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PubMed1984

Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine.

Clear-plaque mutations were induced in the cI and cII genes of lambda by treating lysogenic cells with 9-aminoacridine (9AA). Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII. The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene. Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs. By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.

Aminacrine↗

PubMed1987

Processing of Escherichia coli 16S rRNA with bacteriophage lambda leader sequences.

To test whether any specific 5' precursor sequences are required for the processing of pre-16S rRNA, constructs were studied in which large parts of the 5' leader sequence were replaced by the coliphage lambda pL promoter and adjacent sequences. Unexpectedly, few full-length transcripts of the rRNA were detected after the pL promoter was induced, implying that either transcription was poor or most of the rRNA chains with lambda leader sequences were unstable. Nevertheless, sufficient transcription occurred to permit the detection of processing by S1 nuclease analysis. RNA transcripts in which 2/3 of the normal rRNA leader was deleted (from the promoter up to the normal RNase III cleavage site) were processed to form the normal 5' terminus. Thus, most of the double-stranded stem that forms from sequences bracketing wild-type 16S pre-rRNA is apparently not required for proper processing; the expression of such modified transcripts, however, must be increased before the efficiency of processing of the 16S rRNA formed can be assessed.

Bacteriophage lambda↗

PubMed1980

Promoter for the establishment of repressor synthesis in bacteriophage lambda.

Transcription of the lambda repressor gene (cI) is positively regulated by the phage-encoded proteins cII and cIII. We have isolated and characterized the 5'-terminal region of this RNA and shown that it originates at a promoter (pE) located between genes cro and cII. The DNA sequence of this promoter shows little homology to other known promoters. Initiation of transcription from PE is abolished by the cis-dominant mutations cY; these mutations alter the "-10" and "-35" regions of the promoter. We propose that the "-35" region is the site of activation of PE, possibly via the direct interaction of protein cII.

Bacteriophage lambda↗

PubMed1983

Effects of nonhomology on bacteriophage lambda recombination.

When crosses are performed under conditions severely restricting DNA synthesis, the presence of DNA sequence nonhomologies in the lac region of lambda plac5 limits the parental material contribution to and the yield of phage recombinant in a region bracketed by the nonhomologies. These observations are consistent with the expectation of a role for branch migration in the formation of heteroduplex structures under these conditions. Under conditions permissive for DNA replication, bracketing a region with nonhomologies has an only modest effect on the yield of recombinants within that interval. In addition, recombinants within such a bracketed interval manifest an excess of coincident exchange events in an adjacent region. These observations suggest the possibility that, under conditions permissive for DNA replication, regions of nonhomology can be included in heteroduplex structures.

Bacteriophage lambda↗

PubMed1985

1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator. II. Assignment of the non-exchangeable proton resonances of the OR3 operator.

The 17 base pair operator OR3 oligonucleotide, which is the preferential binding site for the Cro repressor of phage lambda, was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exchangeable base protons and the H1' and the H2'-H2" sugar protons of the OR3 operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the OR3 operator - Cro repressor interaction.

Bacteriophage lambda↗

PubMed1991

The nut site of bacteriophage lambda is made of RNA and is bound by transcription antitermination factors on the surface of RNA polymerase.

The boxA and boxB components of the lambda nut site are important for transcriptional antitermination by the phage N protein. We show here that boxA and boxB RNA in N-modified transcription complexes are inaccessible to ribonucleases and have altered sensitivity to dimethylsulfate. N and NusA suffice to weakly protect boxB, independently of boxA and other factors. However, efficient protection of the entire nut site from ribonucleases requires boxA and boxB, N, NusA, NusB, S10, and NusG. Mutations in RNA polymerase, which inhibit antitermination by N in vivo, disallow protection of the nut site during transcription in vitro; therefore, the surface of RNA polymerase must coordinate the formation of complexes containing the antitermination factors and nut site RNA.

Bacteriophage lambda↗

PubMed1997

Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.

Adenosine Triphosphatases↗

PubMed1988

Mechanism of cos DNA cleavage by bacteriophage lambda terminase: multiple roles of ATP.

