Kinetics of tRNA folding monitored by aminoacylation

  1. John J. Perona1,2,3,4
  1. 1Department of Chemistry and Biochemistry
  2. 2Interdepartmental Program in Biomolecular Science and Engineering, University of California, Santa Barbara, California 93106-9510, USA

    • ↵3 Present address: Department of Chemistry, Portland State University, Portland, OR 97207, USA; Department of Biochemistry and Molecular Biology, Oregon Health and Sciences University, Portland, OR 97239, USA.

    Abstract

    We describe a strategy for tracking Mg2+-initiated folding of 32P-labeled tRNA molecules to their native structures based on the capacity for aminoacylation by the cognate aminoacyl-tRNA synthetase enzyme. The approach directly links folding to function, paralleling a common strategy used to study the folding of catalytic RNAs. Incubation of unfolded tRNA with magnesium ions, followed by the addition of aminoacyl-tRNA synthetase and further incubation, yields a rapid burst of aminoacyl-tRNA formation corresponding to the prefolded tRNA fraction. A subsequent slower increase in product formation monitors continued folding in the presence of the enzyme. Further analysis reveals the presence of a parallel fraction of tRNA that folds more rapidly than the majority of the population. The application of the approach to study the influence of post-transcriptional modifications in folding of Escherichia coli tRNA1Gln reveals that the modified bases increase the folding rate but do not affect either the equilibrium between properly folded and misfolded states or the folding pathway. This assay allows the use of 32P-labeled tRNA in integrated studies combining folding, post-transcriptional processing, and aminoacylation reactions.

    Keywords

    Footnotes

    • Received August 25, 2011.
    • Accepted November 23, 2011.

    Articles citing this article

    • Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology bioRxiv December 13, 2023 0: 2023.12.09.569632v1-2023.12.09.569632
    • Modifications in the T arm of tRNA globally determine tRNA maturation, function and cellular fitness bioRxiv November 15, 2023 0: 2023.11.11.566336v1-2023.11.11.566336
    • Revisiting tRNA chaperones: new players in an ancient game RNA May 1, 2021 27: 543-559
    • The Thermus thermophilus DEAD-box protein Hera is a general RNA binding protein and plays a key role in tRNA metabolism RNA November 1, 2020 26: 1557-1574
    • Insights into the structure-driven protein interactivity of RNA molecules bioRxiv April 13, 2019 0: 436279v1-436279
    • Two for the price of one: RNA modification enzymes as chaperones Proc. Natl. Acad. Sci. USA December 13, 2016 113: 14176-14178
    • RNA modification enzyme TruB is a tRNA chaperone Proc. Natl. Acad. Sci. USA December 13, 2016 113: 14306-14311
    | Table of Contents

    This Article

    1. Published in Advance January 27, 2012, doi: 10.1261/rna.030080.111 RNA 18: 569-580 Copyright © 2012 RNA Society

    Article Category

    Share

    Current Issue

    1. August 2026, 32 (8)
    1. Current Issue
    1. Alert me to new issues of RNA