Faithful initiation of ribosomal RNA transcription from cloned DNA by purified RNA polymerase I

Abstract

A faithful transcription system for ribosomal RNA genes was developed by using components from the small free-living amoeba Acanthamoeba castellanii. The system utilizes protein-free recombinant DNA as a template and in addition requires a crude cell-free extract containing RNA polymerase I and a transcription initiation factor (TIF-I). The transcript is initiated at the same position as the in vivo precursor ribosomal RNA: templates truncated at various sites downstream of the transcription start site give rise to only the predicted runoff RNA transcripts, and the runoff transcript produced has a 5''-terminus identical with the 5''-terminus of the isolated ribosomal RNA precursor. Faithful initiation can be elicited by the DNA sequence extending from -55 to +19 in the template. Subclones containing this sequence yield only the predicted runoff RNA regardless of the orientation of this fragment in the cloning vector DNA; thus, only the in vivo sense strand of the template is specifically transcribed in the in vitro system. The system is specific for the RNA polymerase responsible for the transcription of ribosomal RNA genes in vivo. Faithful transcription, like RNA polymerase I from Acnthamoeba, is insensitive to .alpha.-amanitin inhibition , and transcription is greatly stimualted by highly purified RNA polymerase I but not by RNA polymerases II or III. Conditions of optimal transcription were determined.