Heterogeneous pathways and timing of factor departure during translation initiation

Abstract

The initiation of translation establishes the reading frame for protein synthesis and is a key point of regulation. Initiation involves factor-driven assembly at a start codon of a messenger RNA of an elongation-competent 70S ribosomal particle (in bacteria) from separated 30S and 50S subunits and initiator transfer RNA. Here we establish in Escherichia coli, using direct single-molecule tracking, the timing of initiator tRNA, initiation factor 2 (IF2; encoded by infB) and 50S subunit joining during initiation. Our results show multiple pathways to initiation, with orders of arrival of tRNA and IF2 dependent on factor concentration and composition. IF2 accelerates 50S subunit joining and stabilizes the assembled 70S complex. Transition to elongation is gated by the departure of IF2 after GTP hydrolysis, allowing efficient arrival of elongator tRNAs to the second codon presented in the aminoacyl-tRNA binding site (A site). These experiments highlight the power of single-molecule approaches to delineate mechanisms in complex multicomponent systems.

Figure 1. Pathways leading to 30S PIC formation

a. Single dye-labeled 30S complexes were immobilized on the bottom of zero-mode-waveguide (ZMW) wells and scored by fluorescence (Methods 3). Dye-labeled initiation factors, tRNAs, and 50S subunits were delivered at t = 7 s in all experiments. The appearance of fluorescence signals indicates arrival of the labeled molecules. Fluorescence signal disappears either due to dye-labeled molecule departure or photobleaching. b. Productive initiation events were identified by stable 50S arrival (Methods 4). The order of arrival was determined by the sequence of the fluorescent pulses. Grey portions of the traces represent fluorescence background. c. The ratios of possible 30S PIC formation pathways at different ligand concentrations were measured and plotted. See Methods 4 for an explanation of instrument uncertainty. From left to right, n = 86, n = 51, n = 79, and n = 52.

Figure 2. 50S subunit joining to 30S PIC

a. The appearance of a stable Cy5 signal is used to identify the arrival of the 50S subunit (Methods 5). The time until 50S arrival and the length of the 50S signal are then characterized. b. The arrival times of a 50S subunit to and the observed 50S subunit lifetimes on a 30S PIC are fitted to single-exponential functions and plotted with s.d. error bars. The presence of IF2 is critical for efficiently and stably forming 70S complexes. From left to right for both panels, n = 246, n = 283, n = 451, n = 262, and n = 253.

Figure 3. Timing of IF2 departure and elongator tRNA arrival after 70S complex formation

a. See Methods 6 for experimental setup. The timing of IF2 departure is determined by the disappearance of the Cy5 signal. b. The panels represent postsynchronization plots on 50S subunit arrival and IF2 departure at 1 μM dye-labeled ligand concentrations. With GTP (n = 161), there is a ~2 s overlap between the IF2 and 50S subunit signals with a strong elongator tRNA density. With GDPNP (n = 87), the overlap between IF2 and 50S is longer (~10 s) but there is little elongator tRNA density. c. The exponential lifetimes of the IF2-50S subunit overlap (left panel) in did not depend on IF2 or 50S subunit concentrations. Increasing the elongator tRNA concentration also did not reduce the wait time until tRNA arrival (right panel). From left to right for both panels, n = 169 and n = 161, error bars are s.d. d. The event frequencies per molecule 2 s before and after IF2 departure are similar in both GTP (n = 161) and GDPNP (n = 87). Most events in GDPNP were removed by only counting events >1s. Most of the events in GTP were longer-lived tRNA binding events.

Figure 4. The heterogeneous pathways of translation initiation

Multiple pathways are possible to reach the important stages of initiation as the ribosome converges to an elongation-competent 70S IC. Concentrations of initiation factors, tRNAs, and ribosomal subunits all modulate the flux through the possible pathways that lead to successful initiation. Post 30S subunit binding to the mRNA, IF2 plays a central role in channeling the ribosome towards elongation. At physiological concentrations of initiation factors and tRNAs, the majority of 30S PICs may be formed by IF2 bringing in the initiator tRNA to the 30S. IF2 also guides rapid and stable 50S subunit joining, while GTP hydrolysis by IF2 and its departure from the ribosome gates the stable binding of the first elongator tRNA.