Phage Q protein is a transcriptional antiterminator that is required for late gene expression from the pR' promoter when prophage decides to follow the lytic pathway. Q modifies Escherichia coli RNA polymerase (RNAP) at the early elongation stage. Once engaged, Q maintains itself as a subunit of the elongation complex throughout the elongation. Studies show that Q confers RNAP resistance to both intrinsic and -mediated terminators and less pausing. However, little is known about the interaction between Q and RNAP. In this study, the largest subunits ( and ') of RNAP were examined to map the Q binding site. A stronger binding was observed with than ', and the binding site was narrowed to 501-832. This region was further defined as small as 82 amino acids (600-681) that interacted with Q. In addition, 12 mutant RNAPs were isolated for a reduced Q antitermination within the potential Q binding regions, specifically near the main channel. These results propose that Q modifies the active center of RNAP for antitermination.