Around the bend: A right‐angled bend in the RNA of the hepatitis C virus is stabilized by a core of divalent metal ions (shown as green and violet spheres). Crystal‐structure analysis and fluorescence labeling have been used to investigate the structure and metal‐ion‐dependent stabilization of the RNA domain that plays a key role in viral protein synthesis.
The RNA genome of the hepatitis C virus (HCV) contains an internal ribosome entry site (IRES), which binds to the hostcell 40S ribosomal subunit and initiates protein translation in the absence of most initiation factors. [1,2] Recruitment of the ribosomal subunit to the HCV RNA is driven by the high affinity of the IRES-40S interaction.[3] The IRES sequence adopts a highly ordered secondary structure (Figure 1 a). [4] The three-dimensional architecture of the IRES is dominated by independently folding RNA domains.[5] Structures of IRES-40S complexes have been studied by cryo-electron microscopy (cryo-EM) revealing the overall shape of the RNA. [6,7] Higher-resolution structures of individual subdomains, including II [8] and IIIa-e, [9][10][11][12] have been determined by crystallography and NMR spectroscopy. Cryo-EM studies revealed that the domain II, which plays an important structural role in HCV translation, [13] adopts an L-shaped conformation that directs the apical hairpin loop IIb to overlap with the ribosomal E site in the proximity of the P site.[6] Binding of domain II induces a conformational change in the 40S head [7] and closes the messenger RNA (mRNA) binding cleft.[6] NMR studies suggested that subdomain IIa might be a flexible hinge whose bent state is stabilized by binding of divalent metal ions. [8] To investigate the molecular architecture of the IRES domain II kink and its metal-ion-dependent stabilization, we have used X-ray crystallography and structure-guided incorporation of fluorescent labels. For X-ray crystal structure determination (see Figure S4 and Table S1 in the Supporting Information), an oligonucleotide (IIa-1) was used that contained residues 49-69 and 100-115 of the HCV IRES (Figure 1). The overall structure of the RNA revealed a bent architecture for the IIa subdomain. The two stems that are flanking the internal bulge are arranged at a right angle (Figure 1 b). [14] The upper stem (residues 58-69/100-110) forms a continuous helix that contains both standard and noncanonical base pairs that adopt cis-Watson-Crick geometries. U106 is looped out from the stem to allow continuous stacking of the flanking base pairs. The arrangement of residues of the internal bulge introduces a right-angled bend in the RNA Figure 1. a) Secondary structure of the HCV 5'-nontranslated region that contains the IRES element and the subdomain IIa-1 construct used for crystallization and fluorescence labeling. The position of the IIa-1 construct within the IRES is indicated by a box. The nucleotides shown in red were changed in the construct from the HCV (genotype 1b) sequence to improve RNA stability. Non-Watson-Crick base pairs are indicated by "o" symbols. The numbering scheme for the construct has been adopted from the full HCV RNA. b) The three-dimensional structure of the IIa-1 RNA. 2007, 119, 230 -233 between the base pairs G52-C111 and C58-G110 that is stabilized by a combination of base stacking, hydrogen bonding, and metal-ion participation (see Figure 2 and Figure S5 in the Supporting ...
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