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PubMed2001

The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.

cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.

Bacteriophage lambda↗

PubMed1983

Instability of bacteriophage lambda initiator O and P proteins in DNA replication.

Lambda dv plasmids having an amber mutation in an initiator gene, O or P, were constructed from mutant lambda phages by recombinant DNA techniques and several properties of such derivatives were investigated. These plasmids are perpetuated in suppressor-plus (amber-permissive) cells, but not in non-suppressor cells. The plasmid copy number in the suppressor-plus cells was low as compared to that of the plasmid without the amber mutation. In cells carrying a thermosensitive suppressor 2, raising the temperature is expected to block new production of amber proteins, but should not affect conservation of the protein made prior to heating. It was observed, however, that replication of the plasmids carrying an amber mutation in the O or P gene was abolished soon after raising the temperature, suggesting that neither of the initiator proteins can continue functioning unless replenished. Pulse-chase experiments demonstrated that O protein decays with a half-life of 8 min. Several lines of evidence suggest that this degradation occurs independently of the protein function. On the other hand, P protein was not degraded under the same experimental conditions. These observations are discussed in connection with functional instability of the initiator molecules. It appears that they do not work catalytically.

Bacteriophage lambda↗

PubMed1979

Some properties of site-specific and general recombination inferred from int-initiated exchanges by bacteriophage lambda.

The site-specific recombination at the attachment site for prophage integration might proceed by two general mechanisms: (1) a concerted reaction without a free intermediate; (2) a sequential mechanism differing from typical general recombination only by an inability of the cross-strand intermediate structure to migrate into the region of nonhomology adjacent to the attachment site. The blocked-migration model predicts frequent genetic exchange in the int xis region near the attachment site if Int-mediated recombination occurs between lambda phage with homologous attachment sites. We find such additional int xis exchanges, but only at very low frequency (1% of the Int-mediated recombination). We conclude that the resolution point only rarely moves away from the initial crossover point specified by Int and, therefore, that the Int reaction is mainly concerted. We interpret the rare additional int xis recombinants as indicative of occasional branch migration from an initial Int-mediated crossover. The frequency of the rare int xis recombinants is not simply related to distance from the attachment site to an int- or xis- mutation, suggesting that the heteroduplex distance is often at least a gene in length. The frequency of these additional exchanges is also not a strong function of distance between two mutations; from this we conclude that the resolution to the observed recombinant structure in the sequential cases occurs often by mismatch repair. We have found no marked effect of mutations in the bacterial recA, recB, recC, recF, or recL genes on the frequency of the int xis recombinants; this may indicate that none of these genes specifies a product uniquely required for resolution of a cross-strand intermediate.

Bacteriophage lambda↗

PubMed1989

Heteroduplex substrates for bacteriophage lambda site-specific recombination: cleavage and strand transfer products.

Lambda's Int protein acts as a specific topoisomerase at attachment sites, the DNA segments that are required for site-specific recombination. Int cleaves each strand of an attachment site at a unique place and creates strand exchanges by joining broken ends from two different parents. To study the action of Int topoisomerase in more detail, heteroduplex attachment sites were made by annealing strands that are complementary except for a few base pairs that lie in the region between the points of top and bottom strand exchange in the attachment site core. These heteroduplexes appear to interact normally with Int and its accessory proteins IHF and Xis. Although the heteroduplex sites are specifically cleaved by Int topoisomerase, rejoining of the broken DNA is hindered by the lack of Watson--Crick complementarity adjacent to the break. Because of this, heteroduplexes accumulate broken intermediates which are then processed in novel ways. We have used this feature to provide new information about functional differences between attachment sites, to investigate the way Xis protein controls directionality of site-specific recombination, and to demonstrate that Int protein can join strands indiscriminately and can therefore generate recombinants with either of two genetic polarities.

Bacteriophage lambda↗

PubMed1998

Termination of packaging of the bacteriophage lambda chromosome: cosQ is required for nicking the bottom strand of cosN.

