Scientific Report 2005

Molecular Biology

Nuclear Magnetic Resonance Studies of RNA and RNA-Ligand Complexes in Solution

M. Hennig, N. Kirchner, G.C. Pérez-Alvarado, E.P. Plant,* J.D. Dinman*

* University of Maryland, College Park, Maryland

Viruses constantly threaten human health. Not only are we unable to control infections caused by old enemies such as the influenza virus, but we are continually challenged by new enemies, such as severe acute respiratory syndrome–associated coronavirus (SARS-CoV). Viral mRNAs often contain signals that tell the ribosome to change reading frames during protein synthesis. This recoding event allows viruses to coordinate gene expression from overlapping reading frames such as open reading frames 1a and 1b, which are out-of-frame coding sequences within the SARS-CoV genome. Protein 1a is translated directly from open reading frame 1a; the fused polyprotein 1a-1b is produced by programmed –1 ribosomal frameshifting in which the ribosome slips back 1 nucleotide. Like other viral frameshift signals, the SARS-CoV signal contains 2 cis-acting mRNA elements that make up a slippery heptanucleotide site, X XXY YYZ, followed by an adjacent downstream 3´ pseudoknot, a stable mRNA structure. Pseudoknots generally contain 2 stems of double-stranded RNA and 2 or 3 loops of unpaired nucleotides.

Our biochemical and solution-state nuclear magnetic resonance studies revealed that the pseudoknot in the SARS-CoV frameshift signal contains 3 stems. Mutagenesis studies indicated that specific sequences and structures within the pseudoknot are needed for efficient frameshifting, but the exact role of the extra stem in the SARS-CoV frameshifting signal still remains to be determined. Our current results suggest that the 3 stems form a complex globular RNA structure. The elucidation of this structure via high-resolution nuclear magnetic resonance should facilitate the rational development of therapeutic agents designed to interfere with SARS-CoV programmed –1 ribosomal frameshifting and will increase our understanding of how pseudoknots stimulate frameshifting.

We continue to develop nuclear magnetic resonance techniques to investigate the structural and functional diversity of RNA. Novel approaches were developed to identify and assign 2´-hydroxyl hydrogens that exchange rapidly with the solvent and thus are difficult to detect in aqueous buffers. The ribose 2´-hydroxyl group distinguishes RNA from DNA and is responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2´-hydroxyl protons is essential to understand their respective roles. We provide structural information on 2´-hydroxyl groups in the form of orientational preferences, contradicting the model that the 2´-hydroxyl typically points away from the ribose H-1´ proton.

Publications

Hennig, M., Fohrer, J., Carlomagno, T. Assignment and NOE analysis of 2´-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes. J. Am. Chem. Soc. 127:2028, 2005.

Plant, E.P., Pérez-Alvarado, G.C., Jacobs, J.L., Mukhopadhyay, B., Hennig, M., Dinman J.D. A three-stemmed mRNA pseudoknot in the SARS coronavirus frameshift signal. PLoS Biol. 3:e172, 2005.