Analysis of transcriptional initiation in isolated nuclei

The level of expression of a given gene in a particular cell type is reflected by the concentration of the resultant messenger RNA. This is subject to regulation during a number of processes, including synthesis, processing, export from nucleus to cytoplasm, and degradation. Clearly, the rate of production of nascent transcripts, reflecting the rate of initiation of RNA polymerase at the promoter, is of primary importance. The nuclear run-on assay described here allows labeling of nascent transcripts as they are synthesized, thus allowing measurement of the density of transcription complexes on a gene, which is dependent on the rate of initiation of RNA polymerase at the promoter (1). The essential steps involved in the procedure are outlined in Fig. 1. Fig. 1. Flow diagram indicating the various steps in production and use of radiolabeled nuclear RNA.