Extensive purification of a putative RNA polymerase I holoenzyme from plants that accurately initiates rRNA gene transcription in vitro

被引:51
作者
SaezVasquez, J [1 ]
Pikaard, CS [1 ]
机构
[1] WASHINGTON UNIV,DEPT BIOL,ST LOUIS,MO 63130
关键词
nucleolus; rDNA; gene expression;
D O I
10.1073/pnas.94.22.11869
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
RNA polymerase I (pol I) is a nuclear enzyme whose function is to transcribe the duplicated genes encoding the precursor of the three largest ribosomal RNAs. We report a cell-free system from broccoli (Brassica oleracea) inflorescence that supports promoter-dependent RNA pol I transcription in vitro, The transcription system was purified extensively by DEAE-Sepharose, Biorex 70, Sephacryl S300, and Mono Q chromatography. Activities required for pre-rRNA transcription copurified with the polymerase on all four columns, suggesting their association as a complex, Purified fractions programmed transcription initiation from the in vivo start site and utilized the same core promoter sequences required in vivo, The complex was not dissociated in 800 mM KCl and had a molecular mass of nearly 2 MDa based on gel filtration chromatography. The most highly purified fractions contain approximate to 30 polypeptides, two of which were identified immunologically as RNA polymerase subunits, These data suggest that the occurrence of a holoenzyme complex is probably not unique to the pol II system but may be a general feature of eukaryotic nuclear polymerases.
引用
收藏
页码:11869 / 11874
页数:6
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