C1 repressor of phage P1 is inactivated by noncovalent binding of P1 Coi protein.

The temperate phage P1 encodes two genes whose products antagonize the action of the phage's C1 repressor of lytic functions, namely a distantly linked antirepressor gene, ant, and a closely linked c1 inactivator gene, coi. Starting with an inducible coi-recombinant plasmid, Coi protein was overproduced and purified to near homogeneity. By using a DNA mobility shift assay we demonstrate that Coi protein inhibits the operator binding of the C1 repressors of the closely related P1 and P7 phages. Coi protein (Mr = 7,600) exerts its C1-inactivating function by forming a complex with the C1 repressor (Mr = 32,500) at a molar ratio of about 1:1, as shown by density gradient centrifugation and gel filtration. C1 repressor and Coi protein are recovered in active form from the complex, suggesting that noncovalent interactions are the sole requirements for complex formation. The interplay of repressor and antagonists operating in the life cycle of P1 is discussed.     

A bacteriophage P1-encoded modulator protein affects the P1 c1 repression system

Bacteriophage P1 encodes a tripartite immunity system composed of the immC, immI, and immT region. Their basic genetic elements are the c1 repressor of lytic functions, the c4 repressor which negatively regulates antirepressor synthesis, and the bof gene, respectively. The function of the latter will be described here. We have cloned and sequenced the bof gene from P1 wild type and a P1 bof amber mutant. Based on the position of a TAG codon of the bof amber mutant the bof wild type gene was localized. It starts with a TTG codon, comprises 82 codons, and is preceded by a promoter structure. The bof protein (Mr = 7500) was overproduced in Escherichia coli from a bof recombinant plasmid and was purified to near homogeneity. The N-terminal amino acids predicted from the DNA sequence of the bof gene were confirmed by sequence analysis of the bof protein. Using a DNA mobility shift assay, we show that bof protein enhances the binding of c1 repressor to the operator of the c1 gene. In accordance with this result, in transformants of Escherichia coli, containing both a bof- and a c1-encoding plasmid, c1 expression is down-regulated. We conclude that bof acts as a modulator protein in the repression of a multitude of c1-controlled operators in the P1 genome.     

The c1 repressor inactivator protein coi of bacteriophage P1. Cloning and expression of coi and its interference with c1 repressor function

The immC region of bacteriophage P1 contains the c1 repressor gene and its upstream region with four c1-controlled operators and four open reading frames. A c1 inactivator gene, coi, was defined by mutations in immC that suppress the virulence of the P1virC mutation. The exact location of the coi gene was not known (Scott, J.R. (1980) Curr. Top. Microbiol. Immunol. 90, 49-65). When a variety of P1 immC fragments were inserted into an expression vector, a gene product was inducible for the open reading frame 4 only. We identify this product as the c1 inactivator protein, coi by the following criteria: (a) expression of coi from a recombinant plasmid induces the P1 prophage and inhibits lysogenization of sensitive bacteria by P1; (b) all c1-controlled operator-promoter elements tested in vivo are derepressed by coi; (c) a partially purified coi protein (apparent molecular weight = 4800) interacts with c1 repressor and inhibits its binding to the operator in vitro. Based on these results we refine a model for the regulation of those genes and elements within immC which participate in the decision of P1 to enter the lytic or lysogenic pathway.     

The bof gene of bacteriophage P1: DNA sequence and evidence for roles in regulation of phage c1 and ref genes.

The C1 repressor of bacteriophage P1 acts via 14 or more distinct operators. This repressor represses its own synthesis as well as the synthesis of other gene products. Previously, mutation of an auxiliary regulatory gene, bof, has been shown to increase expression of some C1-regulated P1 genes (e.g., ref) but to decrease expression of others (e.g., ban). In this study the bof gene was isolated on the basis of its ability to depress stimulation of Escherichia coli chromosomal recombination by the P1 ref gene, if and only if a source of C1 was present. C1 alone, but not Bof alone, was partially effective. The bofDNA sequence encodes an 82-codon reading frame that begins with a TTG codon and includes the sites of the bof-1(Am) mutation and a bof::Tn5 null mutation. Expression of ref::lacZ and cl::lacZ fusion genes was partially repressed in trans by a P1 bof-1 prophage or by plasmid-encoded C1 alone, which was in agreement with effects on Ref-stimulated recombination and with previous indirect evidence for c1 autoregulation. Repression of both fusion genes by plasmid-encoded C1 plus Bof or by a P1 bof+ prophage was more complete. When the C1 source also included a 0.7-kilobase region upstream from C1 which encodes the coi gene, repression of both c1::lacZ and ref::lacZ by C1 alone or by C1 plus Bof was much less effective, as if Coi interfered with C1 repressor function.     

