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Nucleic Acids Research, 1990, Vol. 18, No. 6 1541-1548
© 1990

MOLECULAR BIOLOGY

Saturation mutagenesis of the Drosophila tRNAArg gene B-Box intragenic promoter element: requirements for transcription activation and stable complex formation

Bruno A. Gaëta, Stephen J. Sharp1,* and Thomas S. Stewart

School of Biochemistry, University of New South Wales PO Box 1, Kensington, 2033 Australia 1Department of Microbiology and Molecular Genetics, California College of Medicine, University of California Irvine, CA 92717, USA

*To whom correspondence should be addressed

Received November 17, 1989. Revised February 2, 1990. Accepted February 2, 1990.

Transcription of eukaryotic tRNA genes is dependent on the A- and B-Box internal control regions (ICRs) and the upstream transcription modulatory region. The B- Box ICR spans nucleotides 52 to 62 and directs the primary binding of transcription factor C as the first step in the formation of a transcription complex. The conservation of the sequence of the B-Box in all tRNA species reflects its importance in both the expression of the gene and the processing, structure and function of the gene product. In order to identify the nucleotides essential to the promoter function of the B-Box ICR, site-directed mutagenesis was used to generate all the possible single point mutations at positions 52 to 58, 61 and 62 of a Drosophila melanogaster tRNAArg gene. The effect of these mutations on gene transcription was evaluated using in vitro transcription and template exclusion competition assays. Optimal activity was displayed by the wild type tDNAArg B-Box sequence but several other sequences supported in vitro transcription at wild type levels. The majority of mutants, however, showed lower efficiency in the in vitro transcription assay. Of the single point mutations, those at positions 53, 55, and 56 had a critical effect on gene function in Drosophila and HeLa transcription extracts and transcription factor interaction most likely requires base contacts at these positions. Since the effect of several of the point mutations cannot be explained In terms of possible major or minor groove contributions the possibility is raised that local DNA geometry also is an important determinant in specifying B-Box function.


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