Mutations in bacteriophage lambda repressor that prevent RecA-mediated cleavage.

In this paper, we report on the isolation and sequence analysis of mutations that confer an induction-deficient phenotype to lambda repressor. A total of 16 different mutations, which occur at 13 different sites in the repressor gene, have been characterized. For most of the mutant lysogens, frequencies of spontaneous induction in a recA+ strain were reduced dramatically in comparison with those for a wild-type phage, and these mutant lysogens showed little or no prophage induction after UV irradiation. The immunity properties of cells containing the mutant repressors show that all of the mutants but one exhibit operator-binding properties indistinguishable from wild-type repressor.     

A bacterial protein requirement for the bacteriophage lambda terminase reaction.

The bacteriophage lambda terminase enzyme cleaves the cohesive-end sites of lambda DNA to yield the protruding 5'-termini of the mature molecule. In vitro, this endonucleolytic event requires a protein factor which has been isolated and purified from extracts of uninfected E. coli. The terminase host factor (THF) is a heat stable basic protein of M.W. approximately 22,000. The integration host factor (IHF) protein of E. coli can efficiently substitute for THF in the terminase reaction; however, THF can be demonstrated to be physically present in, and isolated with full biological activity from extracts of cells defective or deficient in IHF.     

Sites of contact between lambda operators and lambda repressor.

DNA bearing lambda operator sequences was methylated by dimethyl sulfate (DMS) in the presence or absence of lambda repressor. Under the experimental conditions, DMS methylates only the purine residues. The presence of lambda repressor affects only the methylation of certain G residues in the operators. Repressor blocks the methylation of certain G's and enhances the methylation of other G's. Since the reactive ring-nitrogen of G lies in the major groove of double-stranded DNA, and the reactive ring-nitrogen of A lies in the minor groove, the above results imply that the repressor makes contacts in the major groove of the helix. The repressor effect on G-methylation is sharply confined to the three 17 base pair units within each lambda operator previously proposed as the repressor-binding sites.     

Structure and function of the repressor of bacteriophage lambda

Summary By mutagenizing a cIts (cI857) lysogen, a mutant has been isolated with a wild-type phenotype. This mutant phage lysogenizes with low efficiency and produces a low burst. Though the initial rates of repressor synthesis in Escherichia coli after infection with wild-type and mutant are the same, the maximum level of repressor that is synthesized in the latter case is only about 30% of that synthesized in the former. Virulent plates on the lysogen of mutant with slightly less efficiency producing very tiny plaques. Operator-binding studies made in vitro with purified mutant and wild-type repressors show that the binding curve of the former repressor is a rectangular hyperbola while that of the latter is sigmoid. The half-lives of the complexes of mutant and wild-type repressors with right operator are 133 and 27 min, respectively. All these results suggest that the mutant repressor possibly has a higher affinity for the operators. This mutant has been named cIha (ha=high affinity).     

Structure and function of the repressor of bacteriophage lambda

Summary The high-affinity mutant cI gene of cIha (Nag et al. 1984) was cloned in the multicopy plasmid pBR322. In the resulting plasmid, pMD 102, a lacUV5 promoter was inserted giving the lacUV5-cIha fusion plasmid pMD 205. Bacteria carrying pMD 102 and pMD 205 contain 2.5 and 15 times, respectively, the level of repressor in a monolysogen of cIha. Results of the study of certain properties of the bacteria carrying these plasmids suggest that the ha repressor also has a higher affinity for the virulent mutant operators as well as the prm promoter of .     

Unusual ribosome binding properties of mRNA encoding bacteriophage lambda repressor.

The mRNA encoding repressor cI of phage lambda is the only known E. coli message which starts directly with the initiation AUG codon. The ability of in vitro synthesized cI mRNA fragments (150 or 400 nts) to form ternary initiation complexes has been studied using the toeprint method. In the presence of tRNA(Met)f, these fragments are capable of forming the ternary complexes at the 5'-terminal AUG codon not only with 30S subunits but also with undissociated 70S ribosomes (70S tight couples). In the latter case, no binding at other positions of cI mRNA can be detected at all. The starting region of cI mRNA has a single stranded conformation and is highly enriched in A-residues. This feature of cI mRNA RBS is suggested to be the main factor which allows cI mRNA to form the initiation complex with the ribosome. Unlike 30S subunits, the binding to 70S tight couples is not affected by any of the initiation factors, although it is as efficient as that to 30S subunits supplemented with the factors. 30S subunits prefer to associate with the internal RBSs of the preformed mRNA molecules, provided that they are not sequestered by the secondary structure. In contrast, 70S tight couples tend to avoid extra sequences upstream of the codon directed to the P site and occupy a position as close as possible to the 5'-end of the message. This has been found to be the case both for tRNA(Met)f and for elongator tRNA(Glu)2. The structural features of mRNA RBSs which influence their different binding for 30S subunits and 70S ribosomes are discussed.     

