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PubMed1996

Computer-aided discrimination between active and inactive mutants of the N-terminal domain of the bacteriophage lambda repressor.

Binding of the N-terminal domain of the lambda repressor to DNA is coupled to dimerization. Hydrophobic interactions between helix-5 and helix-5' drive the packing at the dimer interface. We have carried out computations of the conformational energy of packing of the fifth helices (and of the helix-4-loop-helix-5 portions) of variants of the lambda repressor operator binding domain, using an ECEPP/3-based packing algorithm. Here, we report the results for 26 mutants chosen among those that hve been characterized experimentally. We find that the relative orientation of the fifth helices for active mutants is very similar to the wild-type. The fifth helices of the inactive mutants have a significantly different relative orientation. This result illustrates that a unique specific orientation pattern of helix-5 relative to helix-5' is required for dimerization-coupled DNA binding activity. This finding is further supported by computational studies of the whole N-terminal domain of ten variants that showed that the active mutants, including the wild-type protein, have similar values of the number of contacts between the two monomers in the dimer, involving two amino acid residues of the fifth helices (positions 84 and 87 in each monomer). A decrease in the number of such contacts abolishes DNA-binding activity. Furthermore, all active mutants have their "DNA-recognition helices", numbers 3 and 3' positioned so that they can fit in the DNA operator like those of the wild-type protein, while some inactive mutants exhibit a substantial change in the relative orientation of their recognition helices.

Algorithms↗
PubMed1975

Control of gene expression in bacteriophage lambda: suppression of N mutants by mutations of the antirepressor.

The lysogenization and induction properties of phages lambdasusN7CI857Ai7 and lambdasusN53cro27 are described. Both phages, at 32 degrees kill little, but show only a moderate frequency of lysogenization whether an amber suppressor is present or absent in the host bacterium. In the latter case, lysogens for lambdasusN7CI857Ai7 or lambdasusN53CI857cro27 can exist in two different regulatory states, here called P r- and Pr+. The Pr+ phase is characterized by phage release and cell death at 40 degrees; conversely, cells in the Pr- phase are similarly killed but release no or very little phage. Pr- is the phase usually obtained at lysogenization. Each phase may be transmitted at 32 degrees for an unlimited number of generations, however, shifts to the opposite phase take place from time to time with a low probability. Two previously described antirepressor defective mutants. Ai7 and cro27, were found to suppress specifically the growth defect caused by an amber mutation in gene N. This suppression is observed in non-suppressing hosts at 40 or 42 degrees. Apparent revertants of N- mutants were shown to be often (80%) caused by a second mutation, in the Ai gene (also called tof, cro and fed). All the revertants so far examined appeared to be recessive. Lambda phages bearing a double amber mutation in gene N did not acquire full N independence by the acquisiton of an Ai mutation; this could be achieved, however, in the presence of a CII mutation. The above findings are discussed in terms of a direct interaction between the N, Ai and CII products.

Coliphages↗
PubMed1976

Expression of the guanine operon of Escherichia coli as analyzed by bacteriophage lambda induced mutations.

Studies were made on two guanine-requiring strains of Escherichia coli isolated independently as a result of insertion of prophage gamma into one of the structural genes of the guanine operon. These mutants do not exhibit any detectable guaB function but express the guaA function constitutively at a low level, presumably due to transcription from the pI promoter on the prophage. Various types of plaque-forming gua-transducing phages were generated from these lysogens. The approximate location and the mode of substitution of the gua genes in the phage genome were determined. These results clearly indicate that the gene guaB is located closer to the operator-promoter region of the gua operon than is guaA, and the gene order is "operator"-guaB-guaA.

Coliphages↗
PubMed1978

Genetic analysis of two genes, dnaJ and dnaK, necessary for Escherichia coli and bacteriophage lambda DNA replication.

We show that a collection of 93 E. coli mutations which map between thr and leu and which block phage lambda DNA replication define two closely linked cistrons. Work published in the accompanying paper shows that these mutations also affect host DNA replication, so we designate them dnaJ and dnaK; the gene order is thr--dnaK--dnaJ--leu. Demonstration of two cistrons was possible with the isolation of lambda transducing phages carrying one or the other or both of the dna genes. These phages were employed in phage vs bacterial complementation studies which unambiguously show that dnaK and dnaJ are different cistrons.

Coliphages↗