Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR).
A series of disease-related mutations are known to affect the hs mt tRNA(Leu(UUR)) gene, and the molecular-level properties of this tRNA may underlie the effects of pathogenic sequence changes. A combinatorial approach has been used to explore the importance of the D, TPsiC, and anticodon loops of hs mt tRNA(Leu(UUR)) in the structure and function of this molecule. A tRNA library was constructed with 20 randomized nucleotides in the loop regions of hs mt tRNA(Leu(UUR)), and tRNA variants that were aminoacylated by hs mt LeuRS were isolated using an in vitro selection approach. Analysis of 26 selected sequences revealed that a stabilized anticodon stem significantly enhances aminoacylation activity. However, anticodon loop nucleotides were not conserved in the active sequences, indicating that this region of hs mt tRNA(Leu(UUR)) is not involved in recognition by LeuRS. Within the D and TPsiC loops, only two nucleotides conserved their identities, while new sequences were selected that likely mediate interloop interactions. The results indicate that hs mt tRNA(Leu(UUR)), which is known to have structurally weak D and anticodon stems, benefits functionally from the introduction of stabilizing interactions. However, the locations of individual nucleotides that govern discrimination of this tRNA by hs mt LeuRS still remain obscure.
Zagryadskaya
Ekaterina I
EI
Eugene F. Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Kelley
Shana O
SO
D000926:Anticodon / Q000737:chemistry; D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D015723:Gene Library; D006801:Humans; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012356:RNA, Transfer, Leu / Q000737:chemistry*
D000926:Anticodon; D017931:DNA Primers; D012356:RNA, Transfer, Leu