Band 382, Heft 8

Seven purified proteins may be combined to reconstitute regulated, promoterdependent RNA polymerase II transcription: five general transcription factors, Mediator, and RNA polymerase II. The entire system has been conserved across species from yeast to humans. The structure of RNA polymerase II, consisting of 10 polypeptides with a mass of about 500 kDa, has been determined at atomic resolution. On the basis of this structure, that of an actively transcribing RNA polymerase II complex has been determined as well.

In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for γaminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacological and electrophysiological properties. The predominant type, termed GABAA, and a recently identified GABAC type, form ligandgated chloride channels, whereas GABAB receptors activate separate cation channels via G proteins. Based on their homology to nicotinic acetylcholine receptors, GABAC receptors are believed to be oligomeric protein complexes composed of five subunits in a pentameric arrangement. To date up to five different GABAC receptors subunits have been identified in various species. Recent studies have shed new light on the biological characteristics of GABAC receptors, including the chromosomal localization of its subunit genes and resulting links to deseases, the cloning of new splice variants, the identification of GABAC receptorassociated proteins, the identification of domains involved in subunit assembly, and finally structure/function studies examining functional consequences of introduced mutations. This review summarizes recent data in view of the molecular structure of GABAC receptors and presents new insights into the biological function of this protein in the retina.

Mitogen activated protein kinases (MAPK) are important mediators in signal transmission, connecting the perception of external stimuli to cellular responses. MAPK cascades are involved in signalling various biotic and abiotic stresses, like wounding and pathogen infection, temperature stress or drought, but are also involved in mediating the action of some plant hormones, such as ethylene and auxin. Moreover, MAPKs have been implicated in cell cycle and developmental processes. In Arabidopsis mutant screens and in vivo assays several components of plant MAPK cascades have been identified. This review gives an update of recent advances in plant MAPK signalling and discusses the emerging mechanisms of some selected MAPK pathways.

In this review, we summarize the successful interplay between three disciplines, organic synthesis, biophysics and cell biology, in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in molecular detail. Highlighting the example of the Ras proteins, we show how the development of new synthetic methodologies paved the road to the synthesis of lipidated peptides and by a combination of chemical and molecular biological techniques lipidated proteins as molecular tools. We further give an overview of the results of the biophysical properties and biological activities of the molecules synthesized by means of this interdisciplinary approach. This successful combination of different disciplines led to a better understanding of the selective targeting of Ras and related lipoproteins to the plasma membrane.

The generation of a mature tRNA 3end is an important step in the processing pathways leading to functional tRNA molecules. While 5end processing by RNase P is similar in all organisms, generation of the mature 3terminus seems to be more variable and complex. The first step in this reaction is the removal of 3trailer sequences. In bacteria, this is a multistep process performed by endo and exonucleases. In contrast, the majority of eukaryotes generate the mature tRNA 3end in a single step reaction, which consists of an endonucleolytic cut at the tRNA terminus. After removal of the 3trailer, a terminal CCA triplet has to be added to allow charging of the tRNA with its cognate amino acid. The enzyme catalyzing this reaction is tRNA nucleotidyltransferase, homologs of which have been found in representatives of all three kingdoms. Furthermore, in metazoan mitochondria, some genes encode 3terminally truncated tRNAs, which are restored in an editing reaction in order to yield functional tRNAs. Interestingly, this reaction is not restricted to distinct tRNAs, but seems to act on a variety of tRNA molecules and represents therefore a more general tRNA repair mechanism than a specialized editing reaction. In this review, the current knowledge about these crucial reactions is summarized.

We have used an RNA consisting of the potato spindle tuber viroid (PSTVd) and 240 bp of doublestranded RNA derived from the GUS gene as a backbone for scanning force microscope (SFM) studies on RNA binding proteins. The in vitro transcribed RNA forms a rodlike structure of apparent 130 nm in length with a completely base paired central part flanked by the incompletely paired viroid helix with bulges on both sides. The termini of the molecule consist of loops such that no blunt or staggered RNA ends are exposed. Suitable, asymmetrical restriction sites in the construct allow for the insertion of sequences of interest, e. g. protein binding sites. We have inserted the IRE (iron responsive element) sequence into the construct and have used in vitro transcripts to study binding of IREBP. Relative binding frequencies show that 70% of the protein binds to the expected site in the molecule while only a slightly enhanced binding is observed at the termini. In the GUSPSTVdIRE backbone, the orientation of the molecule is easily determined by IREBP binding. It thus provides a versatile tool to study specific as well as preferential interaction of other proteins with sequences or structures inserted into a different part of the molecule.

