase, which acts at a site termed cos. cos consists of cosB, the site where terminase binds lambda DNA, and cosN, the site where nicks are introduced to generate the cohesive ends of virion DNA. cos contains multiple binding sites for gpNu1, the small subunit of terminase, and integration host factor (IHF), an Escherichia coli DNA binding protein. Polyacrylamide gel electrophoresis of circularly permuted segments of cos DNA has been used to locate major bend loci in cos. Two major bends have been located; one bend is ca 146 bp to the left of cosN while the second major bend is located ca 92 bp to the right of cosN. The major bend at 92 coincides roughly with I1, the strongest IHF binding site in cos. The possible roles of static bending in DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1988

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21.

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.

Bacterial Proteins↗
PubMed1988

Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor.

Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells. In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders lambda dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda. Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.

Bacterial Proteins↗
PubMed1988

The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor.

Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda. The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends. Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging. Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF). The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging. A -7 deletion mutation is lethal. Models for the basis of these mutational effects are discussed.

Bacterial Proteins↗
PubMed1988

Partial FI gene-independence of lambda-21 hybrid phages specifying chimeric terminases.

The role of the FI gene in the life cycles of a series of lambda-21 hybrid phages that produce chimeric lambda-21 terminases has been examined. An isogenic series of FI+ and FI- derivatives of the hybrids was constructed, and the growth properties of the phages were examined. It was found that three of the four hybrids (hybrids 51, 67, and 33) are able to form plaques and produce a small burst in the absence of the FI gene product (gpFI), but each of the three phages is much healthier in the presence of gpFI. It is concluded that each of the three chimeric terminases is dependent on gpFI. The ability of the FI- hybrids to grow better than lambda FI- is postulated to be due to a minor qualitative or quantitative difference between the chimeric terminases and lambda terminase. The fourth hybrid (54), known from earlier work to produce an infirm terminase, is more dependent on gpFI than the other hybrids and lambda itself.

Bacteriophage lambda↗
PubMed1988

Domains for protein-protein interactions at the N and C termini of the large subunit of bacteriophage lambda terminase.

The large subunit of phage lambda terminase, gpA, the gene product of the phage A gene, interacts with the small subunit, gpNul, to form functional terminase. Terminase binds to lambda DNA at cosB to form a binary complex. The terminase:DNA complex binds a prohead to form a ternary complex. Ternary complex formation involves an interaction of the prohead with gpA. The amino terminus of gpA contains a functional domain for interaction with gpNul, and the carboxy-terminal 38 amino acids of gpA contain a functional domain for prohead binding. This information about the structure of gpA was obtained through the use of hybrid phages resulting from recombination between lambda and the related phage 21. lambda and 21 encode terminases that are analogous in structural organization and have ca. 60% sequence identity. In spite of these similarities, lambda and 21 terminases differ in specificity for DNA binding, subunit assembly, and prohead binding. A lambda-21 hybrid phage produces a terminase in which one of the subunits is chimeric and had recombinant specificities. In the work reported here; a new hybrid, lambda-21 hybrid 67, is characterized. lambda-21 hybrid 67 is the result of a crossover between lambda and 21 in the large subunit genes, such that the DNA from the left chromosome end is from 21, including cosB phi 21, the 1 gene, and the first 48 codons for the 2 gene. The rest of the hybrid 67 chromosome is lambda DNA, including 593 codons of the A gene. The chimeric gp2/A of hybrid 67 binds gp1 to form functional terminase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes.

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at cosN. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal cosN, rather than any subsequent cosB, during packaging. In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of lambda chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiate packaging of a downstream chromosome, but can also scan and terminate packaging of the downstream chromosome. A model is presented in which processive action of terminase is the basis for sequential packaging of lambda chromosomes.

Bacteriophage lambda↗
PubMed1985

The terminase of bacteriophage lambda. Functional domains for cosB binding and multimer assembly.

