Deliverables Software for RNA structure predictions RNA folding at base-pair resolution and design of optimally folding and switching molecules: Many questions concerning RNA structures cannot be answered without considering RNA folding kinetics explicitly. Despite many attempts no satisfactory and efficient algorithm for RNA folding at sufficiently high resolution was available and thus we decided to conceive and implement a software that can be integrated as a module into the Vienna RNA package. The new folding algorithm is based on the experimentally well justified assumption that formation of an RNA secondary structures can be modeled by a sequence of elementary steps falling into three classes: (i) base-pair closure, (ii) base-pair opening, and (iii) base-pair shift. Structure formation is simulated by computation of a sufficiently large ensemble of individual folding trajectories, commonly a few thousands. The new folding algorithm provides answers to problems that are otherwise inaccessible. Some important examples are: (i) the distribution of folding times and the characterisation of good and bad folders, (ii) the classification of trajectories according to major local minima of conformation space and the detection of long-lived metastable states and (iii) the construction of trees of local minima and the evaluation of lowest barriers between conformations. The result is a software product. One of the major later issues in structure prediction is to proceed from secondary structures to full 3D spatial structures. The new algorithm for kinetic RNA folding allows straightforward extension from base pairing to tertiary interactions since it is not based on dynamic programming in contrast to the conventional algorithms. For this goal sufficiently rich empirical data, mainly free energies of structural elements are required. At present, such data is available for simple pseudoknots (H-type pseudoknots) and preliminary results obtained with an extension of the current folding algorithm indicate applicability without major problems. For other tertiary interactions no sufficient empirical data are available yet. Workshop on RNA structure prediction Within the frame of the project a closed workshop on RNA Structure Prediction was organized at Schlon Wilheminenberg in Vienna, April 26-28, 1999. The main goal of the workshop was to bring together theoreticians and experimentalists to discuss when and how structure prediction methods can be turned into a useful tool for research on the design of tRNA molecules. Participants were the researchers engaged in the current projects and lecturers from a few groups doing closely related research on RNA structures (Alexander Gultyeav, Universiteit Leiden, Paul Higgs, University of Manchester, and Renee Schroeder/Dolly Wittberger, Universität Wien). Several problems of a general nature whose solutions are basic to tRNA design were made specific: 1) What is the role of the L-shaped scaffold of tRNA in the evolution of domains? Can identity elements be varied and better presented at constant scaffold geometry? Are radically different L-shapes with changes in the scaffold possible which allow an optimal presentation of identity elements? 2) Can sequence space concepts based on chain lengths in the range of 75-85 nucleotides be successfully applied to optimization of structure and function of tRNAs? What could be expected from structures derived from longer sequences (85)? 3) Can evolution experiments be successfully simulated in silico and what would we learn from these simulations? 4) Is sequence information itself sufficient to predict structures? 5) Can we analyze and understand the canonical forms of tRNAs found in nature exclusively from their functions? As expected, the answers to these deep questions could not be given instantaneously but they will represent major topics for the cooperation within the projects and for the forthcoming meetings. New Knowledge on combinatorial in vitro approaches related to tRNAs The wide knowledge accumulated over the years on structure and function of transfer RNAs (tRNAs) has allowed one to decipher the rules underlying the function and the architecture of these molecules. These rules will be discussed and the implications for manipulating tRNA properties by structure-based and combinatorial in vitro approaches reviewed. Since most of the signals conferring function to tRNAs are located on the two distal extremities of their three-dimensional L-shape implies that the structure of the RNA domain connecting these two extremities can be of different architecture and/or can be modified without disturbing individual functions(s). This concept is first supported by the existence in Nature of RNAs of peculiar structures having tRNA properties, as well as by engineering experiments on natural tRNAs. The concept is further illustrated by examples of RNAs designed by combinatorial methods. The different procedures used to select RNAs or tRNA-mimics interacting with aminoacyl-tRNA synthetases or with elongation factors, and to select tRNA-mimics aminoacylated by synthetases are presented, as well as the functional and structural characteristics of the selected molecules. Production and characteristics of aptameric RNAs fulfilling aminoacyl-tRNA synthetase functions and of RNAs selected to have affinities for amino acids will also be described. Finally, properties of RNAs obtained by either the structure-based or the combinatorial methods are discussed in the light of the origin and evolution of the translation machinery but also in view to obtain new inhibitors targeting specific steps in translation. The view of a tRNA as a molecular scaffold gives insight into structure and helps to understand the rules underlying function. The scaffold presents chemical groups that are read out by other macromolecules and guide the course of the interaction. For the variety in interaction partners, estimated to be 40 to 50 during the life cycle of a tRNA, the molecule was and is a textbook example for molecular recognition in biology. What are the