Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation.

ABSTRACT: Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA^Phe, with the natural dipeptidyl fMet-Phe-tRNA^Phe in the stopped-flow assay. This analog translocates about two times slower than th

SUBMITTER: Kim C 

PROVIDER: S-EPMC8632105 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature