The specificity loop of T7 RNA polymerase interacts first with the promoter and then with the elongating transcript, suggesting a mechanism for promoter clearance

During the early stages of transcription, T7 RNA polymerase forms an unstab le initiation complex that synthesizes and releases transcripts 2-8 nt in l ength before disengaging from the promoter and isomerizing to a stable elon gation complex. In this study, we used RNA-protein and RNA DNA crosslinking methods to probe the location of newly synthesized RNA in halted elongatio n complexes. The results indicate that the RNA in an elongation complex rem ains in an RNA DNA hybrid for about 8 nt from the site of nucleotide additi on and emerges to the surface of the enzyme about 12 nt from the addition s ite. Strikingly, as the transcript leaves its hybrid with the template, the crosslinks it forms with the RNA polymerase involve a portion of a hairpin loop (the specificity loop) that makes specific contacts with the binding region of the promoter during initiation. This observation suggests that th e specificity loop may have a dual Pole in transcription, binding first to the promoter and subsequently interacting with the RNA product. It seems li kely that association of the nascent RNA with the specificity loop facilita tes disengagement from the promoter and is an important part of the process that leads to a stable elongation complex.