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

• Published in: RNA biology Vol. 18; no. 12; pp. 2363 - 2375
• Main Authors: Kim, Changil, Holm, Mikael, Mandava, Chandra Sekhar, Sanyal, Suparna
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis 02.12.2021
• Subjects: Biological Assay - standards; EF-G; GTP hydrolysis; Guanosine Triphosphate - metabolism; Humans; Kinetics; N-acetyl Phe-tRNA; Peptide Elongation Factors - genetics; Peptide Elongation Factors - metabolism; Protein Biosynthesis; protein synthesis; pyrene mRNA; Research Paper; Ribosome; Ribosomes - genetics; Ribosomes - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Transfer - genetics; RNA, Transfer - metabolism; RNA, Transfer, Amino Acyl - genetics; Spectrometry, Fluorescence; translocation; Ribosome; translocation; GTP hydrolysis; pyrene mRNA; protein synthesis; EF-G; N-acetyl Phe-tRNA
• ISSN: 1547-6286; 1555-8584; 1555-8584
• DOI: 10.1080/15476286.2021.1913312

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.