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


Structure-function relationship in the transcription machinery of           
Pyrococcus furiosus
       
     


1.) Overview and Summary

DNA-dependent RNA polymerases (RNAPs) are multisubunit enzymes that facilitate the transcription of the cellular genome across the tree domains of life. The archaeal transcription system is a simplified version of the eukaryotic RNAPII system in terms of RNAP subunit composition (Fig. 1) and structural organization. Unlike all eukaryotic cells, archaea use only one single RNAP of all, protein endcoding and RNA endcoding genes.

Moreover, archaea possess homologues of eukaryotic basal transcription factors:

  • TATA binding protein (TBP – a subunit of TFIID in eukaryotes)
  • Transcription factor B (TFB – TFIIB in eukaryotes)
  • Transcription factor E (TFE – the N-terminal part of TFIIE subunit
        α in eukaryotes)
  •  

    RNAP subunits
    Fig. 1. RNAP subunits in bacteria, archaea and eukaryotes. Homologous subunits are shown in the same color (modified from doctoral thesis Zeller M.E.)


    2.) Structure-function relationship in the transcription machinery of P.furiosus

    In our lab, we have the possibility to reconstitute highly active P.furiosus RNAP from the 11 RNAP subunits (B, A´, A´´, D, E´, F, H, L, N, P and K) expressed in Escherichia coli (Fig. 2).  This method allows the design of substitution/deletion mutations of RNAP and subsequent specific in vitro analysis. This study contributes to a deeper understanding of the structure-function relationships in archaeal RNAP, and in the eukaryotic RNAP, in which this analysis is, until now, not possible.

     

    RNAP reconstitution

    Fig. 2. 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, endogenous 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

    (Naji S. et al., 2007)

     

    Current structural data on eukaryotic RNAPs therefore serves as a starting point to design mutant enzymes in order to study diverse functional aspects of the transcription cycle (Fig. 3). The design of the mutants is done in collaboration with Patrick Cramer (Gene Center Munich) (http://www.cramer.genzentrum.lmu.de/)

    Fig. 3. The individual steps of transcription.

    The cycle starts with sequential binding of general transcription factors TBP, TFB and RNAP at the promoter DNA (closed complex). This complex, with TFE, melts the DNA double helix (open complex). The polymerase starts to synthesize small RNA oligonucleotides (in red), which are often released (initial transcription: abortive stage). When the RNA reaches a certain length, RNAP enters the elongation phase (here, in the presence of elongation factors TFS and Spt4-5). Finally, transcription is terminated and RNA is released from the dissociated complex. (modified from doctoral thesis Zeller M.E.)

    TL: Trigger loop; T: DNA template strand; NT: DNA non template strand.

    transcription cycle


    a.) Trigger loop

    Conformational changes in the active site of the RNAP are required for correct nucleotide incorporation during transcription elongation. Recent structure-function analysis on yeast RNAPII and bacterial RNAP showed that the highly conserved trigger loop (TL) (Fig. 4) plays a key role at every distinct stage of the elongation cycle (for example, nucleotide positioning and addition, translocation, proofreading) (Sydow J.F. and Cramer P., 2009; Kaplan C., 2010) . However, because of the limited number of experimental assays on RNAPII TL mutants, and a possible functional diversification between yeast RNAPII and bacterial RNAPs, the exact mechanism of TL in each step of eukaryotic transcription remains unclear.

    Therefore, we are investigating the function of TL in the distinct stages of archaeal transcription (initiation, elongation, termination).

    trigger loop

    Fig. 4. Structural element in the active site of RNAP.

    Top, Structural element of RNAPII active site (Kettenberger H. et al., 2003). Trigger loop is highlighted in magenta, the bridge helix in green and the active site metal ion Mg2+ in purple.

    Bottom: Trigger loop domain conservation in tree domains of life. Red indicates 100% of homology, yellow 50-75% sequence similarity. (Pf: Pyrococcus furiosus; Sc: Saccharomyces cerevisiae; Ec: Escherichia coli)


    b.) Transcription initiation factors

    Transcription initiation starts with binding of the TATA binding protein TBP (part of TFIID in eukaryotes) to the TATA box. In the next step archaeal transcription factor B (TFB), which is homologous to eukaryotic TFIIB, binds the B recognition element on the DNA and TBP.  Both factors are required for initiation of gene transcription by RNAP in archaea. Recruitment of the RNAP then occurs and the closed complex is formed, in which the DNA is base paired. DNA is partially melted and the open complex is formed. With the help of our simplified eukaryotic-like transcription machinery, it was shown that various domains of TFB (B-reader and B-linker) have distinct roles during open complex formation (Fig. 5).


    TFB domain organization

    Fig. 5. B-domain organization and sequence conservation in the region connecting the B-ribbon and B-core of TF(II)B.

    Yellow and green highlighting indicates conserved and invariant residues, respectively, between yeast (S. cerevisiae), human (H.  sapiens) and the archaeon P. furiosus. (Kostrewa et al., 2009)


    Furthermore a third factor, transcription factor E (TFE), which is homologous to the N-terminal part of the TFIIE α-subunit in eukaryotes, stabilizes the open complex, depending on the presence of the RNAP subunits RpoE and RpoF. The presence of TFE is, in contrast to TBP and TFB, not essential for promoter dependent transcription in archaea in vitro.
    Currently, we are establishing UV-photocrosslinking experiments with the unnatural amino acid para-benzoyl phenylalanine to study interactions of TFB and TFE with the archaeal transcription machinery and DNA during transition from initiation to elongation (Fig. 6).

    Fig. 6. Schematic overview of the transcription initiation in archaea and the eukaryotic PolII system. (modified from doctoral thesis Zeller M.E.)

    Transcription initiation in archaea and the eukaryotic PolII system