Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Abstract

BackgroundSelenocysteine tRNAs (tRNASec) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNASec-interacting factors are incompletely understood.Methodology/Principal FindingsWe applied rational mutagenesis to obtain well diffracting crystals of murine tRNASec. tRNASec lacking the single-stranded 3′-acceptor end (ΔGCCARNASec) yielded a crystal structure at 2.0 Å resolution. The global structure of ΔGCCARNASec resembles the structure of human tRNASec determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNASec used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2′-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNASec in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn2+-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNASec.Conclusions/SignificanceWe provide the most highly resolved structure of a tRNASec molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNASec support its interaction with proteins.

Noninvasive diagnosis of TRIT1‐related mitochondrial disorder by measuring i6A37 and ms2i6A37 modifications in tRNAs from blood and urine samples

“…The i 6 A modification of mt‐tRNAs is further converted to 2‐methylthio‐ N 6 ‐isopentenyladenosine (ms 2 i 6 A) by Cdk5 regulatory subunit associated protein 1 (CDK5RAP1) (Wei et al, ). The i 6 A modification appears to promote the correct ordering of the anticodon loop, thus contributing to the codon‐anticodon interaction (Ganichkin, Anedchenko, & Wahl, ). Moreover, a structural analysis of the ms 2 i 6 A modification demonstrated that the 2‐methylthio group can directly interact with the first nucleotide of a codon, stabilizing the codon‐anticodon binding and maintaining the reading frame (Jenner, Demeshkina, Yusupova, & Yusupov, ).…”

Tertiary structure of bacterial selenocysteine tRNA

“…We previously reported the crystal structures of human tRNA Sec and the archaeal tRNA Sec •PSTK complex (10,11), and those of the human tRNA Sec •SepSecS complex (12), mouse tRNA Sec (13) and another archaeal tRNA Sec •PSTK complex have also been solved (14). These studies confirmed the ‘9 + 4’ secondary structure, and revealed the characteristic features of their tertiary cores.…”

Mammalian Trit1 is a tRNA[Ser]Sec-isopentenyl transferase required for full selenoprotein expression

“…Murine tRNA [Ser]Sec for activity assays was the same as that crystallized recently [18]. The [ 14 C]DMAPP (dimethylallyl pyrophosphate) reaction was performed as described in [16] with minor modifications.…”