Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation. Issue 12 (2nd December 2021)

• Record Type: Journal Article
• Authors: Kim, Changil; Holm, Mikael; Mandava, Chandra Sekhar; Sanyal, Suparna
• Is Part Of: RNA biology. Volume 18:Issue 12(2021)
• Page Start: 2363
• Page End: 2375
• Publication Date: 2021-12-02
• Subjects: Ribosome -- protein synthesis -- translocation -- pyrene mRNA -- N-acetyl Phe-tRNA -- EF-G -- GTP hydrolysis; RNA -- Periodicals; Molecular biology -- Periodicals; Molecular biology; RNA; Periodicals; 572.8805
• DOI: 10.1080/15476286.2021.1913312
• Languages: English
• ISSNs: 1547-6286

Abstract

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 the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg 2+ concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA. GRAPHICAL ABSTRACT: uf0001