P22 repressor mutants deficient in co-operative binding and DNA loop formation.

We show here, both in vivo and in vitro, that P22 repressor binds co-operatively to operator sites separated by an integral number of turns of the DNA helix. We measure this co-operativity in vivo using an assay in which repression of a promoter requires co-operative binding of P22 repressors to two separated (non-adjacent) operator sites. We report the isolation of mutant repressors that have high affinity for single operator sites, but are defective in co-operative binding. Six different mutants, all bearing single amino acid changes in the carboxyl domain, have been isolated. We purified the two mutants most deficient in co-operative binding, and found that they bind non-co-operatively in vitro to adjacent as well as to non-adjacent pairs of operator sites.     

Proposed alpha-helical super-secondary structure associated with protein-dna recognition

Knowledge of the three-dimensional structure of the bacteriophage λ Cro repressor, combined with an analysis of amino acid sequences and DNA coding sequences for this and other proteins that recognize and bind specific base sequences of double-helical DNA, suggests that a portion of the structure of the Cro repressor that is involved in DNA binding also occurs in the Cro protein from bacteriophage 434, the cII protein from bacteriophage λ, the Salmonella phage P22 c2 repressor and the cI repressor from bacteriophage λ. This α-helical super-secondary structure may be a common structural motif in proteins that bind double-helical DNA in a base sequence-specific manner.     

Electron microscopic study of the repressor of bacteriophage λ and its interaction with operator DNA

The structure of purified phage λ repressor has been examined by high resolution electron microscopy. The repressor molecule appears predominantly as a tetramer of about 95 Å × 120 Å. We have proposed a model to account for the variety of aspects seen on the electron micrographs. Spreading DNA without protein film and use of uranyl formate staining allowed the simultaneous visualization of the DNA and the structure of the repressor molecule bound to it. Mapping the positions of λ repressor bound to whole λ DNA shows preferential binding to the region containing the operators. At high resolution multiple binding of repressor to the operator can be demonstrated. Depending on the amount of repressor present, rows of one to four repressor tetramers are seen on the DNA, confirming the model of the operator containing four binding sites for repressor. The bound repressor can consequently protect against nuclease digestion of operator pieces of approximately 30, 57, 87 and 111 base-pairs. The isolated operator appears in the electron microscope as short double-stranded DNA fragments which can be shown to rebind repressor.     

The gene encoding the periplasmic cyclophilin homologue, PPlase A, in Escherichia coli, is expressed from four promoters, three of which are activated by the cAMP–CRP complex and negatively regulated by the CytR repressor

The rot gene in Escherichia coli encodes PPlase A, a periplasmic peptldyl-prolyl cis-trans isomerase with homology to the cyclophilin family of proteins. Here it is demonstrated that rot is expressed in a complex manner from four overlapping promoters and that the rot regulatory region is unusually compact, containing a close array of sites for DNA-binding proteins. The three most upstream rot promoters are activated by the global gene regulatory cAMP–CRP complex and negatively regulated by the CytR repressor protein. Activation of these three promoters occurs by binding of cAMP–CRP to two sites separated by 53 bp. Moreover, one of the cAMP–CRP complexes is involved in the activation of both a Class I and a Class II promoter. Repression takes place by the formation of a CytR/cAMP–CRP/DNA nucleoprotein complex consisting of the two cAMP–CRP molecules and CytR bound in between. The two regulators bind co-operatively to the DNA overlapping the three upstream promoters, simultaneously quenching the cAMP–CRP activator function. These results expand the CytR regulon to include a gene whose product has no known function in ribo- and deoxyribonucleoside catabolism or transport.     

Cellular levels of the prophage lambda and 434 repressors.

As a prerequisite to a quantitative study of the inactivation of phage repressors in vivo (Bailone et al., 1979), the cellular concentrations of the bacteriophage λ and 434 repressors have been measured in bacteria with varying repressor levels.Using the DNA-binding assay we have determined the conditions for optimal repressor titration. The sensitivity of the λ repressor assay was increased by adding magnesium ions to the binding mixture; this procedure was without effect on the titration of the 434 repressor. The measures of the cellular repressor concentrations varied with the method of cell disruption.The cellular concentration of λ repressor, about 140 active repressor molecules per monolysogen, was relatively constant under specific cultural conditions. The repressor concentration increased with the number of cI gene copies but not in direct proportion.The 434 repressor concentration, hardly detectable in extracts of lysogens carrying an imm434 prophage, was greatly enhanced in bacteria carrying the newly constructed plasmid pGY101, that encodes the 434 cI gene.The cellular repressor level produced by 434 is lower than that produced by λ: this indicates that the maintenance of the prophage state is ensured by a relatively small number of repressor molecules binding tightly to the operator sites.     

Kinetics of RecA protein-directed inactivation of repressors of phage lambda and phage P22

Escherichia coli recA protein directs the inactivation of the repressor of Salmonella typhimurium phage P22 in vitro. As is true for repressor of the E. coli phage λ, inactivation of P22 repressor is accompanied by proteolytic cleavage of the repressor into two detectable fragments.We have investigated the kinetics of inactivation of the λ and P22 repressors in vitro. The fraction of λ repressor inactivated per unit time decreases as its concentration in the reaction is increased. However, high concentrations of λ repressor do not inhibit the inactivation of P22 repressor. Thus, it does not appear that the inactivation system is saturated by λ repressor, but rather that λ repressor is a less efficient substrate at higher concentrations.     

Lipooligosaccharide biosynthesis in Neiseria gonorrhoeae: cloning, identification and characterization of the α1,5 heptosyltransferase I gene (rfaC)

The Escherichia coli arginine repressor (ArgR) is an l -arginine-dependent DNA-binding protein that controls expression of the arginine biosynthetic genes and is required as an accessory protein in Xer site-specific recombination at cer and related recombination sites in plasmids. Site-directed mutagenesis was used to isolate two mutants of E. coli ArgR that were defective in arginine binding. Results from in vivo and in vitro experiments demonstrate that these mutants still act as repressors and bind their specific DNA sequences in an arginine-independent manner. Both mutants support Xer site-specific recombination at cer. One of the mutant proteins was purified and shown to bind to its DNA target sequences in vitro with different affinity and as a different molecular species to wild-type ArgR.     

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.     

Specificity of translational regulation by two DNA-binding proteins

The gene V protein of the filamentous bacteriophages f1, fd and M13, and the gene 32 protein of bacteriophage T4 share the property of binding strongly and co-operatively to single-stranded nucleic acids, especially DNA. Moreover, both are capable of repressing the translation of specific mRNAs (gene 32 protein its own, and gene V protein that of the filamentous phage gene II), both in vivo and in vitro. If the mechanism of repression by either of these proteins were based solely on its ability to bind single strands co-operatively, then the other would be expected to mimic or interfere with its effect in vitro. We have found no such mimicry or interference, even at protein concentrations high enough to have substantial non-specific effects on translation. This suggests that the sites of repression on the mRNAs must offer something other than simple “unstructuredness” for binding and repression to occur.