Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes.
We have prepared three types of RNA polymerase II transcription complexes: a preinitiation complex (complex 0), a complex which has synthesized two phosphodiester bonds (complex 2), and a complex which has synthesized 10-13 bonds (complex 10). We have studied the differential response of these complexes to a variety of disruptions: detergent (Sarkosyl), high levels of KCl, extended incubation at 25 degrees C, proteolysis, and digestion with DNase I. Complex 0 is extremely stable at 25 degrees C in the absence of ATP, but it is sensitive to the other treatments including 25 degrees C incubation in the presence of ATP. Once the complex has made two phosphodiester bonds, the properties almost reverse from those of complex 0; complex 2 remains unstable at 25 degrees C in the presence of ATP but is resistant to high levels of Sarkosyl and KCl, to extensive DNase I digestion, and to brief proteolysis. Addition of 10 or more bases to the growing RNA chain results in a complex completely resistant to all of the treatments used. When DNase I-trimmed complex 0 is allowed to initiate RNA synthesis, chains of about 33 bases are obtained. In contrast, DNase-trimmed complex 2 gives only about 23 base transcripts; DNase-treated complex 10 will elongate its nascent chains by about 21 bases as well (to give, on average, 34 base transcripts).
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000256:Adenoviridae / Q000235:genetics; D002467:Cell Nucleus / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000494:pharmacology; D003850:Deoxyribonuclease I / Q000378:metabolism; D019286:Endopeptidase K; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells / Q000648:ultrastructure; D009711:Nucleotides / Q000378:metabolism; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012313:RNA / Q000096:biosynthesis*; D012319:RNA Polymerase II / Q000378:metabolism*; D012521:Sarcosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014158:Transcription, Genetic
D003838:Deoxyadenine Nucleotides; D009711:Nucleotides; D012313:RNA; C025231:sarkosyl; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D012319:RNA Polymerase II; D003850:Deoxyribonuclease I; D010450:Endopeptidases; D019286:Endopeptidase K; C026600:2'-deoxyadenosine triphosphate; D012521:Sarcosine