Structure of the complete bacterial SRP Alu domain.
Bottom Line: The mammalian Alu domain is a protein-RNA complex, while prokaryotic Alu domains are protein-free with significant extensions of the RNA.The 5' region includes an extended loop-loop pseudoknot made of five consecutive Watson-Crick base pairs.Homology modeling with the human Alu domain in context of the ribosome shows that an additional lobe in the pseudoknot approaches the large subunit, while the absence of protein results in the detachment from the small subunit.
Affiliation: Heidelberg University Biochemistry Center (BZH), INF 328, D-69120 Heidelberg, Germany.Show MeSH
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Mentions: One major determinant of the closed conformation of the Alu RNA is the RNA–RNA tertiary contact (interface of 485 Å2) between helices 3 and 5 connecting the 5′ and 3′ regions (Figure 3 and Supplementary Figure S4). This contact is conserved between bacterial, mammalian and probably also archaeal SRP. Four non-Watson–Crick base pairs in helix 5 (C73/C256, G74/A255, A75/G254 and G77/U252) remodel the minor groove geometry to a flat saddle-like appearance (Figure 3A–C and Supplementary Figure S4A) establishing a unique interaction surface with the perpendicular oriented minor groove of helix 3 (in the following denoted minor-saddle motif (MSM)). While flattening of the minor groove reduces the groove depth up to 1.5 Å, respective cross-strand purine stacking between the G74/A255 and A75/G254 base pairs (Figure 3D) also leads to a decrease of the minor groove width of up to 4 Å (Figure 3E) concomitant with an increased base inclination, base-pair opening toward the minor groove, and helical twist changes. The negative twist induces a helical depression, the characteristic feature of the MSM, which allows the phospho-ribose backbone of helix 5 to align with helix 3 and to form an extensive hydrogen-bonding network between bases, riboses and phosphates (Supplementary Figure S4B–G). Ribose-zippers on both sides and exclusive non-Watson–Crick base pairing with two diametric opposed G-U wobble pairs form the outer rim of the saddle. While Alu107 migrates faster and nearly as a single species in native gel electrophoresis after annealing, only a small portion of a variant with helix 5 shortened beyond the contact interface (Alu87) shows this migration behavior after folding under the same conditions. As most of Alu87 migrates at the same velocity before and after annealing, the MSM seems to be essential for proper folding of the Alu domain (Figure 1D). A ‘similar’ minor groove interaction can be found between helices H2 and H25 of 23S rRNA (Figure 3F and Supplementary Figure S4H and I) (56). However, in this case, the helix geometry is largely maintained and not twisted, thus only allowing for an interaction in which the minor grooves are offset with respect to each other. In summary, the occurrence of a series of conserved non-Watson–Crick base pairs and cross-strand purine stacks results in a shape complementarity of two opposing minor grooves and the formation of a saddle-like RNA–RNA interface, here denoted as the MSM. This conserved interface seems to be formed in all kingdoms of life when the 5′ and 3′ regions are locked in place.
Affiliation: Heidelberg University Biochemistry Center (BZH), INF 328, D-69120 Heidelberg, Germany.