title: Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I.
abstract: Single-point mutations were introduced into the promoter region of the Acanthamoeba castellanii rRNA gene by chemical mutagen treatment of a single-stranded clone in vitro, followed by reverse transcription and cloning of the altered fragment. The promoter mutants were tested for transcription initiation factor (TIF) binding by a template commitment assay plus DNase I footprinting and for transcription by an in vitro runoff assay. Point mutations within the previously identified TIF interaction region (between -20 and -47, motifs A and B) indicated that TIF interacts most strongly with a sequence centered at -29 and less tightly with sequences upstream and downstream. Some alterations of the base sequence closer to the transcription start site (and outside the TIF-protected site) also significantly decreased specific RNA synthesis in vitro. These were within the region which is protected from DNase I digestion by polymerase I, but these mutations did not detectably affect the binding of polymerase to the promoter.
authors.affiliation: Department of Biochemistry, Colorado State University, Fort Collins 80523.
authors.forename: P
authors.initials: P
authors.lastname: Kownin
authors.forename: E
authors.initials: E
authors.lastname: Bateman
authors.forename: M R
authors.initials: MR
authors.lastname: Paule
mesh_terms: D000048:Acanthamoeba / Q000235:genetics*; D000818:Animals; D003850:Deoxyribonuclease I; D005796:Genes; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012318:RNA Polymerase I / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics*; D013698:Templates, Genetic; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D012335:RNA, Ribosomal; D014157:Transcription Factors; D012318:RNA Polymerase I; D003850:Deoxyribonuclease I