Termination of packaging of the lambda chromosome involves completion of translocation of the DNA into the head shell, and conversion of the translocation complex into a cleavage complex. The cleavage reaction introduces staggered nicks into the downstream cosN to generate the right cohesive end of the chromosome. cosQ, a site adjacent to cosN, was found to be required for nicking the bottom strand of cosN; bottom strand nicking was also sequence-specific for bps at the nick site. Nicking of the top strand of cosN (cosNL) was stimulated by cosQ, but fidelity and efficiency of cosNL nicking were largely dictated by other cos subsites (i.e. cosB and I2). Aberrant top-strand cleavage within cosQ was observed in the absence of I2, and nicking at a site 8 nt 5' to the normal cosNL nick site occurred in the absence of cosB. The presence of cosQ was found to be insufficient to arrest DNA translocation in vivo, indicating that cosQ, per se, is not a packaging stop signal. A model is presented in which the role of cosQ is to depolarize the asymmetric arrangement of terminase protomers in the translocation complex so that protomers are configured to match the 2-fold rotational symmetry of cosN.

Bacteriophage lambda↗

PubMed1999

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor.

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.

Amino Acid Substitution↗

PubMed1981

The leftward promoter of bacteriophage lambda. Structure, biological activity, and influence by adjacent regions.

The effect of regions adjacent to the lambda PL promoter was studied using a sequence deleted in an A/T-rich segment immediately upstream from the promoter. High resolution thermal denaturation analysis showed that the undeleted sequence, as isolated on a 360-bp restriction fragment (360-PL) melted in two distinct steps. Since the deleted sequence (230-PL) melts at a higher temperature than any portion of 360-PL, the deleted promoter is more stable to denaturation. This increased stability was also suggested by a transcription assay in which the strong binding of RNA polymerase is inhibited at low temperature. The deleted promoter fragment required a higher temperature to become functional than did the 360-PL fragment. Furthermore, RNA polymerase bound to the 230-PL fragment is displaced by heparin at a faster rate than from 360-PL, and the deleted fragment initiates transcription at a reduced rate. Thus, by several criteria the deleted promoter is less active than the parent sequence. Evidence is presented arguing that the deletion does not alter the sequence directly recognized by RNA polymerase. Therefore, the altered sequence is reducing promoter activity by changing some other level of overall promoter structure. These results suggest that regions outside of the sequence directly recognized by a regulatory protein can affect genetic expression from the site.

Bacteriophage lambda↗

PubMed1984

Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl.

LamB is one of the major cellular proteins when E. coli is grown in the presence of maltose and is localized in the outer membrane. Previous immunolabellings obtained with monoclonal antibodies showed that this protein is a transmembrane protein and led to the detection of 4 epitopes exposed on the cell surface and 2 located on the inner surface of the outer membrane (Scheckman et al., 1983). In the present study, we have used this biological model in order to see whether these two classes of epitopes could be distinguished by immunocytochemical labelling performed on thin sections of E. coli embedded in Lowicryl K4M (Carleman et al., 1982). The optimal conditions of fixation and embedding were first established for labelling with poly- or monoclonal antibodies detected by Protein A-gold complexes. The analysis of gold particle distribution on each side of the outer membrane after labelling with a polyclonal serum or after its adsorption on intact bacteria allowed us to conclude that the resolution of immunolabelling on thin sections was about 20 nm. The use monoclonal antibodies met with difficulties due mostly to the nonspecific labelling of the cytoplasm. Although this nonospecific labelling was decreased by fixing bacteria with paraformaldehyde alone, only one antibody gave a correct specific labelling after high dilution (1/3000). The gold particle distribution obtained with this antibody confirmed the location on the cell surface of this epitope.

Antibodies, Bacterial↗

PubMed1995

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Transcription of downstream genes in the early operons of phage lambda requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of lambda N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.

Bacterial Proteins↗

PubMed1984

1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator. Evidence for a change of the conformation of the OR3 operator on binding.

The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.

