VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

ARP008ATP synthesis above 100°C: structure and function of therotor of the A 1 A O ATP synthase from Pyrococcus furiosusF. Mayer* 1 , J. Langer 2 , V. Leone 3 , J. Faraldo-Gómez 3 , V. Müller 11 Department of Molecular Microbiology & Bioenergetics, Goethe-University Frankfurt, Frankfurt am Main, Germany2 Department of Molecular Membrane Biology, Max-Planck Institute ofBiophysics, Frankfurt am Main, Germany3 Max-Planck Institute of Biophysics, Theoretical Molecular Biophysics,Frankfurt am Main, GermanyATP synthases are the most important enzymes in cellular bioenergetics andpresent in any life form. The ATP synthase of archaea (A 1A O) is veryunusual and shares properties with both the bacterial F 1F O ATP synthasesand the eukaryal V 1V O ATPases. Most of the variation is in the membraneembeddedmotor. Most archaea have an F-type like c subunit in their rotorrings, consisting of one hairpin with one ion binding site. In contrast, somearchaea, e. g. Pyrococcus furiosus, have unusual c subunits [1]. The csubunit from the hyperthermophile P. furiosus is predicted to have fourtransmembrane helices, but only one ion (H + /Na + ) binding site. It wasisolated by chloroform/methanol extraction, purified and the molecular masswas determined with MALDI-TOF-MS. The mature c subunit of P. furiosusis indeed a 16 kDa protein. Labeling of the ion binding sites with theinhibitor N,N’-dicyclohexylcarbodiimide (DCCD) verified that the c subunithas indeed only one ion binding site. The influence of Na + on the labeling isobject of current research. Based on these results and the recent finding thatthe A 1A O ATP synthase of P. furiosus has a rotor with 10 c subunits [2], ahomology model of the c ring is presented, giving first insights into thestructure of an archaeal rotor ring with its ion binding site.[1] Müller, V. (2004): An exceptional variability in the motor of archaeal A1AO ATPases: Frommultimeric to monomeric rotors comprising 6-13 ion binding sites. J Bioenerg Biomembr 36: 115-125.[2] Vonck, J. et al (2009): Three-dimensional structure of A1AO ATP synthase from thehyperthermophilic archaeon Pyrococcus furiosus by electron microscopy. J Biol Chem 284: 10110-10119.ARP009Interaction of transcription factor B with mutants oftranscription activator GvpE of Halobacterium salinarumL. Marschaus*, A. Bleiholder, F. Anderl, F. PfeiferInstitute für Microbiology und Genetics, University of Technology,Darmstadt, GermanyThe GvpE protein of Halobacterium salinarum has been identified astranscriptional activator of the gvp gene cluster involved in gas vesicleformation. The two oppositely oriented promoters P A and P D driving theexpression of the gvp genes are separated by 35 nt only. Both promoters areactivated by GvpE. The putative binding sites of GvpE are adjacent to BRE(transcription factor B recognition element) and the TATA-Box (recognizedby the transcription factor Tbp). The question arises whether GvpE is able tocontact proteins of the basal transcription apparatus. Protein-proteininteractions were found between GvpE and any of the five Tbp proteins [1].In addition, we analyzed the ability of GvpE to interact with the sevendifferent Tfb proteins. All of them were able to interact.The putative secondary structure of GvpE indicates six α-helical regionsincluding the amphiphilic helix AH6 resembling a leucin zipper preceded bya cluster of basic amino acids that could constitute DNA binding site(DNAB). The amphiphilic helix AH4 is highly conserved between the GvpEsequences of different organisms and also contains a cluster of basic aminoacid. Various GvpE mutants are available in these regions [2] and additionalmutants were constructed in the region between the amino acids 50 and 100of GvpE. All of these mutants were unable to activate the P A promoter.The GvpE mutants were analyzed for their ability to interact with TfbC thatappeared to be the strongest transcription factor. The His-tagged TfbCproduced in E. coli was bond to Ni-NTA matrices and tested for interactionwith the GvpE mutants present in lysates of Hfx. volcanii. All of these GvpEmutants interacted with TfbC, whereas the soluble gas vesicle proteinGvpH His (used as control) did not bind these GvpE mutants. These resultsimplied that the regions tested in GvpE are not involved in the binding ofTfbC. However all these regions are required for the activating function ofGvpE. Thus, the lack of activation of these mutants is not due to a lack ofTfb-GvpE binding, but rather due to the lack in DNA binding (DNAB,AH4) or dimerization (AH6).[1] Teufel, K. and F. Pfeifer (2010).