Alanine substitution of four amino acids in two evolutionarily conserved motifs, PSRM and RFGEMIE, near the carboxy terminus of the beta subunit of E. coli RNA polymerase results in a dramatic loss of the enzyme's affinity to substrates with no apparent effect on the maximal rate of the enzymatic reaction or on binding to promoters. The magnitude and selectivity of the effect suggest that the mutations disrupt the substrate binding site of the active center.