The NMR Structure of an Internal Loop from 23S Ribosomal RNA Differs from Its Structure in Crystals of 50S Ribosomal Subunits^,

Abstract

Internal loops play an important role in structure and folding of RNA and in RNA recognition by other molecules such as proteins and ligands. An understanding of internal loops with propensities to form a particular structure will help predict RNA structure, recognition, and function. The structures of internal loops and from helix 40 of the large subunit rRNA in Deinococcus radiodurans and Escherichia coli, respectively, are phylogenetically conserved, suggesting functional relevance. The energetics and NMR solution structure of the loop were determined in the duplex, The internal loop forms a different structure in solution than in the crystal structures of the ribosomal subunits. In particular, the crystal structures have a bulged out adenine at the equivalent of position A15 and a reverse Hoogsteen UA pair (trans Watson-Crick/Hoogsteen UA) at the equivalent of U4 and A14, whereas the solution structure has a single hydrogen bond UA pair (cis Watson-Crick/sugar edge A15U4) between U4 and A15 and a sheared AA pair (trans Hoogsteen/sugar edge A14A5) between A5 and A14. There is cross-strand stacking between A6 and A14 (A6/A14/A15 stacking pattern) in the NMR structure. All three structures have a sheared GA pair (trans Hoogsteen/sugar edge A6G13) at the equivalent of A6 and G13. The internal loop has contacts with ribosomal protein L20 and other parts of the RNA in the crystal structures. These contacts presumably provide the free energy to rearrange the base pairing in the loop. Evidently, molecular recognition of this internal loop involves induced fit binding, which could confer several advantages. The predicted thermodynamic stability of the loop agrees with the experimental value, even though the thermodynamic model assumes a Watson-Crick UA pair.

NMR structure of a 4 × 4 nucleotide RNA internal loop from an R2 retrotransposon: Identification of a three purine–purine sheared pair motif and comparison to MC-SYM predictions

“…The presence of this loop motif in both NMR and crystal structures indicates that it is stabilized by RNA/RNA interactions within the loop. This contrasts with the 59CUAAG/39GAAGC loop, where the structure in crystals of ribosomes Schuwirth et al 2005; Lee et al 2006) is dramatically different from that in the isolated loop (Shankar et al 2006).…”

“…It would not be surprising, however, if the structure of the entire loop could be changed by protein binding. For example, the loop, 59CUAAG/39GAAGC, has adjacent sheared AA and GA pairs in a solution NMR structure (Shankar et al 2006), but in ribosomes Schuwirth et al 2005) the A initially paired with the U is completely bulged out of the helix. Interestingly, the 59AAGC/39AGGA loop studied here has the potential to bulge out an A and a C from the internal loop while forming three tandem GA pairs.…”

“…There are relatively few benchmarks available, however, for testing and optimizing these methods, especially all atom methods. NMR structures of small RNA internal loops in solution (SantaLucia and Turner 1993;Wu and Turner 1996;Heus et al 1997;Butcher et al 1999;Schmitz et al 1999a,b;Znosko et al 2002;Chen et al 2005Chen et al , 2007Shankar et al 2006Shankar et al , 2007Tolbert et al 2007) provide benchmarks for the full range of methods. Comparisons with predictions are straightforward because the structures depend only on interactions within the RNA and between the RNA and solvent.…”

A comparative analysis of the triloops in all high-resolution RNA structures reveals sequence–structure relationships

“…The X-ray crystallographic structures of the 23S rRNA of Deinococcus radiodurans and E. coli contain an interior loop in helix 40. Turner and coworkers have solved this interior loop by NMR and found a lower ground state than that of the X-ray crystals (Shankar et al 2006). An important difference between the two states is the disappearance of a noncanonical A-A base pair in the X-ray crystal structures.…”

Statistical Potentials for Hairpin and Internal Loops Improve the Accuracy of the Predicted RNA Structure

“…The energetic values for other types of structural elements, including helices with dangling ends 8 , hairpin 9 , internal 10; 11 and multi-stem 12 loops, co-axial stacking 13 and other structural motifs, e.g. the UAA/GAN motif 14 , have also been determined.…”