Authors:

Megan Fitzgerald

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About Paper:

Proteins are the building blocks of all organisms. Regulation of protein synthesis defines when and where proteins will be made and thus is central to many cellular and organismal functions. Mistranslation of proteins is related to almost every human disease. Ribosomes sequentially read mRNA during translation, though knowledge of its mechanism is not fully understood. During translation initiation, a small ribosomal subunit moves along mRNA in search of a start codon during a process called scanning. Förster Resonance Energy Transfer (FRET) can be used to determine the distance between two fluorescent dyes. The protein RPS26A was tagged on a small eukaryotic ribosomal subunit with a short peptide (ybbR) tag using CRISPR-Cas to study scanning initiation. It was purified and fluorescently labeled through an enzymatic reaction catalyzed by Sfp transferase. FRET changes when the ribosome's position on mRNA is fixed by adding a tRNA molecule. This system can be used in the future to study scanning initiation, elongation, and frameshifting during translation. In the elongation phase of protein synthesis, ribosomes step precisely by three nucleotides toward the 3′ end of mRNA upon reading each codon. Translocation, a process where the tRNA changes positions in the ribosome, occurs during elongation. A double deletion strain of yeast for fluorescently labeling RPL36A and RPL1B was experimented with to understand intrasubunit conformation during translocation in the future. A Q tag was inserted onto the N-terminal of RPL36A, which is on the large subunit. Labeling experiments with these mutated ribosomes show an active labeling enzyme. Future directions include introducing ybbR on RPL1B, optimizing dual labeling, large-scale purification of ribosomes, and analyzing FRET between dyes.

Source:

Auburn University / College of Sciences and Mathematics / 2025

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Co-authors:

Megan Fitzgerald