D. Reines
2003
Citations
1
Influential Citations
14
Citations
Journal
Methods in enzymology
Abstract
Publisher Summary This chapter discusses the application of 6-Azauracil (6AU) and mycophenolic acid (MPA)-sensitivity as a genetic assay and will provide procedures for the purification of RNA polymerase II for use in in vitro transcription assays. 6-AU is an inhibitor of IMP dehydrogenase (IMPDH), the rate-limiting enzyme in de novo GTP synthesis. Treatment of cells with 6AU, results in depletion of intracellular nucleotide pools. Mycophenolic acid (MPA) has also become useful to assay for mutations in the elongation machinery. This drug is a well-characterized and specific inhibitor of IMPDH. One of the limitations of the 6AU-sensitivity assay is that cells must be tested on uracil-free media since uracil competes with and neutralizes the drug's effect. MPA is effective in rich medium or synthetic dropout medium. However, it is somewhat unstable, so it is recommended that plates be poured as close to use as possible, and that stock solutions are made fresh prior to addition to liquid media. Once a candidate RNA polymerase II mutant has been identified by the drug-sensitive phenotype, the enzyme can be readily purified and subjected to in vitro transcription assays.