Comparison of tRNA gene transcription complexes formed in vitro and in nuclei

Abstract

The nucleoprotein structure of single-copy tRNA genes in yeast nuclei was examined by DNase I footprinting and compared with that of complexes formed in vitro between the same genes and transcription factor C. Transcription factor C bound to both the 5' and 3' intragenic promoters of the tRNA(SUP53Leu) gene in vitro, protecting approximately 30 base pairs at the 3' promoter (B block) and 40 base pairs at the 5' promoter (A block) and causing enhanced DNase I cleavages between the protected regions. Binding to the two sites was independent of the relative orientation of the two sites on the helix and was eliminated by a single point mutation in the 3' promoter. The chromosomal tRNA(SUP53Leu) and tRNA(UCGSer) genes showed a pattern of protection and enhanced cleavages similar to that observed in vitro, indicating that the stable complexes formed in vitro accurately reflect at least some aspects of the nucleoprotein structure of the genes in chromatin.