MOLECULAR-CLONING AND CHARACTERIZATION OF BACTERIOPHAGE-P2 GENE-R ANDGENE-S INVOLVED IN TAIL COMPLETION

The sequences of two previously known tail genes, R and S, of the temp erate bacteriophage P2 and the sequence of an additional open reading frame (orf-30) located between S and V,were determined. Amber mutation s mapping within R and S, Ram3, Ram42, Ram23, Sam75, and Sam89 were se quenced and found to be within their corresponding open reading frames . We constructed overproducing plasmids for R and S and identified the se proteins by SDS-PAGE of whore-cell lysates and Coomassie blue stain ing. The predicted molecular masses of proteins R and S were M(r) 17,4 00 and 17,300, respectively, although both polypeptides migrated more slowly during gel electrophoresis than would be expected from the sequ ence data. orf-30 occupies the strand opposite from RS and V and is pr eceded by several weak potential sigma(70)-RNA polymerase promoters, s ome of which overlap with the V promoter. A construct that had the put ative orf-30 promoter region upstream of the lacZ gene produced low le vers of beta-galactosidase activity in vivo. Expression from the orf-3 0 promoter was not stimulated by the phage P4 transcriptional activato r protein, delta, which acts at all the known P2 and P4 late promoters . insertion mutagenesis showed that orf-30 was not an essential gene f or P2 growth in Escherichia coli. None of the gene or protein sequence s exhibited extensive homology to sequences in the nucleic acid and pr otein databases. However, the R protein contains a small region homolo gous to one in the phage T4 tail protein gp15, which is required for 7 4 tails to bind heads. We propose that R and S are tail completion pro teins that are essential for stable head joining. (C) 1994 Academic Pr ess, Inc.