The goal of the work described in this proposal is to understand the molecular mechanisms involved in the regulation of gene expression at the levels of transcription elongation, termination and antitermination. The level at which genes are expressed, as well as the level at which the resulting transcripts are utilized to produce functional gene products, are both regulated by specific protein-nucleic acid interactions. Events which alter gene regulation can cause inappropriate expression of viral and/or cancerous genes. In addition, some viral proteins reprogram host transcriptional machinery to allow expression of viral genes. If the mechanisms involved in the regulation of gene expression can be characterized, at the level of specific protein-nucleic acid interactions, it then becomes possible to mimic or prevent the action of viral proteins, for example. Detailed analysis of the steps involved in RNA synthesis, or transcription, can eventually lead to strategies for controlling gene expression through specific drug design. This proposal is focused on understanding how a viral protein can reprogram a host RNA polymerase to allow expression of viral genes. Specifically, the mechanism utilized by the bacteriophage lambda to accomplish expression of its late viral genes is being examined. Late gene expression involves modification of the RNA polymerase of the host, Escherichia coli, by the viral antitermination protein Q, to render the polymerase resistant to transcription termination signals. The Q protein recruits at least one host protein, NusA, which normally increases termination efficiency in E. coli. The molecular modifications which occur to reprogram the host polymerase, and to cause a host factor which normally causes termination to participate in antitermination, will be determined by characterizing the specific molecular interactions in the antitermination complex. The bacterium E. coli is an ideal model system for analysis of the molecular mechanisms employed by viral proteins. Transcription can be reconstituted in vitro utilizing purified RNA polymerase and transcription factors. In such a purified system, it is also possible to test methods for inhibiting the action of viral proteins to prevent modification of the transcription process. Molecular interactions are being characterized by using photochemical crosslinking. Photocrosslinker tagged nucleotide analogs will be used to identify interactions with the DNA and/or RNA components in different transcription complexes. Heterobifunctional crosslinkers will be used to characterize the protein-protein interactions. Both unmodified transcription complexes, and those that have been altered by the viral Q protein will be examined and compared, with the goal of better understanding how modification is accomplished. Data obtained in this model system can lead to a greater understanding of the different ways in which viral proteins function, and may lead to greater understanding of mechanisms in more complex systems.
