Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase

The various kinetic and thermodynamic models for transcription elongation a ll require an understanding of the nature of the melted bubble which moves with the RNA polymerase active site. Is the general nature of the bubble sy stem-dependent or are there common energetic requirements which constrain a bubble in any RNA polymerases? T7 RNA polymerase is one of the simplest RN A polymerases and is the system for which we have the highest-resolution st ructural information. However, there is no high-resolution information avai lable for a stable elongation complex. In order to directly map melted regi ons of the DNA in a functionally paused elongation complex, we have introdu ced fluorescent probes site-specifically into the DNA. Like 2-aminopurine, which substitutes for adenine bases, the fluorescence intensity of the new probe, pyrrolo-dC, which substitutes for cytosine bases, is sensitive to it s environment. Specifically, the fluorescence is quenched in duplex DNA rel ative to its fluorescence in single-stranded DNA, such that the probe provi des direct information on local melting of the DNA. Placement of this new p robe at specific positions in the non-template strand shows clearly that th e elongation bubble extends about eight bases upstream of the pause site, w hile 2-aminopurine probes show that the elongation bubble extends only abou t one nucleotide downstream of the last base incorporated. The positioning of the active site very close to the downstream edge of the bubble is consi stent with previous studies and with similar studies of the promoter-bound, pre-initiation complex. The results show clearly that the RNA:DNA hybrid c an be no more than eight nucleotides in length, and characterization of dif ferent paused species suggests preliminarily that these dimensions are not sequence or position dependent. Finally, the results confirm that the terna ry complex is not stable with short lengths of transcript, but persists for a substantial time when paused in the middle or at the (runoff) end of dup lex DNA. (C) 2001 Academic Press.