Identification of the in vivo promoters of bacteriophages S13 and phi X174and measurement of their relative activities

Regions of bacteriophages phi X174 and S13 that contain putative promoter s equences were amplified by the polymerase chain reaction (PCR) and cloned i nto the reporter vector pKO-1. Assays of galactokinase activity revealed in vivo promoter activity in those constructs containing the promoter sequenc es with transcription initiation (+1) sites at nucleotide positions 45, 982 , 1823, and 5211. These were identical in location to sequences with in vit ro promoter activity and to the three known promoters P-A, P-B, and P-D. P5 211 is the location of a new, fourth, promoter. A site with a +1 position a t nucleotide 4876, previously shown to initiate RNA synthesis in an in vitr o run-off transcription assay, had no in vivo promoter activity. To investi gate whether flanking sequences had effects on promoter activity, restricti on fragments of phi X174 and S13 that encompass the in vivo promoters were cloned into the reporter vector pKO-1. The P-A and P5211 promoter construct s showed dramatic effects with increases in activity of up to 7 times that shown with the PCR-generated promoter constructs. The phi X174 P-B promoter construct had a 50% decrease in activity compared with the PCR-generated P -B clone. While the data showed that in most instances promoter activity is affected by the flanking sequences in which the promoter is embedded, no g eneral pattern correlating flanking sequences and promoter activity could b e discerned. Additional evidence that the promoter sequence regions were ac tive in vivo promoters was obtained by S1 nuclease mapping experiments. Ini tiation of RNA synthesis was shown at positions 45, 982, and 5211.