37] DNA-dependent RNA polymerase

Abstract

An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-OP). The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. This chapter details the synthesis of unlabeled MMPR-OP, synthesis of [ 35 S]MMPR-OP. It also discusses affinity labeling DNA-dependent RNA polymerase, enzyme purification, and reduction of the Schiff base to the stable amine. The chapter concludes that in all studies carried out to date on enzymes of nucleic acid metabolism, MMPR-OP has shown specificity for lysine residues. Inhibition of other enzymes of nucleic acid metabolism by MMPR-OP can be taken as presumptive evidence for the presence of lysines in their active centers, and the procedures described above could be modified for individual study.