The right angle (RA) motif: a prevalent ribosomal RNA structural pattern found in group I introns - PubMed (original) (raw)

The right angle (RA) motif: a prevalent ribosomal RNA structural pattern found in group I introns

Wade W Grabow et al. J Mol Biol. 2012.

Abstract

The right angle (RA) motif, previously identified in the ribosome and used as a structural module for nano-construction, is a recurrent structural motif of 13 nucleotides that establishes a 90° bend between two adjacent helices. Comparative sequence analysis was used to explore the sequence space of the RA motif within ribosomal RNAs in order to define its canonical sequence space signature. We investigated the sequence constraints associated with the RA signature using several artificial self-assembly systems. Thermodynamic and topological investigations of sequence variants associated with the RA motif in both minimal and expanded structural contexts reveal that the presence of a helix at the 3' end of the RA motif increases the thermodynamic stability and rigidity of the resulting three-helix junction domain. A search for the RA in naturally occurring RNAs as well as its experimental characterization led to the identification of the RA in groups IC1 and ID intron ribozymes, where it is suggested to play an integral role in stabilizing peripheral structural domains. The present study exemplifies the need of empirical analysis of RNA structural motifs for facilitating the rational design and structure prediction of RNAs.

Copyright © 2012 Elsevier Ltd. All rights reserved.

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Figures

Figure 1

Figure 1

Definition and structural characteristics of the RA motif. (a) Nomenclature and generic sequence signature based on the structural analysis of RA motifs from known X-ray structures (listed in Supporting Information, Table S1). Nucleotides (nt) positions have been numbered from 1 to 13 to facilitate comparison. Tertiary interactions and non-canonical base pairs (bp) are indicated on the 2D diagram according to previously defined nomenclature. The regions colored in blue and pink highlight the “GA-minor” and “along groove” components of the RA motif, respectively. R and Y stand for purine and pyrimidine, respectively; N stands for any nucleotide; X stands for any nucleotide involved in WC:WC bp; lower case nucleotides are less conserved than upper case nucleotides. (b) Topological characteristics of the RA motif. The two adjacent helices H5′ and H3′ are oriented by 90° similarly to the corners of a log cabin. Position N13 at the 3′ end of the motif is in perfect helical continuity with H3′, allowing an additional helix to be stacked in continuity of this helix as previously demonstrated . (c) Superposition and RMSD of the ribose-phosphate backbone of RA motifs from known X-ray ribosomal structures (see Supporting Information, Table S1). (d) Sequence signatures corresponding to RA motifs identified at two distinct locations in the 23S and 16S rRNA sequences of Bacteria, Archaea and Eukaryotes. The sequence signatures were obtained by comparative sequence analysis of non-redundant 23S and 16S rRNA sequence obtained from the European Ribosomal RNA Database –. The sequence space is represented as WebLogo (

http://weblogo.berkeley.edu/

) ,, where the x-axis corresponds to nucleotide positions (as indicated in Figure 1a) and the y-axis corresponds to bits. The larger the letter is, the more conserved it is. (e) Sequence signature of the RA motif at the P2.1-P3 junction determined from 51 group IC1 and ID intron sequences . (f) 2D-diagram of the P2.1-P3-P8 RA junction from the Tetrahymena ribozyme with proposed tertiary interactions. Numbering is according to the one of the Tetrahymena group IC1 ribozyme .

Figure 2

Figure 2

Probing the thermodynamics of minimal RA variant constructs based on tectoRNA assembly attenuation. (a) Schematic illustrating the basic experimental design strategy: RNA molecules containing a GAAA tetraloop, an R1 receptor, and a variant RA motif sequence signatures at the junction between the 5′ and the 3′ hairpin (designated helix X and Y respectively) were evaluated based on their ability to bind to a probe molecule possessing an 11-nt receptor and a GGAA tetraloop. Stronger attenuation corresponds to a more stable RA motif. (b) Sample native PAGE (1x TB) gel-shift assays of titration experiments used to determine relative equilibrium dissociation constant (Kd) in TB 1x at 15mM Mg2+ at 10°C. (c) List of the RA variants tested in the minimal tectoRNA system. The RA sequence in the middle corresponds to the AAAG construct. Construct variants are named after the sequence of their GA minor components (positions 1, 6, 7, 12, and 13 in blue) and the sequence variations (in red) localized in their along-groove component (in pink). Asterisks indicate natural ribosomal RA sequences. (d) Apparent free energy of attenuation of heterodimer formation (ΔΔGAT) for all minimal RA constructs, referenced to the AAAU construct.

