DETERMINANTS OF RNA-POLYMERASE ALPHA-SUBUNIT FOR INTERACTION WITH BETA-SUBUNITS, BETA'-SUBUNITS, AND SIGMA-SUBUNITS - HYDROXYL-RADICAL PROTEIN FOOTPRINTING

Escherichia coil RNA polymerase (RNAP) a subunit serves as the initiat or for RNAP assembly, which proceeds according to the pathway 2 alpha --> alpha(2) --> alpha(2) beta --> alpha(2) beta beta' --> alpha(2) be ta beta' sigma. In this work, we have used hydroxyl-radical protein fo otprinting to define determinants of alpha for interaction with beta, beta', and alpha. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interac tion with beta (i.e., in (alpha(2) beta, alpha(2) beta beta', and alph a(2) beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta ' (i.e., in alpha(2) beta beta' and alpha(2) beta beta' sigma). The pr otected regions are conserved in the alpha homologs of prokaryotic, eu karyotic, archaeal, and chloroplast RNAPs and contain sites of substit utions that affect RNAP assembly. We conclude that the protected regio ns define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon int eraction with beta and the observed maximal magnitude of protection up on interaction with beta' both correspond to the expected value for co mplete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of R NAP interacts with beta and that only one of the two alpha protomers o f RNAP interacts with beta'.