Group of Prof. Dr. M. Thomm
Group member: Mirijam Zeller


Structure-function relation in the transcription machinery of P. furiosus

 

1) Overview and Summary

a.) The archaeal transcription machinery

All living cells require an RNA polymerase (RNAP) for the synthesis of various RNAs. Whereas the bacterial RNAP consists of only 5 subunits, the RNAP of archaea comprises 11 or 12 subunits and thereby mostly resembles the eukaryotic RNAP II (Fig. 1). Unlike all eukaryotic cells, archaea use only a single RNAP for the transcription of all, protein endcoding and RNA endcoding genes.

Moreover, archaea possess eukaryote like general transcription factors: TATA binding protein (TBP – a subunit of TFIID in eukaryotes), transcription factor B (TFB – TFIIB in eukaryotes) and transcription factor E (TFE – the N-terminal part of TFIIE subunit a in eukaryotes).

 
Fig. 1 RNAP subunits in bacteria, archaea and eukaryotes. Homologous subunits are shown in the same color (1)

 

RNAP subunits in bacteria, archaea and eukaryotes

b.) Characterization of the interactions between individual subunits of the P. furiosus RNAP

In our lab, 11 RNAP subunits (B, A’, A’’,D, E’, F, H, L, H, P, N und K) as well as the factors TBP and TFB of the hyperthermophilic Archaeon Pyrococcus furiosus were cloned and expressed in E. coli. This allowed a detailed analysis of protein-protein interactions between all polypeptides of the basal archaeal transcription machinery 1. Fig. 2 summarizes the interactions between archaeal RNAP subunits as determined by Far Western analysis.

Comparison of interaction networks in the archaeal and eukaryotic RNAP

Fig. 2 Comparison of interaction networks in the archaeal and eukaryotic RNAP.
Left, the diagram illustrates the interactions between subunits of the Pyrococcus RNAP based on Far Western analysis. The thickness of the lines estimates the strength of interaction. An interaction emanated by either one (blue line) or both (red line) binding partners.
Right, a modified diagram based on the crystal structure of RNAP II of S. cerevisiae (2,3) is shown. Homologous subunits are marked in the same color

High degree of sequence homology and very similar interactions of RNAP subunits suggest a similar structure for the archaeal and eukaryotic RNAP. Recently, cryo-electron microscopic data of the P. furiosus RNAP could confirm this previous assumption (4).

c.) Reconstitution of the RNA polymerase

In the course of studying the mechanism of archaeal transcription we successfully reconstituted a fully recombinant and highly active RNA polymerase made up of 11 bacterially expressed subunits of P. furiosus RNAP(Fig. 3). Subunits are purified under denturing or non denaturing conditions, mixed in equimolar amounts and are stepwise dialysed against transcription buffer (containing 6, 3 and 0 M urea). After gel filtration, a specific transcription activity at the glutamate dehydrogenase (gdh) promoter of P. furiosus can be detected in the presence of TBP and TFB (5).

Fig. 3. Reconstitution of the P. furiosus RNAP.
Left, 11 purified recombinant RNAP subunits are analyzed on a Coomassie blue stained SDS-PA gel. M, marker, Pfu RNAP, endgenous RNA polymerase of P. furiosus. The respective subunit in each lane is denoted below the gel.
Right, top: gel filtration (Superdex 200) of the reconstituted RNAP.
Right, bottom: in vitro transcription with fractions 18-25


2.) Research focus: structure-function relation in the transcription machinery of P. furiosus 

The high degree of sequence and structural conservation between eukaryotic and archaeal RNAP subunits generally allows to unambigously identify homologous amino acids and distinct structural regions in the P. furiosus enzyme. Moreover, the reconstitution of a fully recombinant P. furiosus RNAP allows the design of point mutants and deletion mutants of the approx. 400 kDa enzyme and subsequent specific in vitro analysis. Until now, this is not possible with any eukaryotic RNAP.

Current structural data on eukaryotic RNAPs therefore serve as starting point to design different mutant enzymes in order to study diverse functional aspects of the transcription cycle (Fig. 4). The design of the mutants is done in a collaboration with P. Cramer (Genecenter Munich).

Individual steps of transcripition
Fig. 4. The individual steps of transcription. The cycle starts with sequential binding of genereal transcription factors TBP, TFB and RNAP at the promoter DNA (closed complex). This complex melts the DNA double helix (open complex). The polymerase starts to synthesize small RNA oligonucleotides, which are often released (abortive stage). When the RNA reaches a certain length, RNAP enters the elongation phase. Finally, transcription is terminated and RNA is released from the dissociated complexes.

a.) Elongation

Our present studies investigate the function of four highly conserved loops in the DNA binding cleft above the active site of the RNAP, comprising Lid, Rudder, Fork Loop 1 and Fork Loop 2 6. Furthermore, we question the roles of three basic residues, which are located in the Switch 2 element (R313, K330) and in Fork Loop 2 (R445).

b.) Initiation of transcription

Up to now, there is no complete preinitiation complex (PIC: TBP-TFB-polymerase-DNA) structure resolved. Nevertheless, detailed crystal structures for TBP-TFB-DNA complexes as well as polymerase-DNA complexes are available. In conjunction with cross-linking data, these complexes are used to design point mutants in TFB and the RNAP in order to study the highly complex transition from initiation to elongation.



References:

Goede, B., Naji, S., von Kampen, O., Ilg, K. & Thomm, M. Protein-protein interactions in the archaeal transcriptional machinery: binding studies of isolated RNA polymerase subunits and transcription factors. J Biol Chem 281, 30581-92 (2006).

Bushnell, D.A., Cramer, P. & Kornberg, R.D. Structural basis of transcription: alpha-amanitin-RNA polymerase II cocrystal at 2.8 A resolution. Proc Natl Acad Sci U S A 99, 1218-22 (2002).

Armache, K.J., Kettenberger, H. & Cramer, P. Architecture of initiation-competent 12-subunit RNA polymerase II. Proc Natl Acad Sci U S A 100, 6964-8 (2003).

Kusser, A. et al. Structure of an archaeal RNA polymerase. J Molecular Biology (2007).

Naji, S., Grunberg, S. & Thomm, M. The RPB7 orthologue E' is required for transcriptional activity of a reconstituted archaeal core enzyme at low temperatures and stimulates open complex formation. J Biol Chem 282, 11047-57 (2007).

Naji, S., Bertero, B., Spitalny, P., Cramer, P. & Thomm, M. Structure-function analysis of the RNA polymerase cleft loops elucidates initial transcription, DNA unwinding, and RNA displacement. Nucleic Acids Res (2007).