A High-Resolution In Vitro Single-Molecule Assay for Eukaryotic Cap-Dependent Initiation Kinetics

Abstract

Protein synthesis rates differ by up to 1000 folds between eukaryotic mRNAs. Only less than half of such variability can be attributed to the difference in mRNA levels, highlighting the importance of translational control on regulating gene expression levels. Cap-dependent initiation is the predominant initiation pathway in eukaryotes and is the main target of translational control mechanisms. There is a lack of high-resolution techniques for kinetic characterization of the cap-dependent initiation pathway. We developed an in vitro single-molecule assay that addressed this lack. Our assay uses reporter mRNAs that code for N-terminal tagged proteins. The initiation kinetics is measured from single-molecule imaging of the binding of fluorescently labeled antibodies to the N-terminal tagged nascent peptides when they emerge from ribosome exit tunnel during active in vitro translation. We used this assay to study Arginine Attenuator Peptide (AAP) dependent translational control. For an AAP-regulated mRNA, its uORF (upstream open reading frame) codes for AAP, a short peptide that can stall ribosome in the exit tunnel at high Arginine concentration and consequently downregulate the main ORF translation. Arginine-regulation of the main ORF translation was well preserved in our assay. The single-molecule observation of the initiation kinetics for both the uORF and main ORF revealed very interesting interplay between the multiple ribosomes traveling on the same mRNA. Instead of a single AAP acting as a switch to turn off the main ORF initiation at high Arginine concentration, we found that each AAP only causes a short stalling of a single ribosome on mRNA. Intriguingly, due to the interplay between ribosomes, a mild stalling event is amplified over multiple ribosomes, which collectively cause a major halt of the main ORF initiation.