Mammalian RNA polymerase I (Pol I) is a multisubunit enzyme that is decorated with accessory proteins, termed PAFs (polymeraseassociated factors). The presence or absence of distinct PAFs may account for the functional differences of distinct fractions of cellular Pol I, and suggests that PAFs could be targets of regulatory pathways. Here we describe and functionally characterize PAF67, a novel 67 kDa protein that is tightly associated with a subpopulation of cellular Pol I. Both PAF67-containing and deficient Pol I transcribe nonspecific templates with similar efficiency, however, only the enzyme that contains PAF67 is capable of specifically transcribing rDNA templates. PAF67 colocalizes with Pol I in the nucleolus at sites of active rDNA transcription, indicating that PAF67 serves a role in rDNA transcription initiation. The results suggest that association of PAF67 with the core enzyme endows Pol I with the capability to assemble into a productive transcription initiation complex at the rDNA promoter.

The partial purification and basic biochemical characterization of the RNase P holoenzymes of two species of methanogenic Archaea, Methanothermobacter thermoautotrophicus (previously Methanobacterium thermoautotrophicum strain δH) and Methanococcus jannaschii, are described. The properties of these enzymes, particularly buoyant density in Cs2SO4 and recent information about the subunit composition of the archaeal enzymes, suggest that RNase P enzymes in Archaea are much more alike than earlier studies in Sulfolobus acidocaldarius and Haloferax volcanii suggested.

Guanylate cyclaseactivating protein 1 (GCAP1) and guanylate cyclaseinhibitory protein (GCIP) are calmodulinrelated Ca2+binding proteins expressed in vertebrate photoreceptor cells. GCAP1 activates photoreceptor guanylate cyclase 1 (GC1) at low free [Ca2+] (<50 nM, in the light), but inhibits it at physiological high [Ca2+](1 M, in the dark). GCIP, a Ca2+binding protein from frog retina, inhibits GC1 at ~1 M [Ca2+], but is unable to stimulate cyclase at low [Ca2+]. In this study, we probed the interaction between GCAP1 and GC1 by producing GCAP1/GCIP chimeras and tested their capability to stimulate GC1. We prepared eight pairs of constructs in which the Nterminal portions of GCIP and GCAP1 were successively replaced by corresponding domains of GCAP1, and GCIP, respectively. The expressed proteins were purified and tested for stimulation of GC1 at 50 nM [Ca2+], and their ability to competitively inhibit GC1 stimulation by a Ca2+insensitive GCAP1 mutant, GCAP1-tm, at high [Ca2+]. While all GCAP1/GCIP chimeras competitively inhibited GC1 stimulation at high [Ca2+] by GCAP1-tm, several of the GCIP/GCAP1 chimeras had no effect. A chimera consisting of residues 120 of GCIP and 21205 of GCAP1 had no effect on GC1 at low [Ca2+], suggesting that the Nterminal region MGNIMDGKSVEELSSTECHQ, which has no sequence similarity to GCIP, is among the key components necessary for GC1 stimulation. A GCAP1/GCIP chimera consisting of residues 143 (including nonfunctional EF1) of GCAP1 and residues 56206 of GCIP stimulated GC1 at low [Ca2+] and inhibited GC1 at high [Ca2+], suggesting that the essential components required to transform an inhibitory to an activating protein are contained within the Nterminal region of GCAP1 (residues 143).

Bone morphogenetic proteins (BMP) are dimeric factors initiating several distinct signaling cascades by binding to two types of transmembrane serine/threonine kinase receptors (BRI and BRII), and are thus regulating several steps in embryonal development and adult tissue homeostasis. BMP-2 contains two symmetrical pairs of juxtaposed epitopes: the wrist epitope with high affinity to BRI consists of residues from both BMP-2 monomers, while the knuckle epitope resembles the low affinity site for BRII and comprises residues from only one monomer. Here we generated heterodimeric BMP-2 muteins with one monomer mutant in either epitope I for BRI (eI) or epitope II for BRII (eII) and the second monomer wild type for receptor interactions (m). These muteins (B2eI/B2m and B2eII/B2m) were analyzed by biosensor analysis as well as by measuring their biological activity and compared to their homodimeric forms (either wild type or mutant). Depletion of only one epitope II results in the loss of biological activity as measured by alkaline phosphatase (ALP) activity and Smad induced reportergene assays. However, depletion of only one epitope I shows a reduction of ALP activity to about 25%, while the activation of the Smad pathway remained normal. Homomeric muteins are nonfunctional for both Smad and ALP activation. This suggests that two functional epitopes II have to be present on one BMP-2 molecule for receptor activation. Futhermore, both pathways (Smad and ALP) are triggered differently by distinct BMPreceptor complexes. Heteromeric BMP-2 mutants therefore allow a distinguishable manipulation of either pathway and thus represent important tools for the generation of specific BMP-2 antagonists or agonists.

