The c4 repressors of bacteriophages P1 and P7 are antisense RNAs.

The c4 repressors of P1 and P7 inhibit antirepressor synthesis and are solely responsible for heteroimmunity of the phages. We show that c4 is a new type of antisense RNA acting on a target, ant mRNA, that is transcribed from the same promoter. Interaction depends on complementarity of two pairs of short sequences encompassing the ribosome binding site involved in ant expression. We demonstrate that heteroimmunity of P1 and P7 is due to just two substitutions in each of the complementary sequences of c4 and ant mRNA. Based on P1-P7 sequence comparison and a mutant analysis, we propose a secondary structure model for c4 RNA, with the complementary regions in loops as important sites for antisense control.     

Effects of mutations in the immunity system of bacteriophage P1.

A mutant of bacteriophage P1 that made an altered c1 repressor is described. The mutant c1 product had two configurations: in lysogens, at high temperatures, it permitted constitutive expression of the normally repressed DNA replication function ban and was insensitive to the action of ant, a product expressed by the virulent mutant P1virs and by the heteroimmune phage P7 (formerly phiamp+) and normally able to overcome c1 repression; in mutant lysogens at low temperatures, the mutant repressor was apparently normal (able to repress ban and sensitive to ant action). Genetic studies of this mutant led to the isolation of a derivative that formed unstable lysogens. These studies suggested that the ban product was normally under c1 control; they further showed that ant overcame c1 repression by inactivating c1 rather than by creating a bypass of repressor activity.     

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 c4 repressor of bacteriophage P1 is a processed 77 base antisense RNA.

The c4 repressors of the temperate bacteriophages P1 and P7 inhibit antirepressor synthesis and are essential for establishment and maintenance of lysogeny. Using in vivo complementation tests we have previously shown that c4 is an antisense RNA acting on a target, ant mRNA, which is transcribed from the same promoter. Here we identify the c4 repressor molecule of P1 as a 77 +/- 1 base RNA by mapping its termini and show that the c4 RNA in P7 lysogens has the same or a similar size. P1 c4 RNA is encoded in a region shown to be sufficient for c4 complementation. It covers exactly the 74 bases previously suggested to fold into a stem-loop secondary structure essential for c4 function. Furthermore, we demonstrate that the 5' end of c4 RNA is generated by processing. Thus, c4 is the first example of an antisense RNA to be processed. A possible mechanism of processing is discussed.     

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.     

Sequence determinants of promoter activity

The bacteriophage P22 promoter for the antirepressor (ant) gene, Pant, in the absence of Arc repressor, directs the synthesis of extremely high levels of antirepressor. Overproduction of antirepressor leads secondarily to the failure to produce progeny phage upon lytic infection. A substantial fraction of revertants of P22 arc-amber phage are pseudorevertants that have acquired additional mutations that decrease the activity of the ant promoter. DNA sequence analysis of 72 independent Pant "promoter-down" mutations reveals more than 25 different alterations that define two regions critical for promoter activity. With few exceptions, these promoter-down mutations decrease the homology of Pant with the consensus promoter sequence, demonstrating that the conserved features among a large number of different wild-type promoters are the determinants of promoter strength.l In general, different substitution mutations at the same site within the promoter have similar effects, resulting in either a severe or a mild reduction in promoter activity.     

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.     

Genetic analysis of the lytic replicon of bacteriophage P1. I. Isolation and partial characterization

Despite the extensive genetic analysis of bacteriophage P1, the region of the viral genome that is responsible for its lytic (vegetative) replication has not been identified. In this paper we describe the identification of various fragments of P1 DNA that can replicate an otherwise replication-defective lambda vector when they are cloned into that vector. The fragments share a 2800 base-pair segment of the P1 genome that is located adjacent to the immI region of the phage. Replication mediated by the cloned P1 fragments is abolished by the product of the P1 c1 gene, the repressor of phage lytic functions. Since these properties resemble those of the P1 lytic replicon, we suggest that the 2800 base-pair segment identified here contains that replicon.     