Terminase is a protein complex involved in lambda DNA packaging. The subunits of terminase, gpNul and gpA, are the products of genes Nul and A. The actions of terminase include DNA binding, prohead binding and DNA nicking. Phage 21 is a lambdoid phage that also makes a terminase, encoded by genes 1 and 2. The terminases of 21 and lambda are not interchangeable. This specificity involves two actions of terminase; DNA binding and prohead binding. In addition, the subunits of lambda terminase will not form functional multimers with the subunits of 21 terminase. lambda-21 hybrid phages can be produced as a result of recombination. We describe here lambda-21 hybrid phages that have hybrid terminase genes. The packaging specificities of the hybrids and the structure of their genes were compared in order to identify functional domains of terminase. The packaging specificities were determined in vivo by complementation tests and helper packaging experiments. Restriction enzyme site mapping and sequencing located the sites at which recombination occurred to produce the hybrid phages. lambda-21 hybrid 51 carries the lambda A gene, and a hybrid 1/Nul gene. The crossover that produced this phage occurred near the middle of the 1 and Nul genes. The amino-terminal portion of the hybrid protein is homologous to gp1 and the carboxy-terminal portion is homologous to gpNul. It binds to 21 DNA and forms functional multimers with gpA, providing evidence that the amino-terminal portion of gpNul is involved in DNA binding and the carboxy-terminal portion of gpNul is involved in the interaction with gpA. lambda-21 hybrid 54 has a hybrid 2/A gene. The amino terminus of the hybrid protein of lambda-21 hybrid 54 is homologous with gp2. This protein forms functional multimers only with gp1, providing evidence that the amino terminus of gpA is involved in the interaction with gpNul. These studies identify three functional domains of terminase.

Bacteriophage lambda↗
PubMed1985

Essential interaction between lambdoid phage 21 terminase and the Escherichia coli integrative host factor.

Lambdoid phage 21 requires the Escherichia coli integrative host factor (IHF) for growth. lambda-21 hybrids that have 21 DNA packaging specificity also require IHF. IHF-independent (her) mutants have been isolated. her mutations map in the amino-terminal half of the 21 1 gene. The 1 gene encodes the small subunit of the 21 terminase, and the amino-terminal half of the 1 polypeptide is a functional domain for specifically binding 21 DNA. Hence changes in the DNA-binding domain of terminase, her mutations, render 21 terminase able to function in the absence of IHF. Three of four her mutations studied are trans-dominant. An in vitro system was used to show that packaging of 21 DNA is IHF-dependent. IHF is directly required during the early, terminase-dependent steps of assembly. It is concluded that IHF is a host factor required for function of the 21 terminase. It is proposed, in analogy to the role of IHF in lambda integration, that IHF facilitates proper binding of 21 terminase to phage DNA. Consistent with this proposal, possible IHF-binding sites are present in the 21 cohesive end site.

Bacterial Proteins↗
PubMed1984

A functional domain of bacteriophage lambda terminase for prohead binding.

Terminase is a multifunctional protein complex involved in DNA packaging during bacteriophage lambda assembly. Terminase is made of gpNul and gpA, the products of the phage lambda Nu1 and A genes. Early during DNA packaging terminase binds to lambda DNA to form a complex called complex I. Terminase is required for the binding of proheads by complex I to form a DNA: terminase: prohead complex known as complex II. Terminase remains associated with the DNA during encapsidation. The other known role for terminase in packaging is the production of staggered nicks in the DNA thereby generating the cohesive ends. Lambdoid phage 21 has cohesive ends identical to those of lambda. The head genes of lambda and 21 show partial sequence homology and are analogous in structure, function and position. The terminases of lambda and 21 are not interchangeable. At least two actions of terminase are involved in this specificity: (1) DNA binding; (2) prohead binding. The 1 and 2 genes at the left end of the 21 chromosome were identified as coding for the 21 terminase. gp1 and gp2 are analogous to gpNu1 and gpA, respectively. We have isolated a phage, lambda-21 hybrid 33, which is the product of a crossover between lambda and 21 within the terminase genes. Lambda-21 hybrid 33 DNA and terminase have phage 21 packaging specificity, as determined by complementation and helper packaging studies. The terminase of lambda-21 hybrid 33 requires lambda proheads for packaging. We have determined the position at which the crossover between lambda DNA and 21 DNA occurred to produce the hybrid phage. Lambda-21 hybrid 33 carries the phage 21 1 gene and a hybrid phage 2/A gene. Sequencing of lambda-21 hybrid 33 DNA shows that it encodes a protein that is homologous at the carboxy terminus with the 38 amino acids of the carboxy terminus of lambda gpA; the remainder of the protein is homologous to gp2. The results of these studies define a specificity domain for prohead binding at the carboxy terminus of gpA.

Bacteriophage lambda↗