rules behind? A priori each specific interaction should require a specific set of chemical groups located on the scaffold. The first information-set imprinted in a tRNA that was identified is the genetic code manifested in the anticodon triplet. The underlying rule is the optimal Watson-Crick base pairing ability with the mRNA-codon interaction. But more information is buried in each tRNA. The most studied is that identifying the tRNAs during aminoacylation. This information is provided by identity elements and correlates real amino acids to the virtual genetic code. The rules constitute a second genetic code and are quite well understood. In principle they rely on the presence or absence of functional groups on bases, the phospho-ribose backbone or even on architectural features, like an extended variable region. The precise location of these signals on the scaffold is monitored by complementary structural features on the protein level and this molecular interplay allows specific aminoacylation of the corresponding tRNA. But even much more information is imprinted on the tRNA scaffold, since many more specific interactions occur during a tRNA-life cycle. Examples are the recognition by elongation factors or by proteins during the maturation process of tRNAs. The in vitro selection of oligonucleotides provided surprisingly specific aptamers to protein targets. Complexes which were formed between translation factors and ribosomes have been studied by RNA-aptamers directed to elongation factor Tu, elongation factor G, initiation factor 2 and release factor 1. The aptamer RNA molecules present rather well defined consensus sequences for each of the protein target against which they were selected, except those directed against initiator factor 2 which have more degenerated sequences with nevertheless G and A-rich motives. These aptamers were used as inhibitors and regulators of translation. The usually high specificity of RNA-aptamers for their ligands and their good binding constants make aptamers also possible reporter molecules. Adequately labelled RNA-aptamers have been synthesised and used as fluorescent probes for detecting protein ligands (Team 5). New knowledge on three-dimensional structures of RNA pseudoknots The structure of 2 pseudoknots have been determined by NMR at atomic resolution (from the tRNA-like structure of TYMV RNA and the frame-shifting signal of SRV-1 RNA). This allowed further studies on the stability and dynamics of RNA pseudoknots. The size of the tRNA-like structure of TYMV RNA has been determined by a detailed aminoacylation study: it corresponds to the 3-terminal 82nts. This size is relevant in view of further structural studies on this domain. The prediction of bulge pseudoknots was introduced in the genetic algorithm program. Surprisingly, no specific role for the pseudoknot in the tRNA-like structure of TYMV RNA could be inferred from in vitro replication studies. A major result was the elucidation at atomic resolution of the 3D structure of the pseudoknot present in the tRNA-like structure of TYMV RNA (with Hilbers, Nijmegen). Apart from establishing the coaxial stacking of the two constitutive stem regions as predicted earlier, the conformation of the loop crossing the shallow groove turned out to have surprising features. An A residue makes two H-bonds with two different G residues of the opposing stem, thereby increasing the stability of this pseudoknot. An identical fold was found in the 3-terminal hairpin loop in the absence of pseudoknot formation. It thereby represents a new RNA folding motif that appears to be suitable for pseudoknot formation. The present structure is a guide for further studies on RNA pseudoknots and tRNA-like structures and for the understanding of their function in virus replication. Interdisciplinary Network on Research on RNAs with 'Transfer RNA properties' An extremely positive aspect of the project after two years of activity was the establishment of many links between partner Laboratories. Some already existed but have been rejuvenated [e.g. between Team 1 (Strasbourg) and Teams 2 (Illkirch), 3 (Leiden) and 5 (Bayreuth) or between subcontractor of Team 1 and Team 7 (Gfttingen)] and more important, many new ones were created. Their birth and the start of actual collaborations were greatly facilitated by the joint and bilateral Meetings and Workshops that were organised in the frame of this contract. Collaboration between Team 3 (Leiden) and Team 4 (Vienna) initiated work for RNA structure prediction and especially for that of metastable structures. Other cooperations were between the Vienna Team and Team 1 (Strasbourg), or Team 5 (Bayreuth) and Team 6 (Rome). For that, a number of RNA structures were provided to the Vienna Team, either wild-type or mutant sequences, for prediction of their most likely folding. Other links, related to common interest in RNA-aptamer research, connected Team 5 (Bayreuth), Team 6 (Rome), and Team 1 (Strasbourg). Cooperations involving the industrial partner (Team 7, Göttingen) were particularly fruitful: they concerned chemical synthesis of modified RNA blocks and tRNA fragments with modified bases (with Team 1, Strasbourg), synthesis of fluorescent nucleotide or dinucleotide analogues (with subcontractor of Team 1, Strasbourg and Team 5, Bayreuth), and synthesis of RNA fragments for studying pseudoknot structures (with Team 3, Leiden). The major result is the implementation of an interdisciplinary network. This network has emphasized the importance of two approaches in modern RNA research, namely: (i) the need of chemical methods for the synthesis of appropriate RNA fragment with well defined properties (e.g. RNAs with novel chemical groups at specific positions in nucleotide and sequence, or containing targeted labels for fluorescence studies), and (ii) the necessity of theoretical tools for structure prediction and structure folding as well as for the prediction of alternate and metastable structures. Scientific Final Report as R & D strategy for further R & D This project is an interdisciplinary approach