Dynamic mechanisms of eukaryotic translation initiation and termination
Protein synthesis starts with the assembly of a ribosomal complex at the start site on the messenger RNA (mRNA). This process involves recruitment of the 40S small ribosomal subunit, numerous translation initiation factors (eIFs), and initiator aminoacyl-tRNA, to the 5′-untranslated region (5′-UTR) of the 7-methylguanosine-capped mRNA, followed by inspection of the 5′-UTR in search of the correct AUG codon as a start site, a dynamic process termed “scanning”. Upon AUG recognition, a series of eIF reorganizations and departures coupled to ribosomal conformational rearrangements occurs, leading to the joining of the 60S large ribosomal subunit and the start of translation elongation. At the conclusion of protein synthesis, release factors collaborate to liberate the completed polypeptide from the 80S ribosome in translation termination. The ribosome, mRNA, and tRNA must then be disassembled during recycling to enable them to participate in subsequent rounds of protein synthesis.
We have reconstituted yeast and human translation in vitro to characterize the compositional and conformational dynamics that orchestrate these fundamental steps of protein synthesis using single-molecule fluorescence microscopy and cryo-electron microscopy. Recently, we have shown that the universally conserved translation initiation factor eIF5B kinetically controls the transition from initiation to elongation in yeast. We have also dissected the mechanism of translation termination in yeast and found that stop codons are recognized rapidly in a regulated process that resembles transfer RNA selection during translation elongation. Our lab is currently seeking to extend these studies to human translation and to further understand how eIFs control early stages of ribosome loading onto the mRNA and scanning.
Figure 1 Single-molecule fluorescence measurements of conformation using FRET. Dynamics are revealed at the single-molecule level showing single red and green fluorophore intensity as a function of time (trace). Forster resonance energy transfer (FRET) occurs when dyes are less than 80Å apart with a distance dependence of 1/R6, where R is the dye separation distance. These dynamics are blurred in a non-synchronized ensemble
Figure 2 Tracking ribosomal conformation and subunit joining in eukaryotic translation using intersubunit FRET. Ribosomes are dye labeled at peptide tags at the termini of ribosomal proteins on the large and small subunit
Figure 3 Correlating structures and dynamics by merging single-molecule fluorescence and cryoEM. The process of eIF5B mediated subunit joining in yeast was monitored using multicolor fluorescence, tracking eIF5B occupancy and 40S and 60S binding to a mRNA. Single molecule data can be synchronized, and it was found that at 40S post mixing, a cryoEM sample should have a significant population of eIF5B-bound 80S subunits, as subsequently shown by detailed cryoEM
Figure 4 CryoEM structure (2.9Å global resolution) of yeast 80S (40S grey, 60S purple) ribosome with eIF5B (red) and initiator tRNA (green) bound, determined in collaboration with Israel Fernandez (St. Judes)
Figure 5 The challenges of 40S ribosomal loading and scanning on a mRNA in initiation, currently under investigation using single-molecule methods. Cutaway of the human 48S initiation complex (40S tan) showing positions of eIF3 subunits (plum), eIF1 (orange), eIF1A (red) and mRNA within the mRNA channel on the 40S. Ribosomes scan directionally 5’ to 3’
Figure 5 Pathway of eukaryotic translation termination determined by single-molecule methods in collaboration with the Green lab (JHU)