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Transcription antitermination by N proteins of lambdoid phages involves specific interactions of the C-terminal domain of N with the elongation complex (EC). The interacting surface of N on the EC is unknown, knowledge of which is essential to understand the mechanism of antitermination. Specific cleavage patterns were generated near the active site Mg Transcription elongation complexes (EC)3 formed by the Escherichia coli RNA polymerase (RNAP) are exceptionally stable and highly processive (1). The stability of the EC arises from nucleic acid-protein interactions in the DNA binding site, RNA-DNA hybrid binding site, and RNA binding site (2-5). The EC dissociates from the template DNA when it encounters specific DNA sequences (intrinsic terminators) (6 -8) or when it is displaced by the nascent RNA-binding protein, Rho (9, 10). Lambdoid phages encode proteins like N and Q, and a cis-acting element like polymerase utilization RNA, which make the EC termination-resistant upon interaction with the elongating RNAP. This phenomenon is called transcription antitermination (11,12).The N protein of bacteriophage activates expression of the late genes by facilitating the read-through of transcription terminators present in the early genes of the phage (11,(13)(14)(15). N is a small basic protein, which binds to a specific stem-loop structure (box B of nut site) of the mRNA through the argininerich motif (ARM) present in its N-terminal domain (16,17), and interacts with the EC through its C-terminal domain via an RNA looping mechanism (18 -20). N requires several hostcoded factors called Nus factors, to form a processive antitermination complex (12). The interacting surface of N on the EC is not yet known, knowledge of which is critical for understanding the mechanism of antitermination.N-mediated antitermination of the lambdoid phage H-19B has reduced requirements for Nus factors compared with ⌵. Efficient antitermination requires only NusA (21) in vivo, and the additional presence of NusG to achieve highly processive antitermination in vitro (22). This minimal requirement for host factors makes it a simpler antitermination system to reconstitute in vitro for biochemical studies.Using a random mutagenesis screen, we have earlier isolated and characterized E. coli RNAP mutants specifically defective for H-19B N-mediated antitermination. These mutations are located very close to important structural elements of the EC, like the RNA exit channel, the lid, and the rudder (22). In this report, we have identified the regions of the EC that come physically close to the C-terminal domain of H-19B N by protein footprinting of the N-modified EC. We have also analyzed the effects of the binding of H-19B N on the interactions around the RNA-DNA hybrid at a class II pause site (23), and the interactions of the flap domain with hairpin RNA near the RNA exit channel at a class I pause site (24 -27). We concluded that the C-terminal domain of H-19B N comes close to the active site Mg 2ϩ and N-induced altered interactions in the active ...