Escherichia coli RNA polymerase (RNAP) α subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2α → α2 → α2β → α2ββ′ → α2ββ′ σ. In this work, we have used hydroxyl-radical protein footprinting to define determinants of α for interaction with β, β′, and σ. Our results indicate that amino acids 30-75 of α are protected from hydroxyl-radical-mediated proteolysis upon interaction with β (i.e., in α2β, α2ββ′, and α2ββ′ σ), and amino acids 175-210 of α are protected from hydroxyl-radical-mediated proteolysis upon interaction with β′ (i.e., in α2ββ′ and α2ββ′ σ). The protected regions are conserved in the α homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of α for direct functional interaction with β and β′. The observed maximal magnitude of protection upon interaction with β and the observed maximal magnitude of protection upon interaction with β′ both correspond to the expected value for complete protection of one of the two α protomers of RNAP (i.e., 50% protection). We propose that only one of the two α protomers of RNAP interacts with β and that only one of the two α protomers of RNAP interacts with β′.
|Журнал||Proceedings of the National Academy of Sciences of the United States of America|
|Состояние||Опубликовано - 17 сент. 1996|
|Опубликовано для внешнего пользования||Да|