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PubMed2004

Role of the RNA polymerase alpha subunits in CII-dependent activation of the bacteriophage lambda pE promoter: identification of important residues and positioning of the alpha C-terminal domains.

The bacteriophage lambda CII protein stimulates the activity of three phage promoters, p(E), p(I) and p(aQ), upon binding to a site overlapping the -35 element at each promoter. Here we used preparations of RNA polymerase carrying a DNA cleavage reagent attached to specific residues in the C-terminal domain of the RNA polymerase alpha subunit (alphaCTD) to demonstrate that one alphaCTD binds near position -41 at p(E), whilst the other alphaCTD binds further upstream. The alphaCTD bound near position -41 is oriented such that its 261 determinant is in close proximity to sigma(70). The location of alphaCTD in CII-dependent complexes at the p(E) promoter is very similar to that found at many activator-independent promoters, and represents an alternative configuration for alphaCTD at promoters where activators bind sites overlapping the -35 region. We also used an in vivo alanine scan analysis to show that the DNA-binding determinant of alphaCTD is involved in stimulation of the p(E) promoter by CII, and this was confirmed by in vitro transcription assays. We also show that whereas the K271E substitution in alphaCTD results in a drastic decrease in CII-dependent activation of p(E), the p(I) and p(aQ) promoters are less sensitive to this substitution, suggesting that the role of alphaCTD at the three lysogenic promoters may be different.

Alanine↗
PubMed1994

Self-assembly of bacteriophage lambda cI repressor: effects of single-site mutations on the monomer-dimer equilibrium.

Dimerization of lambda cI repressor monomers is required for high-affinity binding to bacteriophage lambda operator DNA and is known to involve protein-protein contacts between C-terminal domains of the repressor monomers. In order to address the importance of the C-terminal domain in mediating the oligomeric properties of dimerization and cooperative binding to operator DNA, eight single-site mutant repressors were screened for possible deficiencies in cooperative interactions; all but one of the amino acid substitutions are located within the C-terminal domain. As a prelude to binding studies and the complete characterization of cooperativity mutants of lambda cI repressor (Burz, D. S., & Ackers, G. K. (1994) Biochemistry 33, 8406-8416), the thermodynamics of self-assembly of seven of these mutants was examined from 10(-11) to 10(-5) M total repressor using analytical gel chromatography. Results show that the structural perturbation accompanying single amino acid replacement does not significantly affect the monomer-dimer equilibrium with the exception of that accompanying replacements of serine 228; mutations at that site weaken, by 2-4 kcal/mol, the protein-protein interactions responsible for self-association. An additional mutant repressor, Pro158-->Thr, was also examined and found to associate reversibly from monomers to a species with stoichiometry greater than 2. All mutations increase the apparent Stokes radius of the monomeric form by 2-4.5 A and that of dimers by 1 or 3 A.

Bacteriophage lambda↗
PubMed1990

Transcription of a bacteriophage lambda DNA site blocks growth of Escherichia coli.

The rap mutation in Escherichia coli prevents the growth of bacteriophage lambda. Phage mutations that overcome rap inhibition (bar) have been mapped to loci in the pL operon. We cloned and sequenced three mutations in two of these loci: barIa to the left arm of the lambda attachment site (attP) and barII in the ssb (ea10) gene. The mutations represent single base-pair changes within nearly identical 16-base-pair DNA segments. Each mutation disrupts a sequence of dyad symmetry within the segment. Plasmids carrying a bar+ sequence downstream to an active promoter are lethal to rap, but not rap+, bacteria. The bar sequences isolated from the lambda bar mutants are not lethal. We synthesized a minimal lambda barIa+ sequence, 5'-TATATTGATATTTATATCATT, and cloned it downstream to an inducible promoter. When transcribed, this sequence is sufficient to kill a rap strain.

Alleles↗
PubMed2003

The product of the bacteriophage lambda W gene: purification and properties.

Gene W is one of the 10 genes that control the morphogenesis of the bacteriophage lambda head. The morpho genesis of the phage lambda head proceeds through the synthesis of an intermediate assembly called the prohead. This is an empty shell into which the bacteriophage DNA is introduced--packaged--by the phage enzyme DNA terminase. The product of W (gpW) acts after DNA packaging, but before the addition of another phage product, gene product FII, and before the addition of tails. The role of gpW is unknown. The structure of N- and C-tagged gpW has been previously determined by nuclear magnetic resonance (NMR) spectroscopy. Here we report some of the properties of the native protein. The purification of gpW to homogeneity, overproduced by a plasmid derivative, is described. To obtain large amounts of the protein, the ribosome-binding site had to be modified, showing that inefficient translation of the message is the main mechanism limiting W gene expression. The molecular weight of the protein is in close agreement to the value predicted from the DNA sequence of the gene, which suggests that it is not post-transcriptionally modified. It behaves as a monomer in solution. Radioactively labeled gpW is incorporated into phage particles in in vitro complementation, showing that gpW is a structural protein. The stage at which gpW functions and other circumstantial evidence support the idea that six molecules of gpW polymerize on the connector before the incorporation of six molecules of gpFII and before the tail attaches.

