Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate

Affinity modification of RNA-polymerase II by a phosphorylating analog of t he initiation substrate carrying a zwitterionic 5'-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during sp ecific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modi fication resulted in labeling a polypeptide with molecular weight correspon ding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiati on substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of t he enzyme.