Processing of bacteriophage T4 primary transcripts with ribonuclease III

Abstract

The two promoters (P1 and P2) of the cluster of transfer RNA genes of bacteriophage T4 are situated at distances of 0.95 × 103 and 1.3 × 103 bases, respectively, from the first tRNA gene. Isolated in vitro primary transcripts initiated at these promoters were incubated with highly purified RNase III. The resulting cleavage products were mapped with the use of deletions in the T4 tRNA region. We found three primary RNase III cleavage sites in the leader region of the transcripts at positions about 40 (site 1), 150 (site 2) and 950 (site 3) nucleotides downstream from the promoter P1. Analysis of the kinetics of transcript cleavage indicated that cleavage at sites 1 and 2 occurs simultaneously, presumably through a single act of interaction between RNase III and a double-stranded RNA stem. Cleavage at site 3 just before the first tRNA gene separates the clustered tRNA sequences from the leader part of the transcripts. This cleavage appears to be a major step in the T4 tRNA maturation pathway. © 1982.