Structural Changes in the RNA Polymerase II Transcription Complex during Transition from Initiation to Elongation

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Abstract
We obtained exonuclease III (exoIII) footprints for a series of RNA polymerase II transcription complexes stalled between positions +20 to +51. Downstream advance of the exoIII footprint is normally tightly coordinated with RNA synthesis. However, arrested RNA polymerases slide back along the template, as indicated by exoIII footprints in which the last transcribed base is abnormally close to the downstream edge of the footprint. None of the polymerase II complexes stalled between +20 and +51 were arrested. Nevertheless, the exoIII footprints of complexes with 20-, 23-, or 25-nucleotide RNAs resembled those of arrested complexes, with the last transcribed base very close to the footprint’s front edge. The exoIII footprint of the +27 complex was displaced downstream by 17 bp compared to the footprint of the +25 complex. Many complexes between +27 and +42 also showed evidence of sliding back along the template. We compared the effects of template sequence and transcript length by constructing a new template in which the initial transcribed sequence was duplicated beginning at +98. The exoIII footprints of transcription complexes stalled between +122 to +130 on this DNA did not resemble those of arrested complexes, in contrast to the footprints of analogous complexes stalled over the same DNA sequences early in transcription. Our results indicate that the RNA polymerase II transcription complex passes through a major, sequence-independent structural transition about 25 bases downstream of the starting point of transcription. The fully mature form of the elongation complex may not appear until more than 40 bonds have been made.