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Biomedical subjects

H Echols

Publications and source records attributed to H Echols.

At least 73 records · Page 4Linked to original sources
PubMed1979

Location of the regulatory site for establishment of repression by bacteriophage lambda.

During the lysogenic response to infection by bacteriophage lambda, the phage-specified cII and cIII proteins provide for the coordinate establishment of repression and integration of the viral DNA. One critical regulatory function of cII/cIII is an activation of synthesis of the cI protein, the repressor that maintains lysogeny. The mechanism and site for regulation of the cI gene by cII/cIII have been a subject of controversy. The two principal hypotheses for cII/cIII action are: initiation of new RNA chains in the y region of lambda DNA just to the left of the cII gene; or antitermination of a short leader RNA (4S or oop RNA) initiated to the right of the cII gene. In an effort to distinguish between these hypotheses, we have studied the cII-mediated turn-on of cI protein synthesis in three classes of prophage deletion strains: deletions of the 4S RNA promoter but not the y region, deletions that remove both regions, and deletions that leave both intact. We find that an intact y region is required for normal regulation of the cI gene by cII, but the 4S RNA promoter is not. From experiments with other mutants, we conclude that rightward transcription from the early lytic promoter is also not necessary for positive regulation. Our results suggest that positive regulation by cII/cIII involves initiation of new RNA chains through activation of promoter sites.

Binding Sites↗
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↗
PubMed1977

Cro regulatory protein specified by bacteriophage lambda. Structure, DNA-binding, and repression of RNA synthesis.

The Cro protein specified by bacteriophage lambda is a repressor of the genes expressed early in phage development and is required for a normal late stage of lytic growth. We have purified Cro protein to virtual homogeneity and analyzed its structure and properties as a DNA-binding protein and repressor of RNA synthesis. To confirm that the protein is the product of the cro gene, we have also shown that a missense mutation in the cro gene leads to a product that is more temperature- and salt-sensitive in its DNA-binding property. As purified, Cro protein is a dimer of identical subunits of molecular weight 8600. The purified protein binds to lambda-DNA carrying the specific binding sites (operators oL and oR) with an estimated dissociation constant of 10(-10) M to 10(-11) M; there is also weaker binding to other sites on DNA, as found for other DNA-binding regulatory proteins. In a purified transcription system, the Cro protein is an effective and specific repressor of RNA synthesis from the N and cro genes; thus Cro is an autorepressor which regulates its own synthesis. A comparison of the properties of the two lambda repressor proteins, cI and Cro, indicates that cI is a "strong repressor" specialized for complete turnoff of lytic functions needed for the maintenance of lysogeny, whereas Cro is a "weak repressor" specialized for a gradual turnoff of early viral genes that potentiates the late stage of lytic development.

Amino Acids↗
PubMed1977

Characterization of the integration protein of bacteriophage lambda as a site-specific DNA-binding protein.

The Int protein specified by bacteriophage lambda is required for the recombination event that integrates the viral DNA into the host genome at its specific attachment site. Using a DNA-binding assay, we have partially purified the Int protein and studied some of the features of its binding specificity and regulation. The DNA-binding activity is attributed to Int protein because the activity is eliminated by a nonsense mutation or a deletion in the int gene, and is rendered thermolabile by temperature-sensitive mutations in the int gene. The DNA-binding activity is specific for DNA carrying an appropriate attachment site, suggesting that Int protein directs the sequence-specific recognition essential for integrative recombination. The specific DNA-binding activity is also missing after infection by phage carrying mutations in the cII and cIII regulatory genes of lambda. This finding corroborates the conclusion from other types of experiments that regulation of the int and cI genes by cII/cIII provides for coordinate regulation of both major events of the lysogenic response, establishment of repression and insertion of viral DNA.

Carrier Proteins↗
PubMed1976

Purification and properties of a DNA-binding protein with characteristics expected for the Cro protein of bacteriophage lambda, a repressor essential for lytic growth.

The Cro protein specified by bacteriophage lambda is a repressor essential for normal lytic growth of the virus, thus having a physiological role distinct from that of cI, the repressor that maintains lysogeny. We have purified a lambda-specific DNA-binding protein with the requirements for synthesis and biochemical activities expected for Cro protein from studies in vivo. As isolated, the protein appears to be a dimer of molecular weight approximately 18,000 with DNA-binding properties that are very similar, but not identical, to those of the cI protein. We infer that bacteriophage lambda uses the same regulatory region of DNA for two different DNA-binding repressor proteins with subtle differences in binding activity specialized for different physiological roles.

Carrier Proteins↗
PubMed1976

On the nature of cis-acting regulatory proteins and genetic organization in bacteriophage: the example of gene Q of bacteriophage lambda.

We note the existence of a "partially cis-acting" regulatory protein of bacteriophage lambda: the product of the phage Q gene. We suggest that there may be a complete spectrum from "all cis" to "all trans" for such regulatory proteins. This behavior might arise because a DNA-binding protein either acts at a nearby (cis) site soon after synthesis or becomes "lost" for its trans activity on another genome through nonspecific interactions with DNA. Our proposed explanation provides one evolutionary basis for the linkage of genes for regulatory proteins and the sites at which such proteins act; it also suggests a possible rationale for the "metabolic instability" of certain regulatory proteins.

Coliphages↗
PubMed1975

The gamma protein specified by bacteriophage gamma. Structure and inhibitory activity for the recBC enzyme of Escherichia coli.

The protein encoded by the gam gene of bacteriophage lambda ("gamma protein") is a specific inhibitor of the recBC enzyme of Escherichia coli. The lambda protein has been purified approximately 2,000-fold, and its structure and inhibitory activity have been characterized. It appears to be composed of two identical subunits of 16,500 daltons, inhibits all of the catalytic activities of the recBC enzyme with apparently equal efficiency, but has no effect upon any other E. coli or lambda-DNase tested. Inhibition does not occur unless recBC enzyme is exposed to gamma protein prior to reaction of the enzyme with DNA. The inhibitory activity is independent of temperature, and no catalytic activity has been detected that might fulfill the inhibitory function. It appears instead that the inhibition involves a stoichiometric, rather than a catalytic interaction between gamma protein and the enzyme. Reaction kinetics for the recBC enzyme inhibited by gamma protein show no anomalous protein--only a depressed rate. Inhibition is not competitive and does not appear to affect the enzyme's affinity for DNA. The enzyme remains inhibited after it is separated from "excess" gamma protein by gel filtration or sedimentation in a glycerol gradient, and inhibited enzyme has a reduced electrophoretic mobility compared to that of uninhibited enzyme. Gamma Protein inhibits recBC enzyme which has been reconstituted from cell-free extracts by complementation in vitro, but at least one of the complementing factors present in extracts from recB- cells does not by itself form a complex with gamma protein. The mechanism of inhibition and the implications of these results from gamma replication and recombination are discussed.

Adenosine Triphosphatases↗