Urokinasetype plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the Nterminal region of uPA (uPA19-31). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPARsystem has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPAderived peptide, cyclo19,31uPA19-31, as a lead structure for the development of low molecular weight uPAanalogues, capable of blocking uPA/uPARinteraction [Bürgle et al., Biol. Chem. 378 (1997), 231237]. We now searched for peptide variants of cyclo19,31uPA19-31 withelevated affinities for uPAR binding. Among other tasks, we performed a systematic Damino acid scan of uPA19-31, in which each of the 13 Lamino acids was individually substituted by the corresponding Damino acid. This led to the identification of cyclo19,31uPA19-31 as a potent inhibitor of uPA/uPARinteraction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPARligands uPA and its aminoterminal fragment. Cyclo19,31uPA19-31 not only blocksbinding of uPA to uPAR but is also capable of efficiently displacing uPARbound uPA from the cell surface and to inhibit uPAmediated, tumor cellassociated plasminogen activation and fibrin degradation. Thus, cyclo19,31uPA19-31 represents a promising therapeutic agent to significantly affect the tumorassociated uPA/uPARsystem.

The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants starts with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have expressed and characterized different olfactory receptors with several expression systems. Here we provide the first documentation of functional expression of odorant receptors using the Semliki Forest virus system. The human odorant OR 17-40 receptor and the rat I7 receptor were functionally expressed in vertebrate kidney cells (HEK293) using recombinant Semliki Forest viruses. Receptors were expressed as a fusion protein with the Nterminal membrane import sequence of the guinea pig serotonin receptor. Experiments employing the Ca2+sensitive dye fura-2 revealed a fast, transient increase in the [Ca2+] after application of the specific agonists helional and octanal to HEK293 cells infected with viruses containing RNA for the human odorant OR 17-40 receptor and the rat I7 receptor, respectively.

The interaction of bacterial endotoxins (LPS Re and lipid A, the endotoxic principle of LPS) with the endogenous antibiotic lactoferrin (LF) was investigated using various physical techniques and biological assays. By applying Fouriertransform infrared (FTIR) spectroscopy, we find that LF binds to the phosphate group within the lipid A part and induces a rigidification of the acyl chains of LPS. The secondary structure of the protein as monitored by the amide I band is, however, not changed. Concomitant with the IR data, scanning calorimetric data indicate a sharpening of the acyl chain phase transition. From titration calorimetric and zeta potential data, saturation of LF binding to LPS was found to lie at a : ratio of 1:3 to 1:5 M from the former and 1:10 M from the latter technique. Xray scattering data indicate a change of the lipid A aggregate structure from inverted cubic to multilamellar, and with fluorescence (FRET) spectroscopy, LF is shown to intercalate by itself into phospholipid liposomes and may also block the lipopolysaccharidebinding protein (LBP)induced intercalation of LPS. The LPSinduced cytokine production of human mononuclear cells exhibits a decrease due to LF binding, whereas the coagulation of amebocyte lysate in the Limulus test exhibited concentrationdependent changes. Based on these results, a model for the mechanisms of endotoxin inactivation by LF is proposed.

Zinc ions have both essential and toxic effects on mammalian cells. Here we report the ability of zinc to act as an inducer of apoptosis in C6 rat glioma cells. Incubation with 150 to 300 M ZnCl2 caused cell death that was characterized as apoptotic by internucleosomal DNA fragmentation, formation of apoptotic bodies, nuclear fragmentation and breakdown of the mitochondrial membrane potential. On the other hand, zinc deprivation by the membrane permeable chelator TPEN [N,N,N,N,tetrakis (2-pyridylmethyl)ethylenediamine] also induced programmed death in this cell line, indicating the existence of intracellular zinc levels below and above which apoptosis is induced. Zincinduced apoptosis in C6 cells was independent of major signaling pathways (protein kinase C, mitogen activated protein kinase and guanylate cyclase) and protein synthesis, but was increased by facilitating zinc uptake with the ionophore pyrithione. Lanthanum(III)chloride was also able to increase the net zinc uptake, but nevertheless apoptotic features and zinc toxicity were reduced. Remarkably, lanthanum suppressed the zincinduced breakdown of the mitochondrial membrane potential. We conclude that in C6 cells lanthanum acts in two different ways, as a promoter of net zinc uptake and as a suppressor of zincinduced apoptosis.

