How do we envisage transcription in vitro? Most of the in vitro studies of transcription are performed with purified RNA polymerase that transcribes relatively short DNA fragments. Common sense tells us that in this system RNA polymerase should act as a small spherical object that moves (and rotates) along DNA with a rate of 20-30 base pairs per second. This view derives entirely from our perception of DNA as a long bulky rod and RNA polymerase as a small bead freely moving along the rod. May we be wrong in our assumption? Modern research challenges the in vitro perception of RNA polymerase in several ways. The extensive work done in recent years revealed RNA polymerase as a bulky composite of many different proteins (transcription factors) which come and go, while the polymerase moves across the gene. Moreover, translation of messenger RNA at ribosomes, RNA splicing and processing often occur co-transcriptionally making hundreds of ribosomal and mRNA processing proteins tethered to RNA polymerase in vivo. Years ago, we found that RNA polymerase, which was attached to the polymeric beads through the affinity tag in the protein, performed normally in all steps of the transcription cycle including initiation at promoter, elongation of RNA and termination of transcription (Kashlev, 1993; Sidorenkov, 1998; Kireeva, 2000; Komissarova, 1998; 2002). Transcription through DNA containing nucleosomes was also not affected in this system (Kireeva, 2002). When the enzyme was immobilized, transcription could not proceed by movement and rotation of the polymerase along the DNA. How did the transcription occur? This controversy was relieved by the observation of how and where transcription occurs in the intact eukaryotic cells (Peter Cook, 2001). These experiments showed that RNA synthesis in vivo takes place in the stationary """"""""transcription factories"""""""", containing a pool of immobilized RNA polymerase molecules attached to the solid elements of the nucleus (Peter Cook, 2000). Transcription is believed to occur in these factories by threading the template through the immobilized enzyme, and it may involve temporary recruitment of the genes to these immobile sites. The transcription factories could be visualized with the electron or fluorescent microscopy, but they were never approached biochemically. Transcription Factory: from visualization in vivo to purification and reconstitution in vitro. The project involves isolation and biochemical characterization of the transcription factory, assembled at the unique yeast (S. cerevisiae) or bacterial (E. coli) gene in vivo. For the biochemical isolation of the factory, we developed a novel technology for """"""""halting"""""""" and stabilizing the factory in the cell by specially designed transcription roadblocks, followed by purification of the factory using a special tag, introduced to one of its components. Upon further development, the project will include a comparison of the factories assembled on different yeast and bacterial genes in vivo, and reconstitution of the """"""""minimal"""""""" transcription factory in vitro.
| Komissarova, Natalia; Kireeva, Maria L; Becker, Jodi et al. (2003) Engineering of elongation complexes of bacterial and yeast RNA polymerases. Methods Enzymol 371:233-51 |