Bacteriophage lambda↗

PubMed1989

Structural analysis of the carboxy terminus of bacteriophage lambda repressor determined by antipeptide antibodies.

To analyze lambda repressor function and structure, antibodies were generated with synthetic peptides corresponding to sequences believed to be involved in prophage induction. These site-directed antibodies seemed to recognize preferentially the primary sequence of repressor because they reacted better in competition experiments with the oligopeptide and with the partially denatured forms of repressor than with the native molecules. This information, together with the characteristic ability of the antibodies to immunoprecipitate or react with repressor in immunoblots, allowed us to infer some conformational properties of the specific regions that the antibodies recognized. The antibodies reacted less with some mutant repressors that had a single amino acid substitution within the cognitive sequences. RecA-catalyzed cleavage of repressor was inhibited to different extents in relation to the proportion of repressor that each antipeptide immunoglobulin G (IgG) was able to immunoprecipitate. The antipeptide IgGs did not affect specific binding of repressor to operator DNA, whereas the antirepressor IgG was inhibitory. The three different IgGs competed for binding to repressor in an enzyme-linked immunosorbent assay additivity test, which suggested that the three regions of conserved amino acids are probably located on the same side of the carboxyl domain of repressor and possibly close together in the tertiary structure.

Amino Acid Sequence↗

PubMed1999

Functional importance of regions in Escherichia coli elongation factor NusA that interact with RNA polymerase, the bacteriophage lambda N protein and RNA.

The association of the essential Escherichia coli protein NusA with RNA polymerase increases pausing and the efficiency of termination at intrinsic terminators. NusA is also part of the phage lambda N protein-modified antitermination complex that functions to prevent transcriptional termination. We have investigated the structure of NusA using various deletion fragments of NusA in a variety of in vitro assays. Sequence and structural alignments have suggested that NusA has both S1 and KH homology regions that are thought to bind RNA. We show here that the portion of NusA containing the S1 and KH homology regions is important for NusA to enhance both termination and antitermination. There are two RNA polymerase-binding regions in NusA, one in the amino-terminal 137 amino acids and the other in the carboxy-terminal 264 amino acids; only the amino-terminal RNA polymerase-binding region provides a functional contact that enhances termination at an intrinsic terminator or antitermination by N. The carboxy-terminal region of NusA is also required for interaction with N and is important for the formation of an N-NusA-nut site or N-NusA-RNA polymerase-nut site complex; the instability of complexes lacking this carboxy-terminal region of NusA that binds N and RNA polymerase can be compensated for by the presence of the additional E. coli elongation factors, NusB, NusG and ribosomal protein S10.

Bacterial Proteins↗

PubMed1984

Genetic and biochemical factors affecting the induction of bacteriophage lambda by N-nitroso compounds.

As part of our effort to validate a biochemical (prophage) induction assay (BIA) as a screening test for carcinogens, we have tested more than 100 N-nitroso compounds. An enzyme, beta-galactosidase, is induced as an indirect consequence of DNA damage to the host, as part of the 'SOS' response. Besides the obvious practical importance of detecting this class of carcinogen, there is the question of the mechanism by which these compounds work. Mutagenesis by one compound, N-methyl-N'-nitro-N-nitrosoguanidine, is known to proceed by both SOS (recA)-dependent and SOS-independent pathways. Mispairing due to O6 alkylation of guanine is thought to be responsible for the SOS-independent pathway; however, there has been little consideration of recA-dependent functions, of which phage induction is one. Although nitrosamides could be detected as phage inducers in our assay, N-nitrosamines in the presence of rat liver 9 000 X g supernatant usually gave no response. We found that the use of several mutant strains, particularly a lexA mutant, in combination with hamster liver 9 000 X g supernatant, allowed us to detect most N-nitrosamines reasonably well, in either a spot test or a quantitative tube assay. Induction in a lexA strain was most unexpected, since this mutation usually diminishes the expression of SOS functions induced by ultra-violet light. Because the genetic and biochemical conditions that favour phage induction are different from those that favour mutagenesis, it seems likely that the lesions in DNA leading to the two biological end-points are different.

