TATA-Binding Protein–TATA Interaction Is a Key Determinant of Differential Transcription of Silkworm Constitutive and Silk Gland-Specific tRNA^Ala Genes

Abstract

We have investigated the contribution of specific TATA-binding protein (TBP)–TATA interactions to the promoter activity of a constitutively expressed silkworm tRNA_C ^Ala gene and have also asked whether the lack of similar interactions accounts for the low promoter activity of a silk gland-specific tRNA_SG ^Ala gene. We compared TBP binding, TFIIIB-promoter complex stability (measured by heparin resistance), and in vitro transcriptional activity in a series of mutant tRNA_C ^Ala promoters and found that specific TBP-TATA contacts are important for TFIIIB-promoter interaction and for transcriptional activity. Although the wild-type tRNA_C ^Ala promoter contains two functional TBP binding sequences that overlap, the tRNA_SG ^Ala promoter lacks any TBP binding site in the corresponding region. This feature appears to account for the inefficiency of the tRNA_SG ^Alapromoter since provision of either of the wild-type TATA sequences derived from the tRNA_C ^Ala promoter confers robust transcriptional activity. Transcriptional impairment of the wild-type tRNA_SG ^Ala gene is not due to reduced incorporation of TBP into transcription complexes since both the tRNA_C ^Ala and tRNA_SG ^Ala promoters form transcription complexes that contain the same amount of TBP. Thus, the deleterious consequences of the lack of appropriate TBP-TATA contacts in the tRNA_SG ^Ala promoter must come from failure to incorporate some other essential transcription factor(s) or to stabilize the complete complex in an active conformation.

ACKNOWLEDGMENTS

This work was supported by NIH Public Health Service grants (GM25388 and GM32851) to K.U.S. M.J.M. is an NIH trainee supported by Graduate Training in Genetics grant GM07413.

We thank Diane Hawley, Heather Dunstan, and Fakhruddin Palida for critically reading the manuscript, and we thank Nan Ahnert and Gusti Zeiner for making some of the mutant promoter constructs.