Identification of two middle promoters upstream DNA ligase gene 30 of bacteriophage T4

Bacteriophage T4 DNA ligase gene 30 lies in the cluster of prereplicative genes located counterclockwise from map units 149 to 121. Based on the early transcription studies this gene has been considered as a typical early gene of bacteriophage T4. In agreement with this assignment, two strong T4 early promoters, PE 30.8 (128.6) and PE 30.7 (128.2), located about 3.1 and 2.7 kb upstream from gene 30 have been revealed by promoter mapping and sequence analysis. In addition, the existence of a putative early promoter just upstream of gene 30 was proposed from the sequence data. However, here we show that the putative early promoter just upstream of gene 30 is, in fact, a T4 middle promoter. Furthermore, we detected one more middle promoter located in the genomic region between early promoter PE 30.7 (128.2) and DNA ligase gene 30 in the coding region of gene 30.3. Both new middle promoters have differences from the consensus MotA box, while their −10 regions match the σ70 consensus sequence very well. The 5′ ends of MotA-dependent transcripts directed from these promoters, as well as the kinetics of 5′ end accumulation in the cells, have been determined by primer extension analysis. The results of these analyses indicate that both MotA-dependent and MotA-independent promoters control the transcription of T4 DNA ligase gene 30 in vivo. Moreover, we show that the first transcripts for gene 30 are directed from its own middle promoter, PM 30.