Photochemical cross-linking analysis of protein-nucleic acid interactions in Escherichia coli transcription complexes from lambda PR′ promoter

Abstract

This chapter describes the approaches used to characterize the transcription complex that forms on the bacteriophage λ (A) P R promoter, which is regulated by transcriptional antitermination. λ synthesizes the viral proteins N and Q, which reprogram the host RNA polymerase to allow expression of viral genes. Antitermination by the A Q protein differs from that involving N in several ways. First, Q is a DNA-binding protein and its site of action is inseparable from the P R promoter. Q interacts with promoter DNA from about -31 to -10, and thus binds to DNA at a site overlapping the P R promoter upstream from the transcription start site. Polymerase that initiates transcription from P R pauses after incorporation of the first 15 or 16 nucleotides, where it is modified by Q and NusA to an antitermination form. It is shown that it is the nontranscribed strand of the DNA that is important for the pause at +16. Whether Q modifies the RNA polymerase at the promoter and is then left behind, or whether the Q protein is carried along with the RNA polymerase after modification, as is the case with N, is not known. Similarly, whether any portion of the nascent RNA or promoter DNA remains with the polymerase is also not known.