Transcription initiation of Xenopus 5S ribosomal RNA genes in vitro

We have studied initiation of transcription of 5S RNA genes in extracts of Xenopus laevis oocyte nuclei. To aid in this study we developed a general assay for specificity of transcription initiation that does not require accurate termination of transcription. Following in vitro transcription with gamma-32P-labeled nucleoside triphosphages, the RNA is digested with pancreatic RNAase and fingerprinted by two dimensional chromatography. A 5S RNA gene with a variant sequence, in which the G residue at position +1 is replaced by a C, initiates transcription at an A residue one nucleotide preceding the C. Although Xenopus RNA polymerase form III can initiate transcription at many sites on plasmid DNA, all of the transcripts start with purines. The majority of these purines are triphosphorylated. When a repeating unit of Xenopus 5s DNA is inserted into the plasmid, initiations at the vector start sites are suppressed and the major labeled 5' oligonucleotide is derived from 5S RNA.