Twist-joints and double twist-joints in RNA structure.

Analysis of available RNA crystal structures has allowed us to identify a new family of RNA arrangements that we call double twist-joints, or DTJs. Each DTJ is composed of a double helix that contains two bulges incorporated into different strands and separated from each other by 2 or 3 bp. At each bulge, the double helix is over-twisted, while the unpaired nucleotides of both bulges form a complex network of stacking and hydrogen-bonding with nucleotides of helical regions. In total, we identified 14 DTJ cases, which can be combined in three groups based on common structural characteristics. One DTJ is found in a functional center of the ribosome, another DTJ mediates binding of the pre-tRNA to the RNase P, and two more DTJs form the sensing domains in the glycine riboswitch.

Recurrent RNA motifs as probes for studying RNA-protein interactions in the ribosome.

To understand how the nucleotide sequence of ribosomal RNA determines its tertiary structure, we developed a new approach for identification of those features of rRNA sequence that are responsible for formation of different short- and long-range interactions. The approach is based on the co-analysis of several examples of a particular recurrent RNA motif. For different cases of the motif, we design combinatorial gene libraries in which equivalent nucleotide positions are randomized. Through in vivo expression of the designed libraries we select those variants that provide for functional ribosomes. Then, analysis of the nucleotide sequences of the selected clones would allow us to determine the sequence constraints imposed on each case of the motif. The constraints shared by all cases are interpreted as providing for the integrity of the motif, while those ones specific for individual cases would enable the motif to fit into the particular structural context. Here we demonstrate the validity of this approach for three examples of the so-called along-groove packing motif found in different parts of ribosomal RNA.

G-ribo motif favors the formation of pseudoknots in ribosomal RNA.

Analysis of the pseudoknots existing in the ribosomal RNA showed that four of them are formed with the help of G-ribo, a recently identified RNA recurrent motif. The analysis of these pseudoknots revealed two major aspects in the G-ribo motif structure, which together provide the structural context favoring the formation of two different types of pseudoknots. The first aspect pertains to a particular side-by-side juxtaposition of two double helices that facilitates switches of the polynucleotide chain between different strands. The second aspect deals with the presence of an adenosine at a specific place where it can stabilize a particular arrangement of two quasicoaxial helices required for the pseudoknot formation. Additional analysis shows that the latter aspect is also present in other pseudoknots not related to the G-ribo motif or the ribosome, and thus represents a general structural element favoring the formation of pseudoknots.

G-ribo: a new structural motif in ribosomal RNA.

Analysis of the available crystal structures of the ribosome and of its subunits has revealed a new RNA motif that we call G-ribo. The motif consists of two double helices positioned side-by-side and connected by an unpaired region. The juxtaposition of the two helices is kept by a complex system of tertiary interactions spread over several layers of stacked nucleotides. In the center of this arrangement, the ribose of a nucleotide from one helix is specifically packed with the ribose and the minor-groove edge of a guanosine from the other helix. In total, we found eight G-ribo motifs in both ribosomal subunits. The location of these motifs suggests that at least some of them play an important role in the formation of the ribosome structure and/or in its function.