In the terminus-generating (ter) reaction of phage lambda, the phage enzyme terminase catalyzes the production of staggered nicks within the cohesive-end nicking site (cosN). Although the two nicks are related by a rotational symmetry axis that bisects cosN, the in vitro ter reaction is strikingly asymmetric at the nucleotide level. Nicking of the lambda r strand precedes nicking of the I strand. Furthermore, when the two nicking reactions are uncoupled, they have different nucleotide cofactor requirements. ATP plays critical roles during cos cleavage: First, nicking of both DNA strands is stimulated by the addition of ATP. Second, ATP is required for the correct specificity of r-strand nicking since, in the absence of nucleotide, the r-strand nick is shifted 8 bases to the left. Studies with nonhydrolyzable analogs indicate that ATP hydrolysis is not required for these functions. However, after the two nicks are made, terminase catalyzes a disengagement of the cohered ends in a reaction that requires ATP hydrolysis.

Adenine Nucleotides↗

PubMed1989

Structure and inherent properties of the bacteriophage lambda head shell. VI. DNA-packaging-defective mutants in the major capsid protein.

Some amino acid substitutions in the major capsid protein (gene E product) of lambda phage are found to cause a defect in DNA packaging. These substitutions permit initiation of DNA packaging and expansion of the prohead. However, cleavage of the concatemer DNA at the cos site takes place only to a very small extent, and the capsid eventually becomes empty. Interestingly, the mutations are suppressed by a decrease of the DNA length between the cos sites by 8000 to 10,000 bases. These properties are similar to those of amber mutants in gene D, which codes for the capsid outer-surface protein. Studies on the E missense.D amber double mutant show that the E protein and the D protein contribute additively to the stabilization of the condensed form of the DNA molecule in phage heads.

Bacteriophage lambda↗

PubMed1983

Mapping of the Q-utilization site (qut) required for antitermination of late transcription in bacteriophage lambda.

To locate the site required for transcription antitermination by the gene Q product, we constructed a plasmid containing the p'R promoter, the t'R1 terminator, and gene galK. We measured the galK expression in response to the lambda Q product supplied in trans, while deleting various portions of lambda DNA adjacent to p'R. The presence of the lambda p'R promoter together with the downstream DNA coding for only a 34-bp segment of 5'-proximal 6S RNA permits antitermination to occur, whereas deletions removing this segment abolish antitermination, as measured by galK expression, but do not affect the p'R promoter. Thus the 34-bp segment must contain the p'R-distal (right) boundary of the Q-specific recognition site qut (Fig. 1). The Q-mediated antitermination appears to be p'R-qut specific but not t'R1 specific, since it does not operate with the pp-t'R1 assembly, but is also effective with terminators other than t'R1, e.g., with the combination of the p'R-qut-tL3 modules.

Bacteriophage lambda↗

PubMed1980

The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa.

The amidase genes of Pseudomonas aeruginosa were inserted into a lambda replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant lambdaami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with lambdaami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of lambdaami were made by introducing Q-, S-, mutations. Cultures of E. coli infected with lambdaamiQ-S- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificty, thermal stability and immunological cross-reaction.

Amidohydrolases↗

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↗

PubMed1998

NMR structure of the bacteriophage lambda N peptide/boxB RNA complex: recognition of a GNRA fold by an arginine-rich motif.

The structure of the complex formed by the arginine-rich motif of the transcriptional antitermination protein N of phage lambda and boxB RNA was determined by heteronuclear magnetic resonance spectroscopy. A bent alpha helix in N recognizes primarily the shape and negatively charged surface of the boxB hairpin through multiple hydrophobic and ionic interactions. The GAAGA boxB loop forms a GNRA fold, previously described for tetraloops, which is essential for N binding. The fourth nucleotide of the loop extrudes from the GNRA fold to enable the E. coli elongation factor NusA to recognize the N protein/RNA complex. This structure reveals a new mode of RNA-protein recognition and shows how a small RNA element can facilitate a protein-protein interaction and thereby nucleate formation of a large ribonucleoprotein complex.

Amino Acid Sequence↗

PubMedDate not supplied

[Identification in vivo of promoter activity in the left site of the att region of bacteriophage lambda DNA].

The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.

Bacteriophage lambda↗

PubMed1991

Energetics of subunit dimerization in bacteriophage lambda cI repressor: linkage to protons, temperature, and KCl.

