Quantitative Analysis of Transcription Elongation by RNA Polymerase I In Vitro
The elongation step in transcription has gained attention for its roles in regulation of eukaryotic gene expression and for its influence on RNA processing. Sophisticated genetic analyses have identified factors and/or conditions that may affect transcription elongation rate or processivity; however, differentiation of direct and indirect effects on transcription is difficult using in vivo strategies. Therefore, effective, reproducible in vitro assays have been developed to test whether a given factor or condition can have a direct effect on the kinetics of transcription elongation. We have adapted a fully reconstituted transcription system for RNA polymerase I (Pol I) for kinetic analysis of transcription elongation rate in vitro. The assay described here has proven to be effective in the characterization of defects or enhancement of wild-type transcription elongation by RNA Pol I. Since transcription elongation by RNA Pol I has only recently gained significant attention, this assay will be a valuable resource for years to come.
Key wordsTranscription rRNA Ribosome Elongation rate Pause site RNA polymerase I
The author wishes to thank Professor Masayasu Nomura for his pioneering development of the reconstituted transcription assay for RNA Pol I and for guidance in the adaptation of the system to its current form. This work is supported by the National Institutes of Health grant #GM84946.
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