Comparison of the structures of cro and lambda repressor proteins from bacteriophage lambda

The three-dimensional structures of cro repressor protein and of the amino-terminal domain of lambda repressor protein, both from bacteriophage lambda, are compared. The second and third alpha-helices, alpha 2 and alpha 3, are shown to have essentially identical conformations in the two proteins, confirming the significance of the amino acid sequence homology previously noted between these and other DNA binding proteins in the region corresponding to these helices. The correspondence between the two-helical units in cro and lambda repressor protein is better than the striking agreement noted previously between two-helical units in cro and catabolite gene-activator protein. Parts of the first alpha-helices of repressor and cro show a structural correspondence that suggests a revised sequence homology between the two proteins in their extreme amino-terminal regions. In particular, there is a short loop between the alpha 1 and alpha 2 helices of lambda repressor that is missing from cro. This structural difference may account for the observed differences found with different cros and repressors in the pattern of phosphates whose ethylation prevents the binding of these proteins to their specific recognition sites. Although the two proteins have strikingly similar alpha 2-alpha 3 helical units that are presumed to bind to DNA in an essentially similar manner, stereochemical restrictions prevent the alpha 2-alpha 3 units of the respective proteins aligning on the DNA in exactly the same way.     

Analysis of mutations in the ninR region of bacteriophage lambda that bypass a requirement for lambda N antitermination.

Two mutations in the ninR region of bacteriophage lambda that bypass a requirement for antitermination have been studied. One mutation, byp, has been cloned and mapped by marker rescue to a 417-base-pair segment in the ninR region of the genome. Analysis of the byp mutation by using promoter detection vectors, DNA sequencing, and S1 nuclease analysis showed that the byp mutation created a new promoter that transcribed gene Q. The second mutation analyzed was the deletion nin3. Sequence analysis revealed that 2,485 base pairs of the ninR region were removed, beginning within the ren gene and ending in an open reading frame termed ninG. The tR2 and tR3 terminators, and probably others, were removed by the nin3 deletion, thereby allowing the phage to be N independent and to grow in hosts defective for Nus antitermination factors.     

Recognition sequences of repressor and polymerase in the operators of bacteriophage lambda

Nucleotide sequences in two wild-type and six mutant operators in the DNA of phage λ are compared. Strikingly similar 17 base pair units are found which we identify as the repressor binding sites. Each operator contains multiple repressor binding sites separated by A-T rich spacers. Elements of 2 fold rotational symmetry are present in each of the sites. Superimposed on each operator is an E. coli RNA polymerase recognition site (promoter). Similarities in the sequences of the two λ promoters, a lac promoter, and an E. coli RNA polymerase recognition site in SV40 DNA are noted.     

Construction of a hybrid col E1 plasmid carrying the gene for bacteriophage lambda repressor.

A biologically active hybrid DNA molecule was constructed from plasmid Col E1 and the Eco R1 fragment of lambda DNA containing the gene for lambda repressor. The presence of this gene in the hybrid molecule was demonstrated genetically. The hybrid plasmid contains two closely located targets for restriction endonuclease Hind 111 in the integrated fragment. Thus, the plasmid may be used as a vector not only for Eco R1 fragments but also for Hind 111 fragments.     

C1 and cro repressors of lambda phages. I. Construction of vectors for expression of cro repressor of bacteriophage lambda imm434

Eight derivatives of recombinant plasmid pBRcro434, that consists of pBR322 and fragment of immunity region of phage lambda imm434 have been constructed and characterised. These derivatives contain the deletions in the region adjacent to OR3 operator and in the structural gene of cro-repressor of lambda imm434. The deletions have been produced by the treatment of pBRcro434 with exonuclease III of Escherichia coli and S1 nuclease of Aspergillus orizae and precisely mapped. The unique EcoRI-restriction sites have been reconstructed with the aim of using this deletion plasmids as a vectors for cloning.     

The structure of a repressor: crystallographic data for the Cro regulatory protein of bacteriophage lambda.

Crystals of the bacteriophage λ Cro repressor protein that are suitable for X-ray diffraction studies have been obtained. Preliminary crystallographic analysis reveals that the space group is R32, the cell dimensions in the hexagonal system are $\text{a = b = 91·9}\text{A}\text{?}\text{, c = 268·9}\text{A}\text{?}$, and there are three dimers per asymmetric unit.