Abstract
Aminoacyl-tRNA synthetases are multidomain enzymes that often possess two activities to ensure translational accuracy. A synthetic active site catalyzes tRNA aminoacylation, while an editing active site hydrolyzes mischarged tRNAs. Prolyl-tRNA synthetases (ProRS) have been shown to misacylate Cys onto tRNA(Pro), but lack a Cys-specific editing function. The synthetase-like Haemophilus influenzae YbaK protein was recently shown to hydrolyze misacylated Cys-tRNA(Pro) in trans. However, the mechanism of specific substrate selection by this single domain hydrolase is unknown. Here, we demonstrate that YbaK alone appears to lack specific tRNA recognition capabilities. Moreover, YbaK cannot compete for aminoacyl-tRNAs in the presence of elongation factor Tu, suggesting that YbaK acts before release of the aminoacyl-tRNA from the synthetase. In support of this idea, cross-linking studies reveal the formation of binary (ProRS.YbaK) and ternary (ProRS.YbaK.tRNA) complexes. The binding constants for the interaction between ProRS and YbaK are 550 nM and 45 nM in the absence and presence of tRNA(Pro), respectively. These results suggest that the specificity of trans-editing by YbaK is ensured through formation of a novel ProRS.YbaK.tRNA complex.
Highlights
To clear noncognate amino acids, a double-sieve mechanism was proposed, wherein larger amino acids are rejected by the aminoacylation active site while smaller amino acids are cleared in a second editing active site [18, 19]
In vitro kinetic data suggest that the rate of Cys-tRNACys hydrolysis by YbaK is significantly lower than the rate of Cys-tRNACys synthesis by cysteinyl-tRNA synthetase (CysRS)
H. influenzae YbaK Does Not Recognize the First Base Pair of the Acceptor Stem—We have previously reported that H. influenzae YbaK rapidly deacylates E. coli Cys-tRNAPro, but only weakly hydrolyzes an Ala-tRNAPro G1:C72/U70 variant as well as Ser- and Gly-tRNAAla [28]
Summary
AlaRS, alanyl-tRNA synthetase; BSA, bovine serum albumin; CysRS, cysteinyl-tRNA synthetase; EF-Tu, elongation factor-Tu; ProRS, prolyl-tRNA synthetase; WT, wild type; AF, AlexaFluor 488. Trans-editing by a ProRS1⁄7YbaK1⁄7tRNAPro Complex how specific substrate selection is achieved by a single domain editing module
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
