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In Association with
Analysis of promoters recognized by alternative sigma factors of RNA polymerase using in vitro transcription system in Corynebacterium
2nd World Congress on Biotechnology
Jiri Holatko
Posters: J Microbial Biochem Technol
Abstract
Diff erent sigma subunits of bacterial RNA polymerase (RNAP) recognize diff erent classes of promoters. It is believed that Corynebacterium glutamicum promoters driving transcription of housekeeping genes are recognized by RNAP with the primary sigma factor SigA. Th e primary-like factor SigB is mainly involved in transcription of genes during the transition phase between exponential and stationary growth phases. SigC, SigD, SigE, SigH and SigM of C. glutamicum are members of the Group 4 sigma factors called also ECF (extracytoplasmic function) sigma factors. We characterized promoters of various classes by mapping the respective transcription start points, measuring their activity under various growth conditions by transcriptional fusions with the reporter genes and deriving the consensus sequences of specifi c classes. To determine, which sigma factors are involved in recognition of particular promoters, we have developed the fi rst in vitro transcription system for C. glutamicum. Th e in vitro transcription system consists of the promoter-carrying DNA template and RNAP holoenzyme reconstituted from the purifi ed His-tagged core RNAP and a separately isolated sigma factor. Using the in vitro assay, RNAP+SigA recognized specifi cally the vegetative promoters Pper from the C. glutamicum plasmid pGA1 and Pveg from Bacillus subtilis, whereas no transcriptional activity of RNAP+SigA on the SigH-dependent PdnaK promoter was observed. On the other hand, RNAP+SigH recognized specifi cally PdnaK promoter but not the vegetative promoters. In vitro transcription system may be particularly used for characterization of multiple promoters of diff erent classes and promoters recognized by two or more sigma factors.Biography
JiÃ?Â?i Holátko (born 1979) completed his Ph.D. from Charles University in Prague in 2009. He participates on an international project analyzing sigma factors of RNA polymerase in Corynebacterium glutamicum at postdoctoral position in the Institute of Microbiology of Czech Academy of the Sciences. He has published 6 papers in international journals and is an author of 1 European patent.