The tripartite immunity system of phages P1 and P7

Abstract: Prophages P1 and P7 exist as unit copy DNA plasmids in the bacterial cell. Maintenance of the prophage state requires the continuous expression of two repressors: (i) C1 is a protein which negatively regulates the expression of lytic genes including the C1 inactivator gene coi , and (ii) C4 is an antisense RNA which specifically inhibits the synthesis of an anti-repressor Ant. In addition, C1 repression is strengthened by lxc encoding an auxiliary repressor protein. The repressors C1, C4 and Lxc are components of a tripartite immunity system of the two phages. Here, the mode of action of these regulatory components including their antagonists Coi and Ant is described.     

ban operon of bacteriophage P1. Mutational analysis of the c1 repressor-controlled operator

The repressor of bacteriophage P1, encoded by the c1 gene, represses the phage lytic functions and is responsible for maintaining the P1 prophage in the lysogenic state. The c1 repressor interacts with at least 11 binding sites or operators widely scattered over the P1 genome. From these operators, a 17 base-pair asymmetric consensus sequence, ATTGCTCTAATAAATTT, was derived. Here, we show that the operator, Op72 of the P1ban operon consists of two overlapping 17 base-pair sequences a and b forming an incomplete palindrome. Op72a matches the consensus sequence, whereas Op72b contains two mismatches. The evidence is based on the sequence analysis of 27 operator mutants constitutive for ban expression. They were identified as single-base substitutions at positions 2 to 10 of Op72a (26 mutants) and at position 8 of Op72b (one mutant). We conclude from gel retardation and footprinting studies that two repressor molecules bind to the operator and that positions 4, 5 and 7 to 10 of the operator play an essential role in repressor recognition.     

Bacteriophage P1 Bof protein is an indirect positive effector of transcription of the phage bac-1 ban gene in some circumstances and a direct negative effector in other circumstances

Previous genetic studies have suggested that the Bof protein of bacteriophage P1 can act as both a negative and a positive regulator of phage gene expression: in bof-1 prophages, the ref gene and a putative phage ssb gene are derepressed, but expression of an operator-semiconstitutive variant of the phage ban gene (bac-1) is markedly reduced. An explanation of this apparent duality is suggested by recent reports that Bof is a corepressor of genes that are regulated by the phage C1 repressor, including the autoregulated c1 gene itself. Here we show, by means of operon fusions to lacZ, that the balance points between Bof-mediated decreases in c1 expression and Bof-mediated increases in C1 efficacy are different among various C1-regulated genes. Thus, expression of Bof by P1 prophages affects some genes (e.g., bac-1 ban) positively, and others (e.g., ref) negatively. Even at bac-1 ban, where the positive indirect effect of Bof is physiologically dominant, Bof can be seen to act as a corepressor if C1 is supplied from a nonautoregulated (ptac-c1) source, eliminating the effect of Bof on C1 synthesis.     

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.     

Three additional operators, Op21, Op68, and Op88, of bacteriophage P1. Evidence for control of the P1 dam methylase by Op68

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state. Previously, 11 c1 repressor binding sites or operators scattered over the whole genome of P1 have been found. From sequence analysis an asymmetric, 17-base pair consensus sequence, ATTGCTCTAATAAATTT, was derived. Using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators. We have mapped the operators at the positions 21,68, and 88 of the P1 genome and determined their sequence. These operators are controlled by c1 because corresponding P1 DNA fragments (i) require c1 repressor in vivo in order to be clonable in multicopy plasmids, (ii) exhibit a c1-repressible promoter activity, (iii) are retarded by c1 repressor protein during electrophoresis, and (iv) contain the 17-base pair consensus sequence with one mismatch base each. Furthermore, we suggest that expression of the DNA adenine methylase (dam) encoded by P1 is controlled via Op68.     

Purification and properties of phage P22 c2 repressor.

The c2 repressor of phage P22 has been purified to homogeneity. It specifically binds to lambdaimm21 and P22 DNA. Its affinity for the presumed operator mutant P22 virB is reduced. The initial dissociation rates of the complex between c2 repressor and lambdaimm21 DNA are 0.02 min-1 at 0 degrees C, 0.08 min-1 at 20 degrees C and 0.17 min-1 at 32 degrees C. The dissociation rates of complexes formed between the c2 repressor and the lambdaimm21 operators OR, OL and OR vira were measured and compared to the corresponding rates obtained with 21 cI repressor.     

Dual Regulatory Control of a Particle Maturation Function of Bacteriophage P1

A unique arrangement of promoter elements was found upstream of the bacteriophage P1 particle maturation gene (mat). A P1-specific late-promoter sequence with conserved elements located at positions -22 and -10 was expected from the function of the gene in phage morphogenesis. In addition to a late-promoter sequence, a -35 element and an operator sequence for the major repressor protein, C1, were found. The -35 and -10 elements constituted an active Escherichia coli sigma(70) consensus promoter, which was converted into a P1-regulated early promoter by the superimposition of a C1 operator. This combination of early- and late-promoter elements regulates and fine-tunes the expression of the particle maturation gene. During lysogenic growth the gene is turned off by P1 immunity functions. Upon induction of lytic growth, the expression of mat starts simultaneously with the expression of other C1-regulated P1 early functions. However, while most of the latter functions are downregulated during late stages of lytic growth the expression of mat continues throughout the entire lytic growth cycle of bacteriophage P1. Thus, the maturation function has a head start on the structural components of the phage particle.     

