We localized five rpoC (beta') mutations affecting Escherichia coli RNA polymerase assembly. The Ts4, XH56, and R120 mutations changed beta' residues conserved throughout eubacteria; the JE10092 mutation occurred in the hypervariable region; rpoC1 (TsX) changed a universally conserved residue and corresponds to yeast rpb1-1. Thus, distinct, predominantly conserved beta' residues participate in interactions holding RNA polymerase together.