Bacteriophage lambda↗
PubMed1981

In vivo and in vitro functional alterations of the bacteriophage lambda receptor in lamB missense mutants of Escherichia coli K-12.

lamB is the structural gene for the bacteriophage lambda receptor in Escherichia coli K-12. In vivo and in vitro studies of the lambda receptor from lamB missence mutants selected as resistant to phage lambda h+ showed the following. (i) Resistance was not due to a change in the amount of lambda receptor protein present in the outer membrane but rather to a change in activity. All of the mutants were still sensitive to phage lambda hh*, a two-step host range mutant of phage lambda h+. Some (10/16) were still sensitive to phage lambda h, a one-step host range mutant. (ii) Resistance occurred either by a loss of binding ability or by a block in a later irreversible step. Among the 16 mutations, 14 affected binding of lambda h+. Two (lamB106 and lamB110) affected inactivation but not binding; they represented the first genetic evidence for a role of the lambda receptor in more than one step of phage inactivation. Similarly, among the six mutations yielding resistance to lambda h, five affected binding and one (lamB109) did not. (iii) The pattern of interactions between the mutated receptors and lambda h+ and its host range mutants were very similar, although not identical, in vivo and in vitro. Defects were usually more visible in vitro than in vivo, the only exception being lamB109. (iv) The ability to use dextrins as a carbon source was not appreciably affected in the mutants. Possible working models and the relations between phage infection and dextrins transport were briefly discussed.

Adsorption↗
PubMed1986

Ion etching of bacteriophage lambda: evidence that the right end of the DNA is located at the outside of the phage DNA mass.

Bacteriophage lambda was etched in an Ar+ plasma under conditions in which the capsid and some of the DNA were eroded (by sputtering) from the particle surface. Analysis of the DNA remaining in etched phage demonstrated an enrichment in sequences derived from the left end and middle of the genome; sequences from the right end were selectively lost. The results suggest that the DNA in the mature phage is arranged with its left end toward the center and its right end toward the exterior of the overall DNA mass. Since the left end is the first to enter the phage prohead, the results are most compatible with the view that prohead filling also proceeds from the center to the exterior of the cavity. The suggested arrangement of lambda DNA is comparable to that observed in phage T4 and is consistent with the spiral-fold model of packaged DNA.

Bacteriophage lambda↗
PubMedDate not supplied

Small-scale purification of bacteriophage lambda DNA by an airfuge centrifugation step in cesium chloride gradients.

A rapid and efficient procedure for purifying bacteriophage lambda DNA is described. This small-scale purification involves isolation of bacteriophage particles on cesium chloride gradients. Using an Airfuge ultracentrifuge, the centrifugation step can be readily achieved in 90 minutes. The method allows a 1-day purification of up to 12 independent lambda DNA (20-40 micrograms each). The recovered DNA, essentially devoid of RNA and DNA contaminants, is efficiently cut by restriction endonucleases and can serve as starting material for the ligation of DNA fragments in other cloning vehicles.

Bacteriophage lambda↗
PubMed1988

Helical-repeat dependence of integrative recombination of bacteriophage lambda: role of the P1 and H1 protein binding sites.

The efficiency of site-specific recombination of bacteriophage lambda was found to depend on the spacing between distant protein binding sites. Insertions and deletions of up to 30 base pairs were made in the nonessential regions between the H1 and H2 protein binding sites. Recombination was found to occur in substrates with changes of integral multiples of a DNA helical repeat, whereas recombination was defective in substrates with nonintegral changes. The lambda recombinogenic complex is especially interesting because two different proteins are involved: integration host factor (IHF), which has been shown to bend DNA, and the phage-encoded integrase protein (Int), which has been shown to have two distinct DNA-binding domains. The importance of angular displacement of protein binding sites was confirmed by addition of ethidium bromide to defective substrates. Significant stimulation of recombination was observed when sufficient drug intercalated and unwound the DNA to allow improved orientation of sites. The orientation effects are dependent on supercoiling, as spacing is less important in conditions where supercoiling and the P1-H1 sites are not required for recombination.

