We are focusing on structural and functional studies of biologically important macromolecules. Bacterial transcriptional machinery RNA polymerase (RNAP); transcription factors that are involved in the regulation of bacterial infection to host cells; proteins that play crucial roles in regulation of programmed cell death, cell cycle, etc.; are our major targets currently. Transcription initiation is one of the major issues remaining to be answered in the transcription field. This process includes specific promoter recognition, promoter melting, and promoter clearance. Determining three-dimensional structures of the RNAP holoenzyme and its complex with homoduplex DNA and heteroduplex DNA are essential steps to understand the mechanism. The sigma-54 dependent RNAP holoenzyme represents one of two distinct initiation complexes that have been characterized, the other one being the sigma-70 dependent RNAP holoenzyme. Both sigma factors are structurally and functionally different, although the RNAP involved in these two processes are the same. For the sigma-54 dependent initiation, a preformed holoenzyme complex, containing sigma-54, promoter DNA, and core RNAP, is unable to initiate on its own. Activation, which is coupled with activator multimerization and NTP hydrolysis, triggers the formation of stable open complexes for transcript formation. Since there no structure has been determined for either complex, the transcription initiation mechanism remains unveiled.