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bioRxiv · 10.1101/2024.09.26.615143

An empirical model of aminoacylation kinetics for E. coli class I and II aminoacyl tRNA synthetases

Dykeman, E. C.
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Abstract

Efficient functioning of the prokaryotic translational system depends on a steady supply of aminoacylated tRNAs to be delivered to translating ribosomes via ternary complex. As such, tRNA synthetases play a crucial role in maintaining efficient and accurate translation in the cell, as they are responsible for aminoacylating the correct amino acid to its corresponding tRNA. Moreover, the kinetic rate at which they perform this reaction will dictate the overall rate of supply of aminoacylated tRNAs to the ribosome and will have consequences for the average translational speed of ribosomes in the cell. In this work, I develop an empirical kinetic model for the 20 aminoacyl tRNA synthetase enzymes in E. coli enabling the study of the effects of tRNA charging dynamics on translational efficiency. The model is parametrised based on in vitro experimental measurements of substrate Km and kcat values for both pyrophosphate exchange and aminoacylation. The model also reproduces the burst kinetics observed in class I enzymes and the transfer rates measured in single turnover experiments. Stochastic simulation of in vivo translation shows the kinetic model is able to support the tRNA charging demand resulting from translation in exponentially growing E. coli cells at a variety of different doubling times. This work provides a basis for the theoretical study of the amino acid starvation and the stringent response, as well as the complex behaviour of tRNA charging and translational dynamics in response to cellular stresses. Author summaryElucidating the complex interplay between tRNA charging by aminoacyl tRNA synthetases and the overall ribosomal demand for tRNAs will have important consequences for understanding the effects of amino acid starvation and the stringent response. Here I introduce an empirical kinetic model of the 20 E. coli tRNA synthetases and examine tRNA charging dynamics during exponential growth. The results show that the model is in good agreement with a variety of experimental observations, such as tRNA charging fractions, average translational speed of the ribosome, and measured total cellular tRNA abundances.

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Dykeman, E. C.. 2024-09-27. An empirical model of aminoacylation kinetics for E. coli class I and II aminoacyl tRNA synthetases. https://doi.org/10.1101/2024.09.26.615143

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