Transcription initiation by RNA polymerase II in vitro. Properties of preinitiation, initiation, and elongation complexes.

We have prepared three types of RNA polymerase I1 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 "C, proteolysis, and digestion with DNase I. Complex 0 is extremely stable at 25 "C in the absence of ATP, but it is sensitive to the other treatments including 25 "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 "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).