Bacteriophage lambda↗
PubMed2000

Promoter interference in a bacteriophage lambda control region: effects of a range of interpromoter distances.

The p(R) and p(RM) promoters of bacteriophage lambda direct transcription in divergent directions from start sites separated by 83 phosphodiester bonds. We had previously shown that the presence of an RNA polymerase at p(R) interfered with open complex formation at p(RM) and that this effect was alleviated by the deletion of 10 bp between the two promoters. Here we present a detailed characterization of the dependence of the interference on the interpromoter distance. It was found that the reduced interference between the two promoters is unique to the 10-bp deletion. The relief of interference was demonstrated to be due to the facilitation of a step subsequent to RNA polymerase binding to the p(RM) promoter. A model to explain these observations is proposed. A search of known Escherichia coli promoters identified three pairs of divergent promoters with similar separations to those investigated here.

Bacteriophage lambda↗
PubMed1995

Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein.

The tolerance of bacteriophage lambda morphogenesis for C-terminal additions to the tail tube major protein subunit (the V gene product; gpV) has been investigated. A second modified copy of the lambda V gene, either within a novel phage vector itself or plasmid-borne, was expressed during phage growth. High-level substitution of wild-type gpV by modified gpV bearing a basic C-terminal peptide sequence (RRASV; a target site for cAMP-dependent protein kinase) was possible using multiple repeats of a serine-glycine (SGGG) linker sequence. Highly purified phage bearing copies of gpV-RRASV could be efficiently phosphorylated by the appropriate protein kinase, and the incorporated label was shown to migrate exclusively at the expected size in protein gels. A large tetrameric protein (beta-galactosidase) could be incorporated into active virions in at least one copy, again using a Ser-Gly linker. These studies suggest that with a suitable spacing linker and controlled levels of expression, it is likely that a wide range of protein or peptide substitutents can be fused with gpV at its C terminus and assembled as component subunits of the tail tube.

Amino Acid Sequence↗
PubMed1981

Multilevel regulation of bacteriophage lambda lysogeny by the E. coli himA gene.

Previous experiments have shown that the himA gene of E. coli specifies a protein that is required for bacteriophage lambda integration. lambda Forms clear plaques on himA mutants indicating a possible additional defect in the establishment of repression. We have tested the effects of a himA mutation on the establishment and maintenance of lambda repressor (cl) synthesis and on the synthesis of Int protein. The rate of synthesis of cl and Int after infection by lambda is severely reduced in a strain carrying a himA gene deletion. Synthesis of Int or repressor can occur in the himA- strain if the phage carry constitutive promoters for either the int gene or the cl gene. Maintenance of repression is unaffected by himA mutations as judged by repressor-stimulated transcription of PM fused to the lacZ gene. These results indicate that the himA gene participates in the regulation of the promoter sites specific of the establishment of lysogeny: PE for cl synthesis and PI, for Int production. Since the himA gene product is required also for lambda site-specific recombination, it appears that the himA gene regulates lambda lysogeny at several levels. I discuss the significance of this multilevel regulation to lambda development.

Bacterial Proteins↗
PubMed1980

Cloning of the replication gene O of E. coli bacteriophage lambda and its expression under the control of the lac promoter.

The expression of the replication gene O of bacteriophage lambda was put under the control of the lac promoter-operator region integrated into the pBR322 cloning vehicle. The new plasmid pKK104 was introduced into minicells and the O gene induced by isopropyl-beta-thiogalactoside (IPTG). The O protein could be identified as a major component in extracts from these cells, in association with the cell membrane fractions. The molecular weight of the O protein in SDS gels is about 33 000, and it is metabolically unstable but apparently stable upon isolation as a membrane-associated fraction.

Bacteriophage lambda↗
PubMed1989

Retroregulation of the bacteriophage lambda int gene: limited secondary degradation of the RNase III-processed transcript.

Expression of the int gene of bacteriophage lambda from two promoters, pI and pL, is differentially regulated through RNA processing. Efficient Int protein synthesis from the pL RNA is inhibited by the action of sib, a cis-acting retroregulator downstream from the int gene. We have used mapping procedures with nuclease S1 to study the pL transcripts produced in vivo after phage lambda infection. We have found an RNase III-dependent processing site within the Int coding sequence, 387 nucleotides upstream from the site of the primary cleavage by RNase III at Sib. This secondary processing site is located at the most stable region of secondary structure in the sib int region, as predicted by computer analysis. We suggest that RNase III cleavage at the Sib site allows processive exonucleolytic degradation of the RNA to proceed to a region of secondary structure within the Int coding sequence, which protects the upstream region of the transcript from further degradation.

