Protein-Protein-Wechselwirkungen der Untereinheiten der RNA-Polymerase von Pyrococcus furiosus
Pyrococcus furiosus belongs to the domaine Archaea (Euryarchaeota), formerly known as Archaebacteria and is a hyperthermophilic microorganism with an optimal growth temperature of 100°C. It contains of a DNA dependent RNA polymerase (RNAP) out of 11-12 different subunits (B, A', A'', D, E', E'', F, H, K, L, N, P). All RNAP genes except for the largest subunit B were cloned in E.coli, overexpressed and purified. Medium sized and small subunits (D, E', F, H, K, L, N, P) and the general transcription factors TFB and TBP were subjected to cloning into the vector pET-33b(+) (Novagen Inc.). The rekombinant proteins were specifically labled with radioactivity via Protein kinase A catalytic subunit from heart muscle (HMK) by means of the included 5-amino acid HMK recognition site ArgArgAlaSerVal. Subsequently all 32P-labled proteins were used as probes in Far western blotting analysis to detect direct protein–protein contacts without additional need of antibodies. Specific interactions of subunit D with the small subunits L, N, P and the largest subunit B were shown. Particularly strong interactions became visible between subunits E'-F, A''-H, D-L, D-P and D-B. Binding of subunit K to TFB was less intense as expected from literature. A readily soluble D-L complex and a putative D-L-N complex were detected in size exclusion chromatography experiments. The last-mentioned approach can act as a preliminary experiment for the reconstitution of the P.furiosus RNA polymerase from recombinant subunits. Allready now further experiments with additional subunits are carried out in the workgroup of Prof.Dr.M. Thomm at the University of Regensburg, Germany. Apart from practical experiments a detailed analysis of all RNAP subunit genes by bioinformatic means was done. A computer program was written to facilitate the extraction and the analysis of the genes from the available microbial genomes, to calculate certain informations about proteins and to generate space-saving diagrams. The main focus was a stock-taking of all RNAP subunit genes in the currently accessable 17 completed archaeal genomes. An archaeal consensus sequence was calculated for each subunit via extensive alignments and visualized in the 12 subunit 3D model of the eukaryotic RNAP of S.cerevisiae. A detailed comparision of sequence similarities of the RNAP subunits in Archaea / P.furiosus and S.cerevisiae was worked out and discussed. The analysis of amino acid frequencies within the RNAP genes and all protein genes in the genomes of different microorganisms provides data about amino acid replacement and its importance to thermoadaptation of proteins.