Structure and folding of a rare, natural kink turn in RNA with an A•A pair at the 2b•2n position

Abstract

The kink turn (k-turn) is a frequently occurring motif, comprising a bulge followed by G d A and A d G pairs that introduces a sharp axial bend in duplex RNA. Natural k-turn sequences exhibit significant departures from the consensus, including the A d G pairs that form critical interactions stabilizing the core of the structure. Kt-23 found in the small ribosomal subunit differs from the consensus in many organisms, particularly in the second A d G pair distal to the bulge (2b d 2n). Analysis of many Kt-23 sequences shows that the frequency of occurrence at the 2n position (i.e., on the nonbulged strand, normally G in standard k-turns) is U>C>G>A. Less than 1% of sequences have A at the 2n position, but one such example occurs in Thelohania solenopsae Kt-23. This sequence folds only weakly in the presence of Mg 2+ ions but is induced to fold normally by the binding of L7Ae protein. Introduction of this sequence into the SAM-I riboswitch resulted in normal binding of SAM ligand, indicating that tertiary RNA contacts have resulted in k-turn folding. X-ray crystallography shows that the T. solenopsae Kt-23 adopts a standard k-turn geometry, making the key, conserved hydrogen bonds in the core and orienting the 1n (of the bulge-proximal AdG pair) and 2b adenine nucleobases in position facing the opposing minor groove. The 2b and 2n adenine nucleobases are not directly hydrogen bonded, but each makes hydrogen bonds to their opposing strands.

The plasticity of a structural motif in RNA: Structural polymorphism of a kink turn as a function of its environment

“…The other k-turns shown are those of U4 snRNA (Vidovic et al 2000), H. marismortui Kt-38 (Ban et al 2000), the SAM-I riboswitch (Schroeder et al 2011) and its G2nA variant, and Thelohania solenopsae (T.s.) Kt-23 (Schroeder et al 2012). …”

“…In some k-turns, well-exemplified by Kt-23 (Schroeder and Lilley 2009;Schroeder et al 2012), the G normally found at the 2n position is often replaced by a different nucleotide. However, the adenine at the 2b position is usually retained, and it makes equivalent A-minor interactions with the C helix.…”

“…F is taken from the structure of the SAM-I riboswitch k-turn with a G2nA substitution (Schroeder et al 2011). G is taken from the structure of the SAM-I riboswitch where the normal k-turn has been replaced by Kt-23 of T. solenopsae (Schroeder et al 2012). into the SAM-I riboswitch) (Schroeder et al 2012) that naturally contains an adenine at the 2n position. In contrast to the previous structure, A2b accepts a hydrogen bond at N1 from O2…”

“…The RNA sequence of the SAM-I riboswitch was modified to replace its natural k-turn with that of Kt-7, as we have previously done for other k-turns such as Kt-23 of T. solenopsae (Schroeder et al 2012). The modified species crystallized under very similar conditions to those used for the natural riboswitch, with retention of the P4 3 2 1 2 space group and unit cell parameters (Montange et al 2010).…”

Common themes and differences in SAM recognition among SAM riboswitches

“…The X-ray crystal structure of the Thermoanaerobacter tengcongensis MetF-H2 SAM-I aptamer domain has been determined bound to SAM, SAH and sinefungin [25, 26]; in its ligand-free state[27]; as a scaffold for understanding the kink-turn motifs[28, 29]; and in complex with the kink-turn binding protein YbxF[30]. In addition, the Bacillus subtilis yitJ SAM-I riboswitch crystal structure was determined in complex with SAM [16].…”