Figure 3

Figure 3

Probing the thermodynamics of expanded RA variant constructs based on tectoRNA assembly attenuation. (a) Schematic illustrating the experimental design strategy used to test RA variants in an expanded context. The third helix (helix Z) was added 3′ of the RA motif to either mimic the AAAG_P3_P8 junction from group IC1 and ID introns or the H3-H4-H18 region of bacterial 16S rRNA. (b) Expanded RA variants tested and their resulting Kd values. (c) Sample native PAGE (1x TB, 15 mM Mg2+) gel-shift assays used to determine relative equilibrium dissociation constant (Kd) at 10°C (left) and corresponding apparent free energy variation of attenuation (ΔΔGAT), referenced to the minimal AAAU construct (right) (see also Figure 2).

Figure 4

Figure 4

Monitoring the topology of the P2.1-P3-P8 junction of group I introns and H3-H4-H18 domain of bacterial 16S rRNA by supra-molecular assembly. (a) Schematic illustrating the experimental design and self-assembly strategy used to test expanded RA variants based on XY assembling interface (left) and YZ assembling interface (right). To monitor assembly under the control of the RA motif (left), HIV kissing-loops were placed at the ends of stems X and Y. To monitor assembly under the control of the topology of the stem Y with respect to stem Z, HIV kissing-loops were placed at the ends of the stems Y and Z. (b) Native PAGE gel-shift assays (1x TB at 2mM Mg2+ and 10°C) demonstrate the ability of the AAAG_P3_P8 and AAAU_3U9C_H4 constructs to form tectosquares by XY assembly. (c) Native PAGE gel-shift assays (1x TB at 2mM Mg2+ and 10°C) demonstrate by YZ assembly that stems Y and Z adopt a topology in the group I intron junction (AAAG_P3_P8) that is distinct from the one in the H3-H4-H18 domain from 16S rRNA (AAAU_3U9C_H4).

Figure 5

Figure 5

The RA motif in self-splicing group IC1 and ID introns. (a) Secondary structure diagram of the catalytic core of group IC1 and ID introns. P stands for pairings. P3 is in violet. P8 is in green. Capital letters are for positions conserved at more than 92%. Small letters are for positions conserved at more than 85%. The IC1 and ID subgroups share a similar P2.1-P3-P8 junction (sequence signature in red letters) . (b) Three-dimensional model of the Tetrahymena ribozyme with the stabilizing peripheral RNA belt consisting of P9.1-P13-P2.1 (see Material and Methods). Same color code as in (a). (c) 2D-diagram of the P2.1-P3-P8 RA junction from the Tetrahymena ribozyme with proposed tertiary interactions. Nucleotides in red correspond to the RA motif. The circled adenine positions (A57 and A95) form a UV-induced crosslink in an active form of the Tetrahymena ribozyme (a group IC1 molecule) ,. Boxed nucleotides indicate positions that are protected from Fe(II)-EDTA cleavage in the native Tetrahymena ribozyme . (d) Stereo view of the proposed structure for the P2.1-P3-P8 junction of the Tetrahymena ribozyme. Positions protected from Fe(II)-EDTA cleavage are indicated by blue stars. At the exception of one position (nt 281) likely protected by P2 (not shown), the observed protections are best explained by the formation of the RA motif (in red). UV cross-linked adenines 57 and 95 are indicated in yellow. (e) Molecular dynamics (MD) simulations on the classic RA turn (left) and on the modeled RA-2h_stack motif at the P3, P2.1 and P8 junction of group IC1 Tetrahymena intron (right). The classic RA turn structure was extracted directly from the crystal structure of the ribosome and capped with a GNRA tetraloop. Two trajectories of 35 ns simulated at 300°K are shown for both structures. The AAAG_P3_P8 junction is shown to have a relatively comparable rigidity over the course of the simulation at 300°K.

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