Bacteriophage lambda↗

PubMed1987

Identification of polypeptides encoded by an Escherichia coli locus (hflA) that governs the lysis-lysogeny decision of bacteriophage lambda.

We report the cloning of the Escherichia coli hflA locus, which governs stability of phage lambda cII protein and which has been proposed to encode or regulate a cII-specific protease. The hflA locus was cloned on an 18-kilobase DNA fragment by selecting for plasmids that carry the neighboring purA gene. The boundaries of hflA were delimited by analysis of deletions and insertions constructed in vitro and by use of transposon Tn1000. Maxicell analysis of the proteins encoded by the hflA-containing fragment shows that hflA consists of at least two nonoverlapping genes, hflC and hflK, encoding polypeptides of 37,000 (C) and 46,000 (K) daltons. We observe that insertions into one gene eliminate the corresponding polypeptide and greatly reduce synthesis of the other. We suggest that these two polypeptides (K and C) interact to form a multimeric complex and that free subunits are unstable. We have constructed two types of fusions between hflA and lacZ. One is an hflC-lacZ protein fusion constructed in vitro; the other is an hfl-lacZ operon fusion in which a Mu dX(Apr lac) has inserted into the hflK gene. We have used the operon fusion to infer the direction of transcription of the hflK gene--toward hflC and in the same direction as hflC. Last, we describe evidence that hflA contains an additional gene, hflX, encoding a 50,000-dalton polypeptide.

Autoradiography↗

PubMed1990

Structure and inherent properties of the bacteriophage lambda head shell. VII. Molecular design of the form-determining major capsid protein.

Some mutations in the major capsid protein (gpE) of lambda phage can alter the size and shape of the head shell or block the pathway of head maturation. Previous studies on the classification of such mutants showed that there are at least five functional sites on the gpE molecule. In this study, we determined the amino acid exchanges by DNA sequencing to elucidate the molecular design of the form-determining multifunctional protein gpE. In addition, we characterized the mutated gpE molecules by two-dimensional gel electrophoresis and studied suppression patterns of amber mutants at 43 amino acid residues. Those mutations map at 19 amino acid residues at 22 bases, which are located in three regions, 40 to 91, 222 to 246, and 284 to 324 of the 341 amino acid residues of gpE. These regions seem to be important in the activity of gpE, since amber mutations in these regions are suppressed on the average by less species of suppressors than those outside these regions. The mutations having different phenotypes are not segregated from each other, while some mutations having the same phenotype are separated far apart in the primary structure. This suggests that the functional sites were formed during evolution after the folding pattern of the ancestral gpE polypeptide chain had been established. Many of the mutations are located at serine, glycine and proline residues in predicted beta-turns.

Amino Acid Sequence↗

PubMed1987

Degradation in vitro of bacteriophage lambda N protein by Lon protease from Escherichia coli.

Lon protease from Escherichia coli degraded lambda N protein in a reaction mixture consisting of the two homogeneous proteins, ATP, and MgCl2 in 50 mM Tris, Ph 8.0. Genetic and biochemical data had previously indicated that N protein is a substrate for Lon protease in vivo (Gottesman, S., Gottesman, M., Shaw, J. E., and Pearson, M. L. (1981) Cell 24, 225-233). Under conditions used for N protein degradation, several lambda and E. coli proteins, including native proteins, oxidatively modified proteins, and cloned fragments of native proteins, were not degraded by Lon protease. Degradation of N protein occurred with catalytic amounts of Lon protease and required the presence of ATP or an analog of ATP. This is the first demonstration of the selective degradation of a physiological substrate by Lon protease in vitro. The turnover number for N protein degradation was approximately 60 +/- 10 min-1 at pH 8.0 in 50 mM Tris/HCl, 25 mM MgCl2 and 4 mM ATP. By comparison the turnover number for oxidized insulin B chain was 20 min-1 under these conditions. Kinetic studies suggest that N protein (S0.5 = 13 +/- 5 microM) is intermediate between oxidized insulin B chain (S0.5 = 160 +/- 10 microM) and methylated casein (S0.5 = 2.5 +/- 1 microM) in affinity for Lon protease. N protein was extensively degraded by Lon protease with an average of approximately six bonds cleaved per molecule. In N protein, as well as in oxidized insulin B chain and glucagon, Lon protease preferentially cut at bonds at which the carboxy group was contributed by an amino acid with an aliphatic side chain (leucine or alanine). However, not all such bonds of the substrates were cleaved, indicating that sequence or conformational determinants beyond the cleavage site affect the ability of Lon protease to degrade a protein.

