Paulina Kuwerska, Karolina Bartosik, Katarzyna Kulik, Agnieszka Dziergowska, Justyna Jakubowska, Barbara Nawrot and Grazyna Leszczynska* Pages 1 - 16 ( 16 )
Introduction: 2-Thiouridines with C5-aminomethyl substituents (nm5S2U, mnm5S2U, cmnm5S2U, τm5S2U and inm5S2U, here denoted as xnm5S2U) are widespread at the wobble position (position 34 or first anticodon letter) of several bacterial and mitochondrial tRNAs. Wobble xnm5S2Us are known to recognize purines at the third position of weak and intermediate codons (5’NNA3 and 5’NNG3). Their absence is associated with bacterial virulence defects, mitochondrial dysfunction and human diseases. Previous data suggested that mnm5 in bacterial mnm5S2U34-tRNAs facilitates base pairing with G3 and decreases base pairing with A3. The geometry of mnm5S2U 34-G3 was solved in the crystal mRNA-tRNA-ribosome complex indicating the unique mnm5-mediated zwitterionic nature of wobble mnm5S2U.
Methods: In this work we investigate the hybridization and structural properties of a representative group of naturally existing xnm5-2-thiouridines demonstrating the general tendency of xnm5-substituents to decrease the stability of xnm5S2U-A duplexes.
Results: We found a correlation between an increased abundance of ionized xnm5S2U and decreased base pairing specificity with A, confirming that the zwitterionic state of xnm5S2U facilitates the formation of xnm5S2U-G pair at physiological conditions. These findings suggest that the biological importance of xnm5S2Us relates to the tunning of mRNA codons reading through enhanced specificity of interaction with G3.
Conclusion: In addition, we report the first post-synthetic strategy for xnm5S2U-RNA synthesis, applicable also to Cy3-labeled oligomers.
tRNA, modified nucleosides, 5-aminomethyl-2-thiouridines, zwitterionic form, base pairing, sugar conformation.