N-(1-Pyrenyl)iodoacetamide has been used to introduce fluorescent probes into Escherichia coli RNA polymerase. After an incubation time of 15 min, approximately 2 pyrene equiv was introduced per enzyme molecule. There was no further increase in modification after more extended periods of incubation. Neither calf thymus DNA nor nucleotides protected the holoenzyme from modification. Thus, the sites of modification do not appear to involve the binding sites for polynucleotides or the ribonucleoside triphosphates. From the isolation and analysis of the individual subunits, it was found that sigma contained approximately 1 pyrene equiv, beta contained 0.6, beta' contained 0.6, and alpha less than 0.1. Spectral and Stern-Volmer analyses indicate that the covalently attached pyrene molecules are in comparable apolar microenvironments. On the basis of CD analyses, the introduction of pyrene molecules into RNA polymerase alters its secondary structure. This alteration in secondary structure manifests itself by a reduction in overall enzymatic activity. Transcript analysis of the products obtained by using a linearized plasmid containing the A1 promoter and the Te terminator of bacteriophage T7 indicates that the pyrenyl derivative is capable of producing full-length transcripts and that it has an efficiency of chain termination comparable to the native enzyme. Analysis of tau plots for the interaction of the pyrenyl derivative and the native enzyme, respectively, with the A1 promoter yielded comparable values for the isomerization constant in the conversion of the closed complex to an open one. Comparable values were also obtained for the association constant. The rate of chain elongation for the pyrenyl derivative, however, is approximately 54% of that observed for the native enzyme. Thus, the decrease in overall transcriptional activity observed with the pyrenyl derivative is not due to a decrease in the efficiency of initiation or premature termination, but rather to a decrease in the rate of chain elongation.