Group Prof. Dr. M. Thomm
Group member: Thomas Fouqueau, Robert Reichelt


Analyses of mutated archaeal RNA polymerase subunits in a yeast background with the aim to understand the evolution from a single generalistic enzyme to three specialized RNA polymerases in eukaryotic cells                       


In this approach we benefit from two developed systems in the archaeal and eukaryotic domain of life. First, we study the archaeal system which is, regarding transcription, a simplified version and the evolutionary precursor of the more complex eukaryotic system. In particular the RNA polymerase (RNAP) and the basal transcription machinery of the archaea share many properties with the eukaryotic RNAP II transcription apparatus. Our archaeal model organism is Pyrococcus furiosus (Pfu) and in the basic archaeal system we can take advantage of the in vitro reconstituted RNAP of Pyrococcus furiosus  (Naji et al., 2007) and  a recently developed genetic system for this organism (Waege et al., 2010).


Subunit composition of the RNAPs from the 
three domains of life

Fig. 1
Subunit composition of the RNAPs from the
three domains of life (modified from doctoral thesis Zeller M. E.)


On the other hand, we have the yeast system, which is a well developed genetic system and we can use the information provided by the solved crystal structure of RNAP II.


 

Fig. 2
Overall architecture of RNAPs from bacteria (Thermus aquaticus (1HQM) Minakhin et al., 2001), archaea (Sulfolobus shibatae (2Y0S) Wojtas et al., 2011) and eukaryotes (Saccharomyces cerevisiae (1Y1V) Kettenberger et al., 2004)

Overall architecture of RNAPs from the three domains of life


Previous studies have shown that eukaryotic subunits can functionally replace their archaeal counterparts in the reconstituted RNAP from Pyrococcus furiosus (Rpb12 (archaeal: RpoP) and Rpb5 (archaeal: RpoH) and conversely that the archaeal subunit RpoP works in vivo in the yeast system (Reich et al., 2009; Thomm et al., 2009 and Grünberg et al., 2010). However in contrast, a fusion protein of the archaeal subunit RpoH and Rpb5 doesn´t work in the yeast system.

By converting archaeal subunits to eukaryotic ones through error prone PCR we are studying the evolution from one generalistic, archaeal RNA polymerase to three specialized enzymes in eukaryotic cells.