Group of Prof. Dr.
Group member: Mirijam Zeller
Structure-function relation in the transcription machinery of P. furiosus
b.) Characterization of the interactions between individual subunits of the P. furiosus RNAP
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.
2 Comparison of interaction networks in the archaeal and
|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).
3. Reconstitution of the P. furiosus RNAP.
|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).
|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.|
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
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.
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).