Effects of single-base substitutions within the Acanthamoeba castellanii rRNA promoter on transcription and on binding of transcription initiation factor and RNA polymerase I.
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.
Kownin
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P
Department of Biochemistry, Colorado State University, Fort Collins 80523.
Bateman
E
E
Paule
M R
MR
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
D012335:RNA, Ribosomal; D014157:Transcription Factors; D012318:RNA Polymerase I; D003850:Deoxyribonuclease I