ATP-Dependent Proteases↗

PubMed1996

Mutations affecting the high affinity ATPase center of gpA, the large subunit of bacteriophage lambda terminase, inactivate the endonuclease activity of terminase.

Phage lambda terminase carries out the cos cleavage reaction that generates mature chromosomes from immature concatemeric DNA. The ATP-stimulated endonuclease activity of terminase is located in gpA, the large terminase subunit. There is a high affinity ATPase center in gpA, and a match to the conserved P-loop of known ATPases is found starting near residue 490. Changing the conserved P-loop lysine at residue 497 of gpA affects the high affinity ATPase activity of terminase. In the present work, mutations causing the gpA changes K497A and K497D were found to be lethal, and phages carrying these mutations were defective in cos cleavage, in vivo. Purified K497A and K497D enzymes cleaved cos in vitro at rates reduced from the wild-type rate by factors of 1000 and 2000, respectively. The strong defects in cos cleavage are sufficient to explain the lethality of the K497A and K497D defects. In in vitro packaging studies using mature (cleaved) phage DNA, the K497A enzyme was indistinguishable from the wild-type enzyme, and the K497D enzyme showed a mild packaging defect under limiting terminase conditions. In a purified DNA packaging system, the wild-type and K497D enzymes showed similar packaging activities that were stimulated to half-maximal levels at about 3 microM ATP, indicating that the K497D change does not affect DNA translocation. In sum, the work indicates that the high affinity ATPase center of gpA is involved in stimulation of the endonuclease activity of terminase.

Adenosine Triphosphatases↗

PubMed1994

Multiple molecules of integration host factor (IHF) at a single DNA binding site, the bacteriophage lambda cos I1 site.

Integration host factor (IHF) is an E coli protein that binds DNA sequence-specifically and serves as a cofactor in many intracellular processes including lambda DNA packaging. In gel shift experiments, cos DNA, a DNA fragment containing the recognition signal for lambda DNA packaging, forms multiple protein-DNA complexes when combined with pure IHF. Copper(II)-1,10 orthophenanthroline footprinting of individual IHF-cos DNA complexes shows that multiple complex formation does not result from IHF binding to successive sites on the cos DNA fragment. Instead, the footprinting of DNA from two IHF-cos complexes shows protection at one site alone. DNA in the first complex is only partially protected from nucleolytic cleavage, while DNA in the second, slower-moving, complex is completely protected at the same binding site. Quantitative Western blotting experiments determined the relative stoichiometry of IHF to DNA in the two complexes. The results confirm that two molecules of IHF bind at a single site in the cos fragment. This site, cos I1, has two matches to the IHF consensus sequence, but the two matches overlap by eight of thirteen nucleotides. A search of the DNA sequence around cos, using an expanded IHF consensus sequence, has revealed additional, low-affinity consensus matches, contiguous to these. The extent of the copper(II)-1,10 orthophenanthroline footprint and the stoichiometry of the IHF-cos I1 complexes suggest that either two molecules of IHF bind to overlapping sites, or IHF binds to a site of low affinity contiguous to a strong site. Application of a thermodynamic model to the results of gel shift experiments with IHF and cos DNA suggests that multiple complex formation requires cooperative interaction between the two IHF binding sites.

Autoradiography↗