Group Prof. Dr. M. Thomm
Coworkers: Christoph Reich


Interactions of archaeal and eukaryotic subunits

 


The purpose of this project is to investigate the basics of structure and function-relationships of RNA Polymerases (RNAP).
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 RNAP and the basal transcription machinery of the archaea share many properties with the eukaryal RNAP II transcription apparatus.
Our archaeal model organism is Pyrococcus furiosus (Pfu).

Fig. 1 Amount and size of RNA Polymerase subunits of Eukarya, Archaea and Bacteria
Goede et al., J. Biol. Chem. 2006

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). In the basic archaeal system we can take advantage of the in vitro reconstituted RNAP of Pyrococcus which was established in our group.

Fig. 2 Crystal structure of RNAP II from Saccharomyces cerevisiae (left). Crystal structure of RNAP II without the N-terminal domain of Rpb5 (right).
P. Cramer, Curr. Opin. Struct. Biol., 2002

The main questions in this project are:

1. Do archaeal RNAP subunits work in the yeast?

In this attempt we perform shuffle experiments to find out if archaeal subunits or fusion-proteins consisting of eukaryal and archaeal parts can complement the Saccharomyces RNAPs.
In order to see if these subunits and constructs are incorporated in one of the three RNAPs, immunoprecipitation experiments are performed using antibodies directed against Pfu RNAP subunits.

2. Do eukaryotic subunits work in the archaeal system?

In this approach we purify eukaryotic subunits and incorporate them in a reconstituted Pyrococcus RNAP. Additionally we investigate RNAPs lacking a certain subunit.
These enzymes are characterised in detail, in order to assign functions to the subunits.

Concluding our results so far it seems probable that the archaeal RNAP really is a basal system and a good model for the eukaryotic transcription.
To sum up we want to contribute to an improved understanding of the role played by the various subunits and to the interactions of structure and function in RNAPs.