5' flanking sequence signals are required for activity of silkworm alanine tRNA genes in homologous in vitro transcription systems.

We have used homologous in vitro transcription to examine the nucleotide sequences required for activity of cloned Bombyx mori tRNA2Ala genes. We have compared the transcriptional properties of an intact gene and a truncated derivative of the same gene lacking all but 11 nucleotides of normal 5' flanking DNA, and we find that at least two regions of DNA are required for accurate transcription of tRNA2Ala genes by homologous B. mori extracts. One of these sites is retained in the truncated gene and may be within the tRNA coding sequence. Competition experiments with intact and truncated genes indicate that this site probably acts by binding a factor necessary for specificity in polymerase III-catalyzed transcription. The second required site is removed by the Hind III cleavage used to produce shortened genes and thus must be 11 nucleotides or more upstream from the transcription initiation point. Possible roles for this site are discussed.