to basic and applied research on RNAs with tRNA properties. It was divided into four inter-related parts. The first dealt with recognition of RNA domains by tRNA-recognising proteins, the second concerned RNA and tRNA-like domains from viral RNA genomes, the third comprised selection and structure analysis of RNA domains mimicking tRNAs, and the fourth dealt with biotechnological aspects of RNA chemical synthesis. The feasibility of the project relied on the complementarities between the participating teams in terms of expertise in methodologies (RNA engineering, combinatorial methods, X-ray crystallography, NMR, RNA chemistry, and bio-computing) and availability of a variety of proteins from translation and replication systems. The RNAs that were investigated derived from canonical tRNA (taken as a model) or viral tRNA-like structures. They were obtained either by rational structure-based design or by in vitro selection. A major aim was to understand detailed molecular aspects of the translation machinery, but transcription / replication processes were also considered since tRNA or tRNA-like domains participate in such processes. The final aim was to find small RNAs that specifically block tRNA-target proteins (tRNA maturation endonucleases, aminoacyl-tRNA synthetases, elongation factors, viral replicates), with the hope that our project should lead in the future to the design of new classes of RNA antibiotics and to the proposal of novel anti-viral strategies. A variety of steps in translation (some in replication) were therefore studied and experiments were carried out on RNAs of various biological origins [viruses, prokaryotes (mesophiles or thermophiles), eukaryotes, archaea]. Species specificities between these RNAs, in particular for aminoacylation and elongation, were investigated. The problem of evolution was approached by theoretical methods for the search of RNAs with tRNA characteristics and by a variety of experimental methods including particular in vitro selection procedures. In this way we expected to find RNA structures similar to those retained by natural evolution, but our hope was also to find alternate solutions providing aminoacylation identities or recognition potential by translational factors. An extremely positive aspect of the project after two years of activity was the establishment of many links between partner Laboratories. Some already existed but have been rejuvenated [e.g. between Team 1 (Strasbourg) and Teams 2 (Illkirch), 3 (Leiden) and 5 (Bayreuth) or between subcontractor of Team 1 and Team 7 (Göttingen)] and more important, many new ones were created. Their birth and the start of actual collaborations were greatly facilitated by the joint and bilateral Meetings and Workshops that were organised in the frame of this contract. Collaboration between Team 3 (Leiden) and Team 4 (Vienna) initiated work for RNA structure prediction and especially for that of metastable structures. Other cooperatives were between the Vienna Team and Team 1 (Strasbourg), or Team 5 (Bayreuth) and Team 6 (Rome). For that, a number of RNA structures were provided to the Vienna Team, either wild-type or mutant sequences, for prediction of their most likely folding. Other links, related to common interest in RNA-aptamer research, connected Team 5 (Bayreuth), Team 6 (Rome), and Team 1 (Strasbourg). Cooperations involving the industrial partner (Team 7, Göttingen) were particularly fruitful: they concerned chemical synthesis of modified RNA blocks and tRNA fragments with modified bases (with Team 1, Strasbourg), synthesis of fluorescent nucleotide or dinucleotide analogues (with subcontractor of Team 1, Strasbourg and Team 5, Bayreuth), and synthesis of RNA fragments for studying pseudoknot structures (with Team 3, Leiden) Website and portal with search engine Mamit-tRNA - Compilation of mammalian mitochondrial tRNA genes containing 31 mitochondrial genomes (covering the literature up to September 2000) Mamit-tRNA is a internet based gene compilation aimed at defining typical as well as consensus primary and secondary structural features of mammalian mitochondrial tRNAs (Internet portal with Search engine). It contains presently 679 tRNA gene sequences from 31 fully sequenced mammalian mitochondrial genomes. These are classified into 22 families according to the amino acid specificity as defined by the anticodon triplets. For each family, a vertical alignment based on the search of common secondary structural domains is presented as well as "typical" and "consensus" cloverleaf structures. Discussion on structural characteristics as deduced for each of the 22 tRNA families can be found in reference Helm et al., (2000) RNA 6: 1356-1379. Scientists as well as clinicians will use it as an information source or reference material. The result is a service. Due to the exponential availability of new mitochondrial genomes in genome databases, Mamit-tRNA will be updated regularly and refined consensus sequences displayed. This website was devised to fulfill two main aims. At one side, it is expected to serve as basis for fundamental research on structural properties mammalian mitochondrial tRNAs, a field far behind the present knowledge on bacterial and eukaryotic cytosolic tRNAs and on the protein synthesis machineries in these organelles. At the other side, the consensus sequences are of great importance for the molecular understanding of rare human diseases linked to point mutations in mitochondrial tRNA genes. Indeed, over the last 15 years an increasing number of human neurodegenerative disorders could be correlated to point mutations in mitochondrial tRNA genes (more than 80 different mutations have so far been reported) and the molecular mechanisms remain to be solved. Access to updated consensus sequences will facilitate the work of clinicians dealing with the discovery of new mutation sites as well as to molecular biologists investigating the potential structural and/or functional effects of the mutations. Searching for OpenAIRE data... 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