In vitro initiation and termination of ribosomal RNA transcription in isolated yeast nuclei.

Abstract

Using the Hg-agarose affinity chromatography/gamma-sulfhydryl nucleotide technique of Reeve, et al. (Reeve, A., Smith, M., Pigiet, V., and Huang, R. (1977) Biochemistry 10, 4464-4469) and high resolution electrotransfer of DNA electrophoretograms to diazobenzyloxymethyl paper, we have analyzed the transcription from the 5 S and 35 S ribosomal RNA genes in isolated yeast nuclei. In vitro initiation from these complex gene loci exhibits the same fidelities as in vivo with respect to initiating nucleotide, polymerase activity, and initiating and terminating region of DNA template. The efficiency of the experimental approach is enhanced by the use of RNase-deficient yeast strains. Thus, this system can be used to study structural aspects affecting transcription at these loci.