Evaluation of fluorescence spectroscopy methods for mapping melted regions of DNA along the transcription pathway
This chapter evaluates the fluorescence Spectroscopy technique for mapping melted regions of DNA along the transcription pathway. Fluorescent base analogs provide a relatively non-perturbing probe of very local structural changes within the DNA and/or RNA. They can be used to map melted bubbles in various static states of transcription, either the initially bound promoter complexes, or through walking experiments, in stalled complexes approximating dynamic movement along the DNA. Lists of fluorescent base analogs that have potential utility in probing regions of melted DNA are tabulated. Beyond the mapping of statically melted regions within various transcription complexes, fluorescence is also ideally suited for stopped-flow kinetic measurements of dynamic changes in those complexes. The chapter illustrates the step by step procedure for sample preparation, and analyzes it through fluorescence spectroscopy. Site-specific incorporation of fluorescent probes requires chemical synthesis, but if substantially longer DNA templates are required, these might be introduced by applications of the polymerase chain reaction, in which the probe is introduced in one of the primers. Thus, this approach should increasingly become useful in analyses of the multi-subunit RNA polymerases.