Bidirectional Transcription and the Regulation of Phage λ Repressor Synthesis

Abstract

There are two promoters for transcription of gene cI in phage X, the gene that codes for phage repressor. The promoters, called pre and prm, are located on the distal (pre) and proximal (prm) sides of gene cro, which itself is adjacent to cI. Since ci and cro are transcribed in opposite directions, cl transcription initiating at pre gives rise to an antisense transcript of cro, while cl transcription initiating at prm does not. Pre, active after infection of a sensitive cell, is stimulated by products of phage genes cHi and c'ii, and may be located at the site defined by the rhutant cY. Prm is active in art established lysogen. These conclusions are based on measurements of the rates of synthesis of antisense cro RNA, cI RNA, and repressor protein in infected and lysogenic cells. To measure antisense RNA, an assay based on the formation of nucleaseresistant, double-stranded RNA, specific to the cro region, was developed. These results raise the possibility that bidirectional transcription of cro has a regulatory function in phage X.The genes and transcription pattern of the regulatory region of phage X are shown in Fig. 1. Synthesis of the phage cro gene product depends on rightward transcription from the promoter Pr (1-3), while synthesis of phage repressor requires -leftward transcription of cI (4,5). Furthermore, the ci gene can be expressed by either of two pathways, one active during the establishment of lysogeny and the other maintaining the repressor concentration in immune lysogens (6). Normal cI gene expression after infection requires a DNA site, cY, to the right of cro, and the cdI and cdii gene products (1, 2, 6-8).Although cI expression in a lysogen is independent of these activators and INA site, it appears to depend on active repressor bound to a DNA site, Or, which lies to the left of cro (6,9). If c Y and Or define two promoters for cI gene transcription (6), then antisense RNA should be transcribed from the cro region during the establishment of lysogeny, but not in an established lysogen. This paper reports a test of this prediction. MATERIALS AND METHODSProcedures for growth of cells, pulse-labeling, extraction of RNA, and analysis of labeled RNA by RNA-DNA hybridization have been described (5), as have procedures for phage infection and assay of repressor (6, 9, 10). Leftward, 1, and rightward, r, messenger RNA molecules have their 5' 3' arrows pointing to the left and to the right on the genetic map as conventionally drawn (Fig. 1). Because, by convention (12, 13), the 1 strand of phage X DNA is the template for leftward transcription, 1 RNA is complementary to the 1 strand of X DNA and will hybridize with it. The assay for leftward RNA specific to the cro region (1 cro RNA) is described in Fig. 2. RNA concentrations were measured with orcinol (11). RESULTS Detection of 1 cro RNASince double-stranded RNA is resistant to a mixture of pancreatic and T1 ribonucleases under conditions where singlestranded RNA is completely degraded (14), we suspected that 1 cro RNA could be detected as...

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