We used yeast two-hybrid and in vitro co-immobilization assays to study the interaction between the Escherichia coli RNA polymerase (RNAP) α and β subunits during the formation of α2β, a physiological RNAP assembly intermediate. We show that a 430-amino acid-long fragment containing β conserved segments F, G, H, and a short part of segment I forms a minimal domain capable of specific interaction with α. The α-interacting domain is held together by protein-protein interactions between β segments F and I. Residues in catalytically important β segments H and I directly participate in α binding; substitutions of strictly conserved segment H Asp1084 and segment I Gly1215 abolish α2β formation in vitro and are lethal in vivo. The importance of these β amino acids in α binding is fully supported by the structural model of the Thermus aquaticus RNAP core enzyme. We also demonstrate that determinants of RNAP assembly are conserved, and that a homologue of β Asp1084 in A135, the β-like subunit of yeast RNAP I, is responsible for interaction with AC40, the largest α-like subunit. However, the A135-AC40 interaction is weak compared with the E. coli α-β interaction, and A135 mutation that abolishes the interaction is phenotypically silent. The results suggest that in eukaryotes additional RNAP subunits orchestrate the enzyme assembly by stabilizing weak, but specific interactions of core subunits.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology