Structure Determination of Catalytic RNAs and Investigations of Their Metal Ion-Binding Properties

Abstract

Naturally occurring RNA molecules exhibit many unexpected and fascinating properties in living cells like protein synthesis and transport, regulation of metabolic functions, and catalytic cleavage reactions. To understand this functional diversity, a detailed knowledge of RNA structure and metal ion-binding properties is crucial. In our research group, we address these problems by combining various biochemical, analytical and spectroscopic techniques. A large part of our work is devoted to the structure determination of catalytic RNA molecules, i.e. ribozymes, by NMR. Based on the three-dimensional structure, further experiments are carried out to understand in detail the effects of different metal ions on the local and global structure, as well as catalysis itself. Our research concentrates on two classes of the naturally occurring RNAs: (i) riboswitches and (ii) group II intron ribozymes. The emphasis of this short review is on our structural and analytical NMR studies on group II intron ribozymes; our riboswitch work is only briefly indicated in the following Section. RiboswitchesRiboswitches are highly conserved RNA sequences occurring for example in the 5ʹ-UTR (see Abbreviations) of certain mRNAs. They bind small metabolites like FMN, SAM, TPP, guanine, adenine, and coenzyme B 12 with a high degree of affinity and specificity [6] [7]. Upon binding of the corresponding metabolite, the riboswitch sequence presumably undergoes structural changes [8] and in some cases even cleavage of the RNA strand occurs [4]. Both cleavage and structural changes inhibit binding of the 5ʹ-UTR to the ribosome, and thus prohibit translation to the corresponding protein. As the protein(s) expressed by a mRNA is always involved in either biosynthesis or transport of the metabolite that binds to the riboswitch, this new class of RNA molecules constitutes an elegant way of feedback control within living cells.In our group, we work with the so-called B 12 riboswitch. This riboswitch consists of an about 200 nt long and highly conserved sequence located in the 5ʹ-UTR of mRNAs associated with the metabolism and transport of coenzyme B 12 and vitamin B 12 , respectively [9]. Our investigations encompass structural studies as well as binding studies of coenzyme B 12 and its derivatives to this riboswitch sequence in the presence of different metal ions. Group II Intron RibozymesThe main efforts of our research are presently devoted to group II intron ribozymes. These large molecular machines are self-splicing introns, i.e. capable of cutting themselves out of the precursor RNA and in a second step joining the adjacent exons to yield the final mRNA -all without the aid of proteins (Fig. 1). In addition, these amazing molecules can insert themselves again into RNA and even DNA. The occurrence, reaction mechanisms, and the different aspects of metal ion binding to group II introns have lately been reviewed [10][11], thus only a few important points are summarized below:

From nucleotides to ribozymes—A comparison of their metal ion binding properties

“…in the case of the hammerhead ribozyme [45][46][47]. In addition, there are several examples of both naturally occurring and in vitro designed ribozymes and DNAzymes [8,[48][49][50][51][52], which show a fast decrease in reaction kinetics if trace amounts of other metal ions are present aside from the inherent one. It is therefore evident that not only Mg 2+ but other metal ions as well need to be considered in order to understand the structure and functioning of nucleic acids in general, and of ribozymes specifically.…”

Metal Ion–RNA Interactions Studied via Multinuclear NMR

“…For example, by 25 Mg NMR line shape analysis of RNAs titrated with isotopically enriched Mg 2+ it was possible to estimate the numbers and the association constants of weakly bound Mg 2+ to tRNAs (11). However, in order to localize and quantify RNA-metal ion interactions by NMR, 1 H, 13 C, 15 N and 31 P are the nuclei normally applied.…”

NMR Spectroscopy in Bioinorganic Chemistry

“…However, they are modular in nature, and hence offer an optimal platform for the study of metal ion-RNA binding properties. [12,13,32] The single parts can be transcribed in large amounts, [33] studied independently, and their properties translated to the whole intron. For this reason we largely focus on single parts or domains of the yeast mitochondrial group II intron Sc.ai5γ being one the best investigated introns.…”

“…Nuclear magnetic resonance (NMR) spectroscopy is a crucial technique that we use not only to obtain structural information on our molecules at an atomic level [6][7][8][9][10][11] but, even more important, to elucidate in detail their metal ion binding. [2,9,10,[12][13][14][15][16][17][18] Fig. 1 shows three examples of recently solved structures, belonging to the three families of biomolecules we are working with: the exon/intron binding site 1 (EBS1/IBS1; PDB ID 2K65) [19] of the yeast mitochondrial group II intron Sc.ai5γ, a silver modified duplex DNA (PDB ID 2KE8), [8] and the C-terminal domain of the seed-specific wheat MT Zn 6 E c -1, denoted as Zn 4 β E -E c -1 (PDB ID 2KAK).…”

“…[10] The chemical institutes at the University of Zurich run a central NMR facility currently equipped with one 700, one 600, three 500, two 400, and one 300 MHz spectrometer. Three of the high-field machines (500, 600, and 700 MHz) are equipped with z-axis pulsed field gradient Cryoprobes TM (Bruker), and are used for classical structural biology experiments (2D and 3D experiments on 1 H, 13 C, 15 N nuclei, as well as 31 P). One 500 MHz spectrometer is dedicated to variable-temperature and multinuclear NMR studies.…”