The cloning of a T4 transfer RNA gene cluster

Abstract

Bacteriophage T4 codes for eight tRNAs that are clustered between genes e and 57. In order to obtain a well-defined T4 fragment containing only the tRNA genes and their promoter(s) for the study of tRNA biosynthesis, recombinant DNA technology was applied. However, in numerous attempts the T4 fragment carrying a cluster of seven T4 tRNA genes had not been cloned, while other T4 genes were cloned repeatedly. This led us to believe that there must be a segment of DNA whose expression was detrimental to bacterial viability contained within this region. In this paper we describe the successful cloning of the T4 fragment that contained the DNA segment “lethal” to the host. Three kinds of λ-T4 hybrid phages containing the tRNA gene cluster were isolated. Some had a deletion in the T4 fragment. Using our restriction map of the cloned fragment, the deletions were mapped. All the deletions encompass a common region, about 1000 base-pairs upstream from the tRNA cluster. Deletion of this DNA segment allowed us to locate a T4 tRNA promoter. λ-T4 hybrid phages that had a full-length fragment produced T4 tRNAs, while little T4 tRNA synthesis was detected in cells infected with λ-T4 hybrid phages deleted in the lethal region. RNA polymerase binding experiments confirmed this conclusion that there was a promoter in the deleted region. These results strongly support the idea that T4 tRNAs are synthesized in a large transcriptional unit. T4 tRNAs are then matured mainly by the host enzymes. However, the fact that three of the T4 tRNAs were not produced in any infections with these λ-T4 hybrid phages indicates that some T4 factor(s) is involved in the biosynthesis of some of the T4 tRNAs. Having cloned and mapped the T4 fragment containing the tRNA gene cluster, we now can proceed toward obtaining an understanding of the biosynthesis of the tRNAs at the molecular level.