We have studied specific transcription by RNA polymerase I in vitro using a mouse tissue-culture cell extract and cloned mouse rDNA containing the transcription initiation region of a 45S rRNA gene. In vitro, transcription initiates at a unique site on the rDNA, which we have located using runoff transcription and S1 nuclease mapping. This site is several hundred base pairs upstream from the presumptive initiation sites previously identified by others. Using runoff transcription and pulse-chase experiments, we further show that our primary transcript is specifically processed in vitro to generate a smaller RNA species. This defines a new rRNA-processing site within the external transcribed spacer of 45S RNA. Ribosomal RNA made in vivo also is longer than previously thought. The farthest upstream, in vivo 5′ end we detect by S1 nuclease mapping is at the in vitro initiation site; the only other in vivo 5′ end we detect maps at the in vitro processing site. This suggests that in vitro, both the initiation of rDNA transcription and the initial processing of this primary transcript mimic the in vivo situation. Both in vivo and in vitro, transcription extends through several large clusters of T residues (37 of 40 and 15 of 15) located approximately 450 and 350 nucleotides downstream from the initiation site. This demonstrates that T clusters, which have a central role in specifying termination for RNA polymerase III and E. coli RNA polymerase, are not the sole determinants of RNA polymerase I termination. In addition, there is a striking sequence homology between the nucleotide sequences immediately surrounding the initiation sites of mouse and frog rRNA genes.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)