A new gene of bacteriophage P22 which regulates synthesis of antirepressor

Two new mutants of bacteriophage P22 are described which define a new regulatory gene, arc (for antirepressor control). The properties of the arc mutants and of 31 phenotypic revertants indicate that the arc gene codes for a trans-acting protein whose primary role is to depress synthesis of P22 antirepressor protein during the lytic cycle of infection. Failure to regulate antirepressor production apparently leads secondarily to a lethal defect (i.e. failure to produce progeny phage).Although under certain conditions the arc function can be expressed by P22 prophages and can act as a weak barrier to superinfecting homologous phage, the arc product is neither necessary nor sufficient for maintenance of the prophage state or superinfection immunity in lysogens. Instead, as shown previously by others (Levine et al., 1975; Botstein et al., 1975), the prophage mnt gene product is responsible for repressing antirepressor synthesis, both by the prophage and by superinfecting phage.     

The Bof protein of bacteriophage P1 exerts its modulating function by formation of a ternary complex with operator DNA and C1 repressor.

Bacteriophage P1 encodes several regulatory elements for the lytic or lysogenic response, which are located in the immC, immI, and immT regions. Their products are the C1 repressor of lytic functions with the C1 inactivator protein Coi, the C4 repressor of antirepressor synthesis and the modulator protein Bof, respectively. We have studied in vitro the interaction of the components of the immC and immT regions with C1-controlled operators using highly purified Bof, C1, and Coi proteins. Bof protein (M(r) = 9,800) does not interact with C1 repressor alone, but as shown by DNA mobility shift experiments, in the presence of C1 repressor Bof binds to all operators tested by forming a C1.Bof-operator DNA ternary complex. The effect of this complex formation was studied in more detail with the operator of the c1 gene. Here, Bof only marginally alters the C1 repressor footprint at Op99a,b, but nevertheless considerably influences the repressibility of the operator.promoter element: (i) the autoregulated c1 mRNA synthesis is further down-regulated and (ii) the ability of Coi protein to dissociate the C1.operator DNA complex is strongly inhibited. We suggest that Bof protein functions by modulating C1 repression of many widely dispersed operators on the prophage genome.     

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.     

Isolation of Lambda Transducing Phage with the bio Genes Inserted between Lambda Genes P and Q

Plaque-forming, biotin-transducing phages were constructed with the bio genes inserted between lambda genes P and Q. These phages were isolated for the eventual aim of fusing the lambda Q gene to the bio operon. The following steps were used to construct these phages: A defective temperature-sensitive lysogen was constructed with the bio genes adjacent to and to the left of lambda genes beta NcI857OPQSRA. Heat-resistant survivors were screened for deletions with endpoints in the bio operon and to the right of lambda P and to the left of lambda A. Five of approximately 1,600 heat-resistant survivors had these properties. Two had the gene order bioAB .... lambda QSRA. When these two strains were lysogenized with lambda cI857b221 and heat induced, the desired transducing phages were obtained. We characterized these phages and studied one in detail. Two-thirds of the plaque-forming transducing phages isolated carried the entire bioB gene and only part of the bioA gene, and one-third carried the entire bioA and bioB genes. The phages isolated lost the bio genes upon propagation, indicating that they contain a partial duplication of phage genes. The duplication was shown not to involve the entire lambda Q gene in one of these phages, lambda bioq1b221. A recombinant of this phage, lambda Nam7am53c17b221, failed to form plaques under biotin-derepression conditions. We conclude that if the lambda Q gene was fused to the bio operon in this phage, not enough lambda Q gene product was made to allow phage propagation.     

Polarity suppressors increase expression of the wild-type tryptophan operon of Escherichia coli

Three new polarity suppressors, selected to relieve the polar effect of nonsense mutations in the tryptophan (trp) and lactose (lac) operons of Escherichia coli, increase expression distal to nonsense mutations in both operons to a greater extent than suA. These suppressors relieve the polarity created by amber, ochre and frameshift mutations with equal efficiency.Two of the three polarity suppressors elevate enzyme synthesis in the wildtype trp operon two- and fivefold, respectively. The increase in enzyme levels is in each case correlated with increased levels and rates of synthesis of structural gene trp messenger RNA. Since expression of all genes is elevated, these findings suggest the existence of a site early in the wild-type trp operon that affects the extent of operon expression. We located the site affected by these two polarity suppressors between the operator and the first structural gene, trpE. Although the third polarity suppressor also relieves mutational polarity efficiently, it has no detectable effect on expression of the wild-type trp operon.