Imaging and analysis of transcription on large, surface-mounted single template DNA molecules

Abstract

A surface-based approach is described for the transcriptional analysis of large, single DNA molecule templates and their imaged reaction products using RNA polymerase (RNAP). Results demonstrated that surfaces with a charge density supporting stretching of single DNA molecules to 70–80% of their full contour length were ideal for analysis of T7 RNAP transcription complexes on bound single template DNAs. Such DNA molecules were shown to sustain efficient transcription reactions and analysis, which enabled localization of transcription complexes on templates at kilobase resolution. Direct labeling of nascent RNA transcripts by the incorporation of a second fluorochrome into DNA templates promotes more robust and sensitive detection of punctates. Further characterization by RNase digestions, atomic force microscopy studies, and fluoro-immunolabeling revealed a “supercomplex” structure within a punctate where elongation complexes aggregate through entanglement of DNA and RNA strands from individual ternary elongation complexes. We have proposed mechanisms that underlie the supercomplex formation process. Whereas supercomplexes develop naturally in free solution, spatial constraints involved in a topologically limited system where template DNA is bound to the surface may facilitate the assembling process by stalling transcriptional elongation.