The c1 repressor of bacteriophage P1 operator-repressor interaction of wild-type and mutant repressor proteins.

The c1 repressor gene of bacteriophage P1 and the temperature-sensitive mutants P1c1.100 and P1c1.162 was cloned into an expression vector and the repressor proteins were overproduced. A rapid purification procedure was required for the isolation of the thermolabile repressor proteins. Identification of the highly purified protein of an apparent molecular weight of 33,000 as the product of the c1 gene was verified by (i) the coincidence of partial amino acid sequences determined experimentally to that deduced from the c1 DNA sequence, and (ii) the temperature-sensitive binding to the operator DNA of the thermolabile repressor proteins. Analysis of the products of c1-c1.100 recombinant DNAs relates the thermolability to an unknown alteration in the C-terminal half of the c1.100 repressor. Binding to the operator DNA of c1 repressor is sensitive to N-ethylmaleimide. Since the only three cysteine residues are located in the C-terminal half of the repressor it is suggested that this part of the molecule is important for the binding to the operator DNA. This assumption is supported by the findings that a 14-kDa C-terminal repressor fragment obtained by cyanogen bromide cleavage retains DNA binding properties.     

Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences.

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super-repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer-DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co-ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.     

Bending of synthetic bacteriophage 434 operators by bacteriophage 434 proteins.

The extent of DNA bending induced by 434 repressor, its amino terminal DNA binding domain (R1-69), and 434 Cro was studied by gel shift assay. The results show that 434 repressor and R1-69 bend DNA to the same extent. 434 Cro-induced DNA bends are similar to those seen with the 434 repressor proteins. On approximately 265 base pair fragments, the cyclic AMP receptor protein of Escherichia coli (CRP) produces larger mobility shifts than does 434 repressor. This indicates that the 434 proteins bend DNA to a much smaller extent than does CRP. The effects of central operator sequence on intrinsic and 434 protein-induced DNA bending was also examined by gel shift assay. Two 434 operators having different central sequences and affinities for 434 proteins display no static bending. The amount of gel shift induced by 434 repressor on these operators is identical, showing that the 434 repressor bends operators with different central sequences to the same extent. Hence, mutations in the central region of the operator do not influence the bent structure of the unbound or bound operator.     

Effects of physical, ionic, and structural factors on the binding of repressor of mycobacteriophage L1 to its cognate operator DNA

To determine the factors influencing the binding of L1 repressor to its cognate operator DNA, several gel shift as well as bioinformatic analyses have been carried out. The data show that time, temperature, salt, and pH each greatly affect the binding. In order to achieve optimum operator binding of L1 repressor in Tris buffer, the minimum requirements of time, temperature, salt, and pH were estimated to be 1 min, 32 degrees C, NaCl (50 mM), and 7.9, respectively. Interestingly Na+ but not NH4+, K+, or Li+ was found to augment significantly the binding activity of CI protein above the basal level. Anions like Cl-, citrate-, acetate-, and H2PO4- do not alter the binding of L1 repressor to its operator. We also show that an in frame deletion mutant of L1 repressor which does not carry the putative HTH motif (at its N-terminal end) fails to bind to its cognate operator DNA even at very high concentrations. The putative HTH motif was found highly conserved and evolutionarily very close to that of regulatory proteins of Y. pestis, H. marismortui, A. tumefaciens, etc. Taken together we suggest that N-terminal end of L1 repressor carries a HTH motif. Further analysis of the putative secondary structures of mycobacteriophage repressors reveals that two common regions encompassing more than 90% of primary sequence are present in all the four repressor molecules studied here. The results suggest that these common regions are utilized for carrying out identical functions.     

Crystallographic analysis of Lac repressor bound to natural operator O1

Previous structures of Lac repressor bound to DNA used a fully symmetric "ideal" operator sequence that is missing the central G-C base-pair present in the three natural operator sequences. Here we have determined the X-ray crystal structure of a dimeric Lac repressor bound to a 22 base-pair DNA with the natural operator O1 sequence and the anti-inducer ONPF, at 4.0 A resolution. The natural operator is bent in the same way as the symmetric sequence, due to the binding of the hinge helices of the repressor to the minor groove at the central GCGG sequence of O1. Comparison of the structures of the repressor bound to the natural and symmetric operators shows very similar overall structures, with only slight rearrangements of the headpiece domains of the repressor. Analysis of crystals with iodinated DNA shows that the operator is uniquely positioned and allows for the sequence registration of the DNA relative to the repressor to be determined. The kink in the operator is centered between the left half-site and the central G-C base-pair of O1. Our results are most consistent with a previously proposed model in which, relative to the complex with the symmetric operator, the repressor accommodates binding to the natural operator sequence by shifting the position of the right headpiece by one base-pair step towards the center of O1.     

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage lambda repressor and its interaction with operator sites OL1 and OR3.

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.