Project

Metal Ions; RNA; NMR; ribozymes & riboswitches; ribozymes; bioinorganic chemistry

Lead
Lay summary
Metal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are poorly understood. For living systems, it is usually assumed that Na(I) and Mg(II) ions are the sole metallic cofactors involved with nucleic acids, both being most abundant and freely available in the cell. However, in living organisms, a multitude of other metal ions is present, although tightly regulated. Furthermore, in biochemical experiments many different metal ions are applied. The recognition of specific metal ions by nucleic acids is very poorly understood as is their effect on structure, folding, and catalytic activity. This study focuses on the thermodynamic and structural characterization of these interactions in large RNAs, i.e. group II intron ribozymes and Mg(II) riboswitches. We are applying a multidisciplinary approach by using a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology. Thus, our results will not only contribute to the understanding of the global structure and function of these naturally occurring RNAs, but they also promise to have a significant impact on the Bioinorganic Chemistry of RNAs and on RNA Biochemistry in general.

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Last update: 21.02.2013

Active participation

Title	Year

Number	Title	Start	Funding scheme

SUMMARY AND CENTRAL AIMS OF THE STUDYMetal ions are necessary for folding and function of catalytic RNA molecules. However, the structural and mechanistic roles of these ions are poorly understood. For living systems, it is usually assumed that Na(I) and Mg(II) ions are the sole metallic cofactors involved with nucleic acids, both being most abundant and freely available in the cell. However, in living organisms, a multitude of other metal ions is present, although tightly regulated. Furthermore, in biochemical experiments many different metal ions are applied. The recognition of specific metal ions by nucleic acids is very poorly understood as is their effect on structure, folding, and catalytic activity. This study focuses on the thermodynamic and structural characterization of these interactions in large RNAs, i.e. group II intron ribozymes and Mg(II) riboswitches. We are applying a multidisciplinary approach by using a combination of tools from Coordination and Analytical Chemistry as well as Structural Biology. Thus, our results will not only contribute to the understanding of the global structure and function of these naturally occurring RNAs, but they also promise to have a significant impact on the Bioinorganic Chemistry of RNAs and on RNA Biochemistry in general.AIM A: Understanding the First Step of Group II Intron Folding Group II introns are among the largest occurring RNAs in Nature and are closely related to the eukaryotic spliceosomal machinery. These autocatalytic self-splicing introns exhibit a straight folding pathway to the active structure devoid of kinetic traps. Folding is initiated by Mg(II) coordination, whereby the kappa-zeta region within domain 1 (D1) is the key element for the first phase of folding. We will solve the NMR solution structure of this key region of the yeast mitochondrial Sc.ai5gamma intron and characterize the Mg(II) binding as well as the structural changes associated. Our results will lead to the detailed understanding of the first folding step, which guides the assembly of the remaining domains to the catalytically active structure. AIM B: Understanding the Main Step of Catalytic Core FormationThe kappa-zeta region exhibits a second crucial function in group II introns: It constitutes the main docking site for domain 5, comprising together the largest part of the catalytic core. We will determine the structure of the about 80 nucleotide long kappa-zeta/D5 complex by NMR, investigate the structural changes of the two (sub)domains, and characterize the crucial role of metal ions associated with this docking event. A special focus will be addressed to the different effects and binding modes of various metal ions, especially Mg(II) and Ca(II), as the latter ion strongly perturbs the folding pathway and thus inhibits splicing. Surface Plasmon Resonance spectroscopy (SPR) will allow us to characterize in detail the effect of these M(n+) on the D1-D5 docking, i.e. the final step of the catalytic core assembly.AIM C: Recognizing DNA for Retro-HomingThe 5'-splice site is recognized and defined by base pairing between the exon binding site 1 in domain 1 and the complementary intron binding site 1 on the 5'-exon. Alternatively, also a corresponding sequence on DNA can be recognized, thus initiating homing or transposition. A divalent metal ion has thereby been shown to promote the final structure of this recognition complex, presumably stabilizing a strong kink in the backbone just opposite the scissile phosphodiester. Based on a recently solved NMR structure of this d3'-EBS1·IBS1 complex, we will now determine the detailed coordination sphere of this crucial divalent metal ion and investigate its exact role in the splice site recognition as well as in the RNA·DNA complex standing at the beginning of the homing/retrotransposition pathway. Part of this work also includes the characterization of the influence of different metal ions on this binding event by SPR. Hence, our results will lead to the understanding of the role of metal ions in substrate recognition.AIM D: Metal Ion Coordination at Atomic ResolutionNucleic acids show a surprising selectivity and specificity for a given kind of metal ion. The binding of natural metal ions to nucleic acids is governed by fast ligand exchange as well as a combination of inner- and outer-sphere interactions, making the characterization of the metal ion-coordination sphere highly challenging. Aside from X-ray crystallography, we will use NMR to determine the binding pockets and the corresponding intrinsic metal ion-binding constants of the investigated RNAs as well as to elucidate the inner- and outer-sphere coordinating atoms. This will allow us to understand the coordination-chemical basis of the accelerating or inhibiting influence of metal ions in ribozymes as well as their structure-defining influence in all RNAs.AIM E: Triggering and Characterizing Structural Changes in a Mg(II) RiboswitchCytoplasmic Mg(II) levels are confined to a narrow range, whose regulation is poorly understood. It has recently been discovered that in bacteria like S. typhimurium or E. coli, two alternating stem-loop structures within a conserved riboswitch sequence in the 5'-UTR of the mgtA gene regulate the expression of a Mg(II) transporter by directly sensing Mg(II) in the cytoplasm. It is the goal of our study to investigate the alternating structures at low and high Mg(II) concentrations and their interconversion, to identify the Mg(II) binding sites, to determine the affinity constants as well as the intrinsic coordination chemistry responsible for the discrimination of Mg(II) from other divalent metal ions.To summarize, the results of this study will reveal metal ion-binding motifs and their direct roles in the function of two large RNAs. This characterization will provide an important basis for research related to either structure, folding, and/or catalysis of RNAs in general as well as for the biomedical application of group II introns and riboswitches.