In Escherichia coli, the ribosomeassociated Trigger Factor (TF) cooperates with the DnaK system in the folding of newly synthesized cytosolic polypeptides. Here we investigated the functional relationship of TF and DnaK by comparing various functional properties of both chaperones. First, we analyzed the ability of TF and DnaK to associate with nascent polypeptides and fulllength proteins released from the ribosome. Toward this end, we established an E. coli based transcription/translation system containing physiological ratios of TF, DnaK and ribosomes. In this system, TF can be crosslinked to nascent polypeptides of σ32. No TF crosslink was found to fulllength σ32, which is known to be a DnaK substrate. In contrast, DnaK crosslinked to both nascent and fulllength σ32. DnaK crosslinks critically depended on the type of chemical crosslinker. Crosslinks represent specific substratechaperone interactions since they relied on the association of the nascent polypeptides with the substrate binding pocket of DnaK. While DnaK is known to be the major chaperone to prevent protein aggregation under heat shock conditions, we found that TF did not prevent aggregation of thermally unfolded proteins in vitro and was not able to complement the heatsensitive phenotype of a δnaK52 mutant in vivo. These data indicate that TF and DnaK show strong differences in their ability to prevent aggregation of denatured proteins and to associate with native like substrates, but share the ability to associate with nascent polypeptides.

Active site reactivity and specificity of RhdA, a thiosulfate:cyanide sulfurtransferase (rhodanese) from Azotobacter vinelandii, have been investigated through ligand binding, sitedirected mutagenesis, and Xray crystallographic techniques, in a combined approach. In native RhdA the active site Cys230 is found persulfurated; fluorescence and sulfurtransferase activity measurements show that phosphate anions interact with Cys230 persulfide sulfur atom and modulate activity. Crystallographic analyses confirm that phosphate and hypophosphite anions react with native RhdA, removing the persulfide sulfur atom from the active site pocket. Considering that RhdA and the catalytic subunit of Cdc25 phosphatases share a common threedimensional fold as well as active site Cys (catalytic) and Arg residues, two RhdA mutants carrying a single amino acid insertion at the active site loop were designed and their phosphatase activity tested. The crystallographic and functional results reported here show that specific sulfurtransferase or phosphatase activities are strictly related to precise tailoring of the catalytic loop structure in RhdA and Cdc25 phosphatase, respectively.

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and Nterminal sequences that are not consistent with simple posttranslational modifications. Furthermore, their behaviour differs at high temperatures (42 C47 C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catAderived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.

Asp83 is a highly conserved residue in the second transmembrane domain of visual pigments and many members of other G proteincoupled receptor subfamilies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho BSI Lumi Meta I Meta II). Meta II represents the active state of rhodopsin, which binds and activates the G protein transducin. Evidence has been presented that Asp83 participates in the formation of Meta II and undergoes a change in Hbonding. To investigate whether this role of Asp83 requires its protondonating capacity and/or its Hbonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating that Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and the pH sensitivity of the Meta I Meta II transition. In general, D83C behaves much more like wildtype than D83N. We conclude that the structural role of Asp83 also involves the acidic nature of its carboxyl group. In addition, the participation in Meta II formation of Cys83 in D83C manifests itself as a change in the vibrational properties of the sulfhydryl group, demonstrating that the SH group can be used as a noninvasive probe for local structural changes.

Photochemically induced dynamic nuclear polarisation (photoCIDNP) in intact bacterial reaction centres has been observed by 13Csolid state NMR under continuous illumination with white light. Strong intensity enhancement of 13C NMR signals of the aromatic rings allows probing the electronic ground state of the two BChl cofactors of the special pair at the molecular scale with atomic selectivity. Differences between the two BChl cofactors are discussed. Several aliphatic 13C atoms of cofactors, as well as 13C atoms of the imidazole ring of histidine residue(s), show nuclearspin polarisation to the same extent as the aromatic nuclei of the cofactors. Mechanisms and applications of polarisation transfer are discussed.

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases that have been implicated in various disease processes. Different classes of MMP inhibitors, including hydroxamic acids, phosphinic acids and thiols, have been previously described. Most of these mimic peptides and most likely bind in a similar way to the corresponding peptide substrates. Here we desccribe pyrimidinetriones as a completely new class of metalloprotease inhibitors. While the pyrimidinetrione template is used as the zincchelating moiety, the substituents have been optimized to yield inhibitors comparable in their inhibition efficiency of matrix metalloproteinases to hydroxamic acid derivatives such as batimastat. However, they are much more specific for a small subgroup of MMPs, namely the gelatinases (MMP-2 and MMP-9).