Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes

phi 29 DNA directs the synthesis of three major proteins of Mr = 22,400, 13,900, and 10,500 in a cell-free transcription-translation system derived from Bacillus subtilis. We have determined the locations of the coding regions for these early proteins on the phi 29 genome, and our results are in agreement with genetic evidence that the 22.4-kilodalton protein is the product of cistron 17 (p17) and the 13.9-kilodalton protein is the product of cistron 6 (p6). The 13.9-kilodalton and 10.5-kilodalton proteins are encoded on a polycistronic mRNA previously designated G3b RNA. We have determined the nucleotide sequence of a HindIII restriction fragment of phi 29 DNA, the H fragment, that encodes the 13.9-kilodalton protein and contains two early promoters, G3a and G3b. The nucleotide sequence of the ribosome binding site contains a polypurine region capable of forming a very stable complex with nine bases on the 3' end of B. subtilis 16 S rRNA. This strong Shine-Dalgarno complementarity supports the hypothesis that an extensive mRNA . rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291). The nucleotide sequences of both promoters are very similar to the "consensus" sequence for Escherichia coli promoters. While E. coli RNA polymerase initiates transcription from both of these promoters in vitro, only the G3b promoter is utilized by B. subtilis RNA polymerase under the same conditions.