The crystal structure of Rpo26 in the context of RNA polymerase II highlights a network of intramolecular side chain contacts entailing salt bridge, hydrogen bonding, and π-cation interactions (Figure 6A). Here, we performed a structure-guided alanine scan of eight residues that comprise this network: Arg79, Glu89, Arg97, Glu124, Arg135, Arg136, Asp145, and Glu150. The alanine mutations were introduced into the biologically active RPO26-(78-155) gene on 2-µ plasmids under the control of the TPI1 promoter and tested for complementation of rpo26∆ by plasmid shuffle. Four of the alanine mutations were lethal: E89A, E124A, R135A, and R136A. Three of the alanine mutants—R79A, D145A and E150A—were viable and grew as well as “wild-type” RPO26-(78-155) at 18°, 25°, 30°, and 37° (Figure 6B). R97A cells grew at 25° and 30° but displayed cs and ts defects, whereby they failed to grow at 18° and grew slowly at 37°, as gauged by colony size (Figure 6B). We interpret the mutational data in light of the crystal structure, as follows.