Gp2, a 7 kDa protein encoded by T7 bacteriophage, is a potent inhibitor of Escherichia coli RNA polymerase (RNAp), the enzyme responsible for transcription of all bacterial genes and early viral genes. A prominent feature in the structure of Gp2 is a contiguous strip of seven negatively charged amino acid residues (negatively charged strip or NCS), located along one side of the molecule. The role of the NCS in Gp2 function is not known. Here, the in vivo and in vitro properties of altered forms of Gp2 with amino acid substitutions in the NCS are described. While mutations in the NCS do not compromise the folding or the ability of Gp2 to bind to the RNAp β′ subunit, disruption of the NCS significantly attenuates Gp2 function in vivo and its ability to inhibit RNAp in vitro. Efficient inhibition of the RNAp by Gp2 also involves the amino terminal region 1 domain of the RNAp promoter specificity subunit σ70, located in the vicinity of the primary Gp2 binding site in β′. The results are discussed in the context of hypothetical molecular mechanisms of RNAp inhibition by Gp2.