Investigation of the role of TBP-TATA interaction in differential transcription of two alanine tRNA genes in silkworm Bombyx mori

The TATA-binding protein (TBP) is a general transcription factor required by all three eukaryotic nuclear RNA polymerases. I have cloned TBP cDNA from a silkworm cDNA library in order to investigate the contribution of specific TBP-TATA interactions to the promoter activity of silkworm tRNAAla genes. Silkworm TBP is encoded by a single copy gene and contains a highly conserved C-terminal DNA-binding domain as do the TBPs cloned from other organisms. By comparing TBP binding, TFIIIB-promoter complex stability (measured by heparin-resistance) and transcriptional activity in a series of tRNA AlaC (c&barbelow;onstitutively-expressed) promoter mutants, I have found that the wild-type promoter contains two overlapping functional TBP binding sequences (TATA elements), and that specific TBP-TATA contacts are important for both TFIIIB-promoter interaction and transcriptional activity. The lack of similar interactions accounts for the low promoter activity of a tRNA AlaSG (s&barbelow;ilkg&barbelow;land-specific) gene. The wild-type tRNA AlaSG promoter lacks a TBP binding site in the correct place, but provision of either of the TATA elements derived from the tRNA AlaC promoter confers robust transcriptional activity. The low promoter activity of the wild-type tRNA AlaSG promoter is not due to reduced ability to incorporate TBP into transcription complexes, however, since transcription complexes formed on the tRNA AlaSG promoters contain the same amount of TBP as do those formed on the tRNA AlaC promoters. Therefore, I propose that the absence of appropriate TBP-TATA contacts in the tRNA AlaSG promoter impairs function either because it prevents the incorporation of some other essential transcription factor(s), or because it de-stabilizes a conformation that is required for activity.;This dissertation includes both my previously published and unpublished co-authored materials.