This research program deals with basic mechanisms of transcription and with transcriptional regulation of the activity of genes. Its implications for human health relate to the application of precise and specific interventions in gene expression based on a broad and fundamental understanding of transcription mechanisms and their associated protein-nucleic acid interactions. Two specific systems will be analyzed: One project area deals with eukaryotic nuclear RNA polymerase (pol) III and its core transcription initiation factor (TF) IIIB and includes the following objectives: 1) Protein-protein and protein-DNA interactions in the TFIIIB-DNA complex will be mapped, and the structure of the TFIIIB-DNA complex will be determined (the latter in collaboration). 2) The mechanism by which TFIIIB participates with RNA polymerase III and promoter opening will be elucidated. 3) The competition between the pol III transcription initiation factors and nucleosomes for formation of promoter complexes will be analyzed. 4) The retrovirus-like Ty3 transposition element integrates DNA into the promoters of RNA polymerase III-transcribed genes through a direct interaction with the TFIIIB-DNA complex. This homing mechanism will be dissected (in collaboration). 5) The protein contacts of nascent RNA and template DNA with RNA polymerase III will be determined at high resolution. 6) The mechanism of RNA chain elongation past DNA-bound and obstructing transcription factors and nucleosomes will be analyzed (partly in collaboration). 7) RNA polymerase III makes short RNA chains and recognizes simple RNA chain-termination signals. The mechanism of transcriptional termination will be analyzed (in collaboration). A parallel group of projects focuses on the sliding clamp of the bacteriophage T4 DNA polymerase, which activates transcription of a large group of viral genes and couples their expression to DNA replication. Specific objectives include: 1) a kinetic analysis of transcriptional initiation to determine the sliding clamp's activation mechanism; 2) elucidation of key protein-protein and protein-DNA interactions in this transcriptional activation system.