Identification of the in vivo promoters of bacteriophages S13 and ϕX174 and measurement of their relative activities

Abstract

Regions of bacteriophages ϕX174 and S13 that contain putative promoter sequences were amplified by the polymerase chain reaction (PCR) and cloned into the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequences with transcription initiation (+1) sites at nucleotide positions 45, 982, 1823, and 5211. These were identical in location to sequences with in vitro promoter activity and to the three known promoters PA, PB, and PD. P5211 is the location of a new, fourth, promoter. A site with a +1 position at nucleotide 4876, previously shown to initiate RNA synthesis in an in vitro run-off transcription assay, had no in vivo promoter activity. To investigate whether flanking sequences had effects on promoter activity, restriction fragments of ϕX174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The PA and P5211 promoter constructs showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The ϕX174 PB promoter construct had a 50% decrease in activity compared with the PCR-generated PB clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no general pattern correlating flanking sequences and promoter activity could be discerned. Additional evidence that the promoter sequence regions were active in vivo promoters was obtained by S1 nuclease mapping experiments. Initiation of RNA synthesis was shown at positions 45, 982, and 5211.Key words: promoters, phage S13, phage phiX174, in vivo.