Molecular cloning, sequence analysis and in vitro expression of a rat tRNA gene cluster

Abstract

A rat genomic DNA fragment containing a tRNA gene cluster was isolated from a lambda phage library. Hybridization and nucleotide sequence analysis revealed the presence of a 83 bp tRNALeuCUG gene and a 72 bp tRNAAspGUG gene. Both genes possessed intact coding regions and putative transcription termination signals at their respective 3' ends. In vitro transcription analysis of the two subcloned genes in a HeLa cell S-100 system demonstrated the specific synthesis of a number of RNAs by RNA polymerase III. Studies carried out in the presence of alpha-amanitin showed that the larger RNAs are precursors for the final processed transcripts of the tRNALeu and tRNAAsp genes, respectively. Further nucleotide sequence analysis of the cluster revealed the presence of tRNAGly and a tRNAGlu pseudogenes with missing areas within their coding regions which are essential for transcription by RNA polymerase III. Within the region of DNA between the tRNALeu and tRNAAsp genes is a sequence which is 65% homologous to a region of the rat B1 element. The significance of this latter structure within the gene cluster is unknown.