A prolonged pause in transcription elongation at positions +16 and +17 of the phage lambda late gene operon has an important role in the modification of RNA polymerase by the lambda gene Q transcription antiterminator. Mutations included in the transcription bubble of the paused transcription complex, particularly at +2 and +6, abolish pausing and the ability of Q protein to modify RNA polymerase. By transcribing heteroduplex templates made in vitro, we show that the sites identified by these mutations act through the nontranscribed strand of DNA. This result suggests unexpected regulatory functions of the nontranscribed DNA strand in transcription.