A common feature of gene regulatory systems is the linkage between reversible protein oligomerization and DNA binding. Experimental dissection using temperature dependence of the subunit-subunit energetics and their linkage to processes such as ion binding and release is necessary for characterization of the chemical forces that contribute to cooperativity and site specificity. We have therefore studied the effects of temperature, proton activity, and monovalent salt on monomer-dimer assembly of the lambda cI repressor using a recently developed gel chromatographic procedure. This technique has made possible studies in the previously inaccessible picomolar concentration ranges where the assembly reactions occur. Upon formation of the dimer interface in the range pH 5-9, we find an overall absorption of protons which is temperature-dependent. The dimerization reaction displays a large negative enthalpy of association at all conditions studied (pH 5, 7, and 9). The reaction is also dependent on monovalent salt concentration: subunit association is weaker at low-salt conditions. The results suggest that a repulsive interaction between negatively charged side chains (i.e., aspartates and glutamates) on each monomer surface is attenuated by increasing concentrations of KCl. Formation of the dimer interface may be mediated by absorption of cations which stabilize the complex.

Bacteriophage lambda↗

PubMed1988

Analysis of transcription termination signals in the nin region of bacteriophage lambda: the roc deletion.

Deletions in the region, nin, between the P and Q genes of phage lambda remove a portion of the phage genome that includes signals for termination of transcription. These deletions were selected because they permit growth of lambda derivatives defective in the N-mediated transcription antitermination system; i.e., the deletions confer N independence (nin). Thus nin phages (e.g., lambda nin5) grow in most Escherichia coli nus mutants. The nus genes encode functions necessary for N action. We report the isolation of a deletion in the nin region delta roc that confers a partially N-independent phenotype; lambda derivatives with delta roc can grow under normally nonpermissive conditions, 32 degrees C, in a host with the rpoB-nusC60 mutation. The roc deletion also partially suppresses the inhibitory effects of other nus mutations at higher temperatures. Delta roc, which extends from base pairs 41883 to 43825, overlaps the nin5 deletion, which extend from base pairs 40501 to 43306. Unlike the nin5 deletion, the sequences deleted by delta roc do not include a stem-loop structure, tR2, previously shown to have terminator activity. Using promoter and terminator testor vectors, we demonstrate that a 2,400-base-pair fragment that includes the wild-type roc region but excludes tR2 has terminator activity. Thus, delta roc permits a functional division of the transcription termination signals in the nin region.

Bacteriophage lambda↗

PubMed1998

Characterization of bacteriophage lambda Q- mutant for stable and efficient production of recombinant protein in Escherichia coli system.

We previously demonstrated that the lambda system integrated into the host chromosome can overcome the instability encountered in continuous operations of unstable plasmid-based expression vectors. High stability of a cloned gene in a lysogenic state and a high copy number in a lytic state provide cloned-gene stability and overexpression in a two-stage continuous operation. But the expression by the commonly used S- mutant lambda was only twice as high as that of the single copy. To increase the expression in the lambda system, we constructed a Q- mutant lambda vector that can be used in long-term operations such as a two-stage continuous operation. The Q- mutant phage lambda is deficient in the synthesis of proteins involved in cell lysis and lambda DNA packaging, while the S- mutant is deficient in the synthesis of one of two phage proteins required for lysis of the host cell and liberation of the progeny phage. Therefore, it is expected that the replicated Q- lambda DNA containing a cloned gene would not be coated by a phage head and would remain naked for ample expression of the cloned gene and host cells would not lyse easily and consequently would produce larger amounts of cloned-gene products. The beta-galactosidase expression per unit cell by the Q- mutant in a lytic state was about 30 times higher than that in a lysogenic state, while the expression by the commonly used S- mutant in a lytic state was twice as high as that in a lysogenic state. The optimal switching time of the Q- mutant from the lysogenic state to the lytic state for the maximum production of beta-galactosidase was 5.3 h, which corresponds to an early log phase in the batch operation.

Bacteriophage lambda↗

PubMed1988

Bacteriophage lambda DNA packaging. The product of the FI gene promotes the incorporation of the prohead to the DNA-terminase complex.

Lambda DNA packaging in vitro can be examined in stages. In a first step, lambda DNA interacts with terminase to form a DNA-enzyme complex, called complex I. Upon addition of proheads, in a second step, a ternary complex, complex II, containing DNA, terminase and the prohead is formed. Finally, upon addition of the rest of the morphogenetic components, complete phages are assembled. We have investigated the effect of the FI gene product (gpFI) in these reactions and found that a stimulation in phage yield is observed when gpFI is included early in the reaction, at the time when DNA, terminase and proheads interact to form complex II. Measurements of complex II formation revealed that gpFI stimulated the rate of formation of this intermediate. gpFI was further shown to stimulate the addition of proheads to preformed complexes I to give complex II, but the protein did not stimulate complex I formation.

Bacteriophage lambda↗