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

ABSTRACT: 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.

SUBMITTER: Citron M

PROVIDER: S-EPMC312442 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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

Citron M M Schuster H H

Nucleic acids research 19920601 12

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 ...[more]

PMID: 1620606

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Project description:The coding regions of six putative open reading frames (ORFs) identified near the phage phi31 late promoter and the right cohesive end (cos) of lactococcal bacteriophage phi31 were used to develop antisense constructs to inhibit the proliferation of phage phi31. Two middle-expressed ORFs (ORF 1 and ORF 2) and four late-expressed ORFs (ORF 3 through ORF 6) were cloned individually between the strong Lactobacillus P6 promoter and the T7 terminator (T(T7)) to yield a series of antisense RNA transcripts. When expressed on a high-copy-number vector from a strong promoter, the constructs had no effect on the efficiency of plaquing (EOP) or the plaque size of phage phi31. To increase the ratio of antisense RNA to the targeted sense mRNA appearing during a phage infection, the antisense cassettes containing the late-expressed ORFs (ORF 3 through ORF 6) were subcloned to pTRK360, a low-copy-number vector containing the phage phi31 origin of replication, ori31. ori31 allows for explosive amplification of the low-copy-number vector upon phage infection, thereby increasing levels of antisense RNA transcripts later in the lytic cycle. In addition, the presence of ori31 also lowers the burst size of phage phi31 fourfold, resulting in fewer sense, target mRNAs being expressed from the phage genome. The combination of ori31 and P6::anti-ORF 4H::T(T7) resulted in a threefold decrease in the EOP of phage phi31 (EOP = 0.11 +/- 0.03 [mean +/- standard deviation]) compared to the presence of ori31 alone (EOP = 0.36). One-step growth curves showed that expression of anti-ORF 4H RNA decreased the percentage of successful centers of infection (75 to 80% for ori31 compared to 35 to 45% for ori31 plus anti-ORF 4H), with no further reduction in burst size. Growth curves performed in the presence of varying levels of phage phi31 showed that ori31 plus anti-ORF 4H offered significant protection to Lactococcus lactis, even at multiplicities of infection of 0.01 and 0.1. These results illustrate a successful application of an antisense strategy to inhibit phage replication in the wake of recent unsuccessful reports.

Dataset Information

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

Publications

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

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