A new single molecule system -Transchip -was developed for analysis of transcription products at their genomic origins. The bacteriophage T7 RNA polymerase and its promoters were used in a model system, and resultant RNAs were imaged and detected at their positions along single template DNA molecules. This system, Transchip, has drawn from critical aspects of Optical Mapping, a single molecule system that enables the construction of high resolution, ordered restriction maps of whole genomes from single DNA molecules. Through statistical analysis of hundreds of single molecule template/transcript complexes, Transchip enables analysis of the locations and strength of promoters, the direction and processivity of transcription reactions and termination of transcription. These novel results suggest that the new system may serve as a high-throughput platform to investigate transcriptional events on a large, genome-wide scale.
KeywordsSingle molecule; transcription; elongation complex; T7 RNA polymerase; promoter mapping; automated fluorescence microscopy
Introductory StatementThis study aims to develop an integrated system for transcriptional analysis of single template DNAs, with the ultimate goal being analysis of transcriptional regulation for genes on a whole genome basis. Large template DNA molecules can be queried to probe for regulatory elements and for transcriptional events. The Transchip system described in this manuscript aims to produce large and meaningful datasets for transcriptional analysis, all based around single molecule measurements. Experiments based on single molecules are direct and rapid, and can generate a read-out of thousands of data points in a short time period.The development of single molecule approaches to biochemical analysis has enabled a broad range of techniques that visualize the details of and that elegantly probe individual steps involved in transcription [1][2][3][4][5][6]. These elegant biochemical studies performed on single molecules have brought numerous insights into the mechanisms of transcription, but they do not lend themselves to high throughout transcriptional analysis. Visualization and mapping of RNAs transcribed from single template molecules has long been possible by electron + To whom correspondence should be addressed: dcschwartz@wisc.edu, 608-265-0546; 608-265-6743 (FAX) # current position, Amgen, Inc., One