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Real‐time Imaging of Transcriptional Elongation

Transcription is a key regulatory step in gene expression that involves initiation, elongation, cotranscriptional modifications, termination and posttranscriptional modification. Gene expression has long been known to be regulated at transcription with recruitment of RNA polymerase II (RNAPII) to the transcription initiation complex. But, there is also evidence that the rate of elongation varies between genes. The purpose of this study is to develop a new technology to measure the elongation rate of RNAPII in vivo and in real‐time. For this technology, IMAGEtags (Intracellular MultiAptamer Genetic tags), encoded in the initial and/or terminal regions of a gene that is expressed from a promoter of choice, are utilized as markers. IMAGEtags are strings of RNA aptamers that have high affinity and specificity for their ligands. For imaging, the cells are incubated with aptamer ligands that are labeled with fluorescent dyes. To increase signal/noise, ligands have been separately conjugated with the FRET pairs, Cy3 and Cy5. To measure the elongation rate, IMAGEtags have been inserted in the initial and/or terminal regions of YLR454 gene after the GAL1 promoter. IMAGEtags are then used to monitor RNAPII passage through defined positions in the gene as it transcribes in living cells in culture. This work was supported by the NIH and the Biological Systems Science Division, Office of Energy Science Research of the U. S. DOE.