Transcription of tRNA gene fragments by HeLa cell extracts

Promoter elements for tRNA genes from several eukaryotes have been identified in the coding regions of the DNA. There are two non-contiguous sequences, an A-block or D-control region and a B-block or T-control region, located in the 5'- and 3'-halves of the tRNA sequence respectively. Both sequences are about 12 bp in length and are strongly conserved in all tRNA genes. We and others have recently shown that some tRNA genes from yeast and insects have a third control region located in the 5'-flanking sequences adjacent to tDNA. The tRNALeu3 genes from yeast have such a sequence. It is strongly conserved in non-allelic copies of tRNALeu3 genes as well as several other yeast tRNA genes. This 5'-flanking sequence is indispensable for transcription of the gene in an in vitro system derived from yeast cells. Further, the transcription apparatus from yeast will recognize and transcribe gene fragments including the 5'-flanking sequence in conjunction with either the A or B-blocks. Neither the 5'-flanking sequence alone nor the A and B-blocks lacking the 5'-flanking region can act as promoters in the yeast system. We have used these tRNALeu3 gene fragments to analyze the promoter activity of the three control regions with a Hela cell extract which actively transcribes class III genes. We find that the Hela cell system requires the presence of both A and B-block sequences and is insensitive to 5'-flanking DNA.