Specificity of RNA-RNA helix recognition - PubMed (original) (raw)

Specificity of RNA-RNA helix recognition

Daniel J Battle et al. Proc Natl Acad Sci U S A. 2002.

Abstract

Functional RNAs often form compact structures characterized by closely packed helices. Crystallographic analysis of several large RNAs revealed a prevalent interaction in which unpaired adenosine residues dock into the minor groove of a receptor helix. This A-minor motif, potentially the most important element responsible for global RNA architecture, has also been suggested to contribute to the fidelity of protein synthesis by discriminating against near-cognate tRNAs on the ribosome. The specificity of A-minor interactions is fundamental to RNA tertiary structure formation, as well as to their proposed role in translational accuracy. To investigate A-minor motif specificity, we analyzed mutations in an A-minor interaction within the Tetrahymena group I self-splicing intron. Thermodynamic and x-ray crystallographic results show that the A-minor interaction strongly prefers canonical base pairs over base mismatches in the receptor helix, enabling RNA interhelical packing through specific recognition of Watson-Crick minor groove geometry.

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Figures

Figure 1

The P4–P6 domain of the Tetrahymena group I intron is held together by five A-minor interactions. (a) The two primary types of A-minor interactions are identified by the position of the 2′-OH group of the adenosine relative to the 2′-OH groups of the receptor base pair. The first position of the receptor base pair is defined as the residue closest to the 2′-OH of the adenosine. The buried surface area is the area buried between the adenosine and receptor base pair. Buried surface area was calculated with the program

cns

(5, 6) using a 1.4-Å radius probe and subtracting the surface area of the base triple from the sum of the surface area of the receptor base pair and the adenosine. (b) A-minor interactions in the P4–P6 domain. Donor adenosine residues are shown in blue, and the receptor base pairs are orange. A-minor interactions are represented by dashed lines.

Figure 2

P4–P6 folding assay. (a) Constructs used in the assay. The J5/5a-bp construct contains mutations causing the J5/5a hinge region between the P5abc subdomain and the P456 region to base pair, forming a linear molecule unable to make tertiary contacts between P5abc and P456 at any magnesium concentration (8). (b) P4–P6 domain variants with mutations of the C109-G212 base pair to all 16 possible base-pair combinations were incubated at various magnesium concentrations and subjected to native gel electrophoresis at constant temperature. As magnesium concentration increased, the molecules folded, resulting in increased mobility relative to the J5/5a control molecule. (c) Plots of electrophoretic mobility of P4–P6 domain variants with Watson–Crick base pairs at the 109–212 receptor position relative to the J5/5a-bp unfolded control molecule vs. magnesium concentration.

Figure 3

Analysis of the P4–P6-GC A-minor interaction. (a) 2Fo − Fc omit map contoured at 1.2 σ in which A184, G109, and C212 were omitted from electron density calculation. (b) Structural alignment of the P4–P6-Δ210 (gray) and P4–P6-GC (blue and gold) structures. The P4–P6-Δ210 and P4–P6-GC structures were aligned on 150 backbone phosphorous atoms to an rms difference of 0.85 Å by using the program

lsqman

(14). (c) Diagram of the refined interaction highlighting the 190 Å2 of buried surface area and the network of hydrogen bonds bridging the interaction. For comparison with the wild-type interaction, see Fig. 1.

Figure 4

Native gel folding assays conducted with P4–P6 domain variants. P4–P6 domain variants with mutations of the C109-G212 base pair to all 12 possible base mismatches were incubated at various magnesium concentrations and subjected to native gel electrophoresis at constant temperature. Shown are plots of electrophoretic mobility relative to the J5/5a-bp unfolded control molecule vs. magnesium concentration. Values for the Hill coefficient (n), the apparent equilibrium magnesium concentration required for folding of one-half of the molecules ([Mg2+]1/2) and ΔGapparent were calculated as described. (a) Plots of relative electrophoretic mobility vs. magnesium concentration for P4–P6 domain variants with pyrimidine–pyrimidine mismatches at the 109–212 position. (b) Plots of relative electrophoretic mobility vs. magnesium concentration for P4–P6 domain variants with purine–purine mismatches at the 109–212 position. (c) Plots of relative electrophoretic mobility vs. magnesium concentration for P4–P6 domain variants with wobble base pairs at the 109–212 position. (d) Model for the effects of mutation of the receptor base pair to G-U or U-G. Wobble base pairs adapted from an oligonucleotide crystal structure (PDB entry code 485D) (16) were superimposed on the P4–P6 wild-type structure by alignment on the phosphate backbone with the program

(12). Arrows represent some likely areas of steric conflict between the model base pairs and A184.

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Specificity of RNA-RNA helix recognition - PubMed (original) (raw)