Structure of HCV IRES domain II determined by NMR

Abstract

Complex RNA structures regulate many biological processes, but are often too large for structure determination by NMR methods. The 5' untranslated region (5' UTR) of the hepatitis C viral (HCV) RNA genome contains an internal ribosome entry site (IRES) that binds to 40S ribosomal subunits with high affinity and specificity to control translation. Domain II of the HCV IRES forms a 25-kDa folded subdomain that may alter ribosome conformation. We report here the structure of domain II as determined using an NMR approach that combines short- and long-range structural data. Domain II adopts a distorted L-shape structure, and its overall shape in the free form is markedly similar to its 40S subunit-bound form; this suggests how domain II may modulate 40S subunit conformation. The results show how NMR can be used for structural analysis of large biological RNAs.

Structure of the full-length HCV IRES in solution

“…The molecule contains tertiary fold motifs, the IIIabc four-way junction and the IIIef/IV pseudoknot 4,14,15 , and adopts a defined ion-dependent fold under physiological conditions 16 , although it does not form a compact, globular structure 13,16,17 . The structure at atomic resolution has been determined for several autonomous folding regions, including domain II, the III abc junction and, more recently, the III ef /IV pseudoknot [18][19][20][21][22][23][24] . The presence of several hairpin loops and regions predicted to be single-stranded likely confers dynamic flexibility to the entire IRES molecule 13,16,23 and has so far hampered structure determination of the entire molecule by X-ray crystallography or electron microscopy.…”

“…The structure at atomic resolution has been determined for several autonomous folding regions, including domain II, the III abc junction and, more recently, the III ef /IV pseudoknot [18][19][20][21][22][23][24] . The presence of several hairpin loops and regions predicted to be single-stranded likely confers dynamic flexibility to the entire IRES molecule 13,16,23 and has so far hampered structure determination of the entire molecule by X-ray crystallography or electron microscopy. As the IRES is essential for viral replication and its sequence is well conserved among all HCV genotypes, its structure represents a novel target for drug design 25 .…”

Resolution-optimized NMR measurement of 1DCH, 1DCC and 2DCH residual dipolar couplings in nucleic acid bases

“…RDCs are particularly useful in nucleic acids, where the number of NOE restraints is typically relatively low, especially those involving long-range contacts. The fact that dipolar restraints provide information on internuclear bond vectors relative to a single axis system, that of the alignment tensor, allows for the study of global properties such as helix bending or relative helix orientations in multi-subunit molecules (Tjandra et al, 2000;Vermeulen et al, 2000;Al-Hashimi et al, 2002;Bondensgaard et al, 2002;MacDonald and Lu, 2002;Barbic et al, 2003;Lukavsky et al, 2003;Stefl et al, 2004). However, most applications have relied on a relatively small number of couplings per nucleotide, far fewer than the number of variable torsion angles.…”

“…NMR spectroscopy provides the opportunity to study the structure and dynamics of oligonucleotides as large as 30 kD Cabello-Villegas et al, 2002;Lawrence et al, 2003;Leeper et al, 2003;Lukavsky et al, 2003;D'Souza et al, 2004). Traditionally, many of the structural restraints have relied on quantitative interpretation of 1 H-1 H NOEs, supplemented by 3 J HH and 3 J HP couplings.…”

Solution NMR of Large Molecules and Assemblies

“…However, paralleling the advances in NMR of larger proteins, isotope labeling strategies have allowed investigation of RNAs whose spectral complexity would have previously been prohibitive. For example, by implementing segmental and residue-specific isotope labeling strategies, the solution structure of domain II of the hepatitis C virus IRES (internal ribosome entry site) RNA was determined using both conventional and RDC restraints ( Figure 10) (17,68). Although the domain adopts an extended structure, inclusion of the orientation information from RDC restraints enabled precise definition of the orientation of the top and bottom of the domain.…”