[2] Plößer, P. and F. Pfeifer (2002).ARP010Hot Trehalose: The Unusual Bifunctional TPSP Pathwayof Thermoproteus tenaxA. Hagemann* 1 , M. Zaparty 2 , C. Bräsen 1 , B. Siebers 11 Department of Molecular Enzymetechnology and Biochemistry, UniversityDuisburg-Essen, Essen, Germany2 Institute for Molecular and Cellular Anatomy, University of Regensburg,Regensburg, GermanyThe multifunctional disaccharide trehalose is a widespread molecule,occurring in all three domains of life, where it plays a major role as storagecompound and in stress protection (thermoadaptation, osmoregulation) [1].The most common pathway for trehalose synthesis is the OtsA/OtsBpathway, where UDP-(ADP-)glucose and Glucose-6-phosphat istransformed into trehalose-6-phosphat by TPS and subsequentlydephosphorylated by TPP into trehalose and P i [2]. In the genome of thehyperthermopilic crenarchaeon Thermoproteus tenax a gene coding for atrehalose-6-phosphate synthase/phosphate (tpsp) was identified, encoding aprotein with a C-terminal TPS- and N-terminal TPP-domain [3]. This geneis part of an operon harboring a putative glycosyl transferase (gt) and aputative small conductive mechanosensitve channel (msc). The two-domainTPSP structure has already been described for plants (e.g. Selaginellaleptophylla, Arabidopsis thaliana) and for Saccharomyces cerevisiae, butthe TPSPs only possess one activity, either TPS or TPP. Only recently abifunctional TPSP activity has been reported from Cytophaga hutchinsonii[4]. For the archaeon T. tenax, biochemical studies of the recombinantprotein revealed a bifunctional TPSP, however the enzyme posses onlyminor TPS and full TPP activity. Surprisingly, a significant increase oftrehalose formation in T. tenax was observed in the presence of the putativeGT. In our current model, we suggest that GT activates TPS by complexformation. The MCS might function as the emergency valve which allowsthe maintenance of the cell turgor in order to respond to environmental cues(e.g. osmotic stress).[1] Jain, N. K. and I. Roy (2008): Protein Science 18:24-36.[2] Avonce, N. et al. (2006): BMC Evolutionary Biology. 6:109.[3] Siebers B. et al. (2004): J. Bacteriol. 186:2179-2194.[4] Avonce, N. et al (2010): Mol. Biol. Evol. 27(2):359-369.ARP011The role of Trigger-loop in archaeal transcriptionT. Fouqueau* 1 , P. Cramer 2 , M. Thomm 11 Department of Microbiology & Archaea Center, University of Regensburg,Regensburg, Germany2 Gene Center and Department of Biochemistry, Ludwig-Maximilians-University, Munich, GermanyThe archaeal RNAP is closely related to eukaryotic RNAPII in terms ofsubunit composition and architecture, promoter elements and basaltranscription factors required for the initiation and elongation phase oftranscription. The possibility to reconstiute archaeal RNAP from singlesubunits expressed in Escherichia coli has been used for analyses ofstructure-function relationships in archaeal RNAP [1; 2]Conformational changes in the active site of the RNAP are required forcorrect nucleotide incorporation during transcription elongation.The highlyconserved trigger loop (TL) plays a key role at every distinct stage of thenucleotide addition cycle. Two residues of the TL, A´´ Leu83 (S. cerevisaeRpb1 Leu1081; E.coli β´ Met1238) and A´´ His87 (S. cerevisae Rpb1 His1085; E.coli β´ His1242), interact, recognize and select the correct incomingNTP. Recent analyses of the bacterial TL showed that it is also required indifferent steps of transcription as selection of the correct nucleotide, intrinsicRNA cleavage and proofreading. Substitution of Leu83 and His87, andpartial deletion of TL were introduced for this study.Employing in vitro assays with reconstituted RNAP of Pyroccocus furiosuscontaining mutated subunit A´´, we investigated the role of TL in archaealtranscription. Mutations within the TL affect promoter-dependenttranscription and nucleotide addition activity. Interestingly, substitution ofHis87 has only minor effects on RNA intrinsic cleavage, suggesting that theinvariant His, which has a crucial role in bacterial intrinsic cleavage [3], isnot required for intrinsic cleavage activity of the archaeal RNAP. Takentogether, our results confirm the essential role of TL in transcriptionelongation.[1] Naji, S. et al (2008): Structure-function analysis of the RNA polymerase cleft loops elucidatesinitial transcription, DNA unwinding, and RNA displacement. Nucl. Acids Res. 36, 676-687.[2] Kostrewa, D. et al (2009): RNA polymerase II - TFIIB structure and mechanism of transcriptioninitiation. Nature, 462, 323-330. (*These authors contributed equally to this work)spektrum | Tagungsband 2011