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Molecular and Cellular Biology, February 2000, p. 1329-1343, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

TATA-Binding Protein-TATA Interaction Is a Key Determinant of Differential Transcription of Silkworm Constitutive and Silk Gland-Specific tRNAAla Genes

Ching Ouyang,1,2 M. Juanita Martinez,2,3 Lisa S. Young,2 and Karen U. Sprague2,4,*

Department of Physics,1 Department of Chemistry,3 Department of Biology,4 and Institute of Molecular Biology,2 University of Oregon, Eugene, Oregon 97403

Received 11 October 1999/Returned for modification 8 November 1999/Accepted 23 November 1999

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

* Corresponding author. Mailing address: Institute of Molecular Biology, University of Oregon, Eugene, OR 97403-1229. Phone: (541) 346-6094. Fax: (541) 346-5891. E-mail: ksprague{at}molbio.uoregon.edu.

Molecular and Cellular Biology, February 2000, p. 1329-1343, Vol. 20, No. 4
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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