Characterization of factors and DNA sequences required for accurate transcription of the Saccharomyces cerevisiae 5 S RNA gene.

Cell-free extracts prepared from yeast cells have previously been shown to direct selective and accurate in vitro transcription of tRNA and 5 S RNA genes. We have further analyzed the transcription factors and DNA sequences required for in vitro transcription of the yeast 5 S RNA gene. Fractionation of a yeast extract has identified a 5 S RNA gene-specific factor required, in addition to the two previously described tRNA factors (Klekamp, M. S., and Weil, P. A. (1982) J. Biol. Chem. 257, 8432-8441), for accurate transcription of the 5 S RNA gene by RNA polymerase III. Transcription of variant 5 S RNA genes has indicated that a region of the gene extending from residue +57 to residue +99 is essential for directing specific initiation of transcription. Although the 5' flanking and initial coding sequence is not absolutely required for transcription of the gene, some of the variant genes which have substitutions in this region are less actively transcribed than the wild-type gene. Transcription initiates on some of the variant genes at a position equivalent to +1 in the substituted sequence, while on other variant genes transcription initiates further upstream.