REGULATION OF TRANSFER-RNA GENE TRANSCRIPTION IN THE SILKWORM, BOMBYX MORI

Transcriptional regulation plays a primary role in differential gene expression in eukaryotes. The alanine tRNA genes of the silkworm, Bombyx mori, provide an attractive system for investigating the molecular basis of transcriptional control, because they are tissue-specifically expressed in vivo. In this work, I have examined the structure and activity of a cloned bombyx tRNA(,2)('Ala) gene. Using partially deleted versions of this gene, I have localized control regions that are required for its transcription in vitro. Both 5' flanking and intragenic sequence elements are essential and may have distinct roles in promoting initiation by Bombyx RNA polymerase III.;To investigate the functions of the 5' flanking and internal control regions, I have tested the ability of genes that lack one or both of these regions to compete with wild-type genes for binding to the Bombyx RNA polymerase III transcription machinery. The essential coding region can interact with at least one transcription factor to form a stable complex that persists through several rounds of transcription. Sequences in the 3' half of the gene can affect the rate of formation of this complex, even though they are not essential for transcription. The upstream control region does not affect either the stability or the rate of formation of this complex. These results suggest a possible model for the interactions between tRNA(,2)('Ala) gene control regions and the Bombyx RNA polymerase III transcription apparatus.;The presence of required sequences in 5' flanking DNA distinguishes RNA polymerase III control elements in Bombyx from those in certain other eukaryotes in which all essential sequences appear to be internal. The crucial upstream signal for the Bombyx tRNA gene lies within 34 nucleotides preceding the transcription initiation site. This region may function as a general recognition signal for Bombyx RNA polymerase III since it contains sequences that are conserved among tRNA and 5S RNA genes in Bombyx, and is also required for Bombyx 5S RNA gene activity (D. Morton, unpublished). In contrast to the absolute requirement for 5' flanking sequences, much of the tRNA coding region is dispensable. The gene retains 60% of wild-type activity even after replacement of the 3' half of the coding sequences. Sequences in the 5' half of the coding region appear to be important however, because deletions that enter this region abolish transcription.