Footprinting of ribosomal RNA genes by transcription initiation factor and RNA polymerase I

Abstract

The binding of a species-specific transcription initiation factor (TIF) and purified RNA polymerase I to the promoter region of the 39S ribosomal RNA gene from Acanthamoeba were studied by using DNase I "footprinting." Conditions were chosen such that the footprints obtained could be correlated with the transcriptional activity of the TIF-containing fractions used and that the labeled DNA present would itself serve as a template for transcription. The transcription factor binds upstream from the transcription start site, protecting a region extending from around -14 to -67 on the coding strand, and -12 to -69 on the noncoding strand. The protein that binds to DNA within this region can be competed out by using wild-type promoters but not by using mutants which do not stably bind the factor. RNA polymerase I can form a stable complex in the presence of DNA and transcription factor, allowing footprinting of the complete transcription initiation complex. RNA polymerase I extends the protected region obtained with TIF alone to around +18 on the coding strand, and to +20 on the noncoding strand. This region is not protected by polymerase I in the absence of TIF. The close apposition of the regions protected by TIF and polymerase provides evidence that accurate transcription of the ribosomal gene may be achieved through protein-protein contacts as well as through DNA-protein interactions.