Bacteriophage lambda↗
PubMed1986

Role of homology and pathway specificity for recombination between plasmids and bacteriophage lambda.

To determine the minimum amount of homology required for efficient recombination in Escherichia coli, we measured recombination frequencies between bacteriophage lambda and pBR322 derivatives containing lambda DNA fragments of various sizes by assaying for phages that could transduce the bla and ori genes of pBR322. Efficient recombination required about 40 bp of homology; increases in homology above 40 bp resulted in proportionate increases in recombination, while decreases below 40 bp resulted in precipitous decreases in recombination. The recA+ gene stimulated recombination over the entire range of homologies tested. Restriction enzyme digests of several recombinant DNA molecules indicated that they contained the complete plasmid DNA inserted in the lambda genome as expected for a reciprocal crossover. Analysis of recombination frequencies in different recombination-deficient mutant strains indicated that the formation of lambda-plasmid cointegrates by homologous recombination proceeded predominantly by the RecBC pathway and very inefficiently, if at all, by the RecE and RecF pathways.

Alleles↗
PubMed1985

"N" transcription antitermination proteins of bacteriophages lambda, phi 21 and P22.

Comparison is made among the amino acid sequences of three transcription antitermination proteins, based upon the DNA sequences of their genes in bacteriophages lambda, phi 21 and P22. The three proteins are all small (about 100 amino acids), hydrophilic and basic, but otherwise show little homology. A basic region near the amino terminus has several amino acid positions common to all three proteins and is the locus of mutations that alter six different amino acid positions inactivating the lambda N protein. A less basic region near the center is the locus of three mutations affecting the interaction of lambda N with host nusA protein. The N gene of phi 21 has an amino terminus more like that of P22, and a carboxy terminus clearly related to that of lambda.

Amino Acid Sequence↗
PubMed1982

Bacteriophage lambda initiators: preparation from a strain that overproduces the O and P proteins.

A recombinant plasmid was constructed which carries bacteriophage lambda initiator genes O and P under control of tandemly arranged PL and PR promoters. These promoters were repressed by a thermosensitive repressor, cI857, at low temperature, but became active when the culture was incubated at 42 degrees C. Upon elevation of the temperature, the O and P proteins were overproduced to the extent that they constituted several per cent of the total E. coli cellular proteins. Both the O and P proteins have been purified to apparent homogeneity, and were shown to consist of 298 and 233 amino acid residues, respectively. The amino acid composition and the terminal partial amino acid sequence of each protein were determined. Through these analyses, the locations of the O and P genes in the known lambda DNA sequence were determined. The termination codon for the O gene overlaps with the initiation codon for the P gene. The purified O protein binds specifically to the replication origin of lambda (lambda ori) in accordance with our previous observations. The purified P protein inhibits an ATPase activity of dnaB protein.

Adenosine Triphosphatases↗
PubMed1980

Site-specific recombination functions of bacteriophage lambda: DNA sequence of regulatory regions and overlapping structural genes for Int and Xis.

Site-specific recombination in bacteriophage lambda is mediated by two phage-encoded proteins, Int and Xis. The structural genes encoding these proteins are located immediately to the right of their site of action, the phage att site. The DNA sequence for both the structural and regulatory regions of these genes has been determined. The location and reading frame of the xis gene were ascertained by sequence comparisons with the b538 deletion (that ends within xis) and with the xis6 amber mutation. From the DNA sequence Xis has a molecular weight of 8630; it is rich in basic amino acids with lysine and arginine comprising 25% of the 72 amino acids. Identification of the int reading frame was also unambiguous. From the DNA sequence, Int has a molecular weight of 40,330; of the 356 amino acids, 69 are basic and 46 are acidic. In the NH(2)-terminal portion of Int, 35% of the first 20 amino acids are basic. The site-specific recombination functions form a very tight cluster (att-int-xis) on the lambda chromosome. The combined protein-encoding sequences of xis and int start 1347 base pairs, and terminate 84 base pairs, from the center of the phage att site. The two genes overlap one another by 20 base pairs (xis is upstream of int) and a possible means of controlling the relative synthesis rates of Int and Xis at the level of translation is proposed. Control at the level of transcription is also considered. The mutation intc226 leads to constitutive production of Int, independent of cII/cIII activator proteins normally required for transcription from the p(I) promoter. It is shown that this mutation is the result of a single base change (in the fMet codon of the xis gene) that generates an improved promoter heptamer sequence. This result, in conjunction with comparisons with other promoter sequences and other sequences responding to cII/cIII action, leads to a tentative identification of the p(I) promoter and site of cII/cIII action.

Bacteriophage lambda↗