Taming the ribosome

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The ribosome is the centerpiece of gene expression, the molecular machine that translates the genetic code within an mRNA into protein. The global mechanism of translation has been outlined over the past 40 years; here, the focus will be on bacterial translation, but the process of peptide synthesis is generally conserved (1). Aminoacylated tRNAs are brought to the ribosome as a ternary complex with a protein elongation factor (EF-Tu in bacteria), a GTPase. Correct (cognate) tRNAs pair with their corresponding 3-nucleotide mRNA codon in the ribosomal A site on the small 30S subunit, and this correct interaction stimulates the GTPase function of EF-Tu, releasing the GDP form of the factor from the 3′ end of the aminoacyl-tRNA, which then can accommodate into the peptidyl transferase center on the large 50S subunit. Within this RNA-only active site, the amino acyl tRNA attacks the growing peptide chain held on the P-site tRNA that is bound to the 5′ adjacent codon. This results in a peptidyl-tRNA extended by the next amino acid located in the A site and a now-deacylated tRNA in the P site. As proposed by Noller (2), these two tRNAs start the process of translocation (directional movement by the ribosome in the 5′ to 3′ direction along the mRNA) by moving their 3′ ends to the exit (E) site and P site, with a corresponding intersubunit counterclockwise small subunit ratchet or rotation (3). This hybrid state pretranslocation intermediate is resolved by second GTPase EF-G that accelerates greatly the translocation of the two tRNAs to the E and P sites, resetting the ribosomal intersubunit conformations and moving the ribosome 3 nucleotides in the 3′ direction on the mRNA (4). The translation elongation cycle is complete, and the ribosome is now prepared to add the next amino acid in the encoded protein.

Decades of biochemistry, biophysics, and structural biology have established this central mechanism, timescales, and structural pathways of translation elongation. Rodnina, Wintermeyer, and Ehrenberg determined the kinetic mechanisms of rapid tRNA selection and translocation, consistent with in vivo elongation rates (20-40 amino acids/s) and coding fidelity (5,6,7). Myriad structural studies by both crystallography and cryoelectron microscopy have provided molecular snapshots of key states in the translation elongation cycle, most recently using time-resolved cryoelectron microscopy (8). Single-molecule fluorescence investigations by many groups have woven these snapshots into dynamic trajectories, providing timescales for ribosomal and ligand conformational changes during translation elongation (9,10). Critically for the work described here, Bustamante and co-workers performed elegant optical trapping experiments to determine the forces imparted by the ribosome during translocation (11). These groundbreaking experiments provided the first support for the concept that translocation is a Brownian ratchet-type process (as opposed to a power stroke function of the translocation motor).

What has been missing is a global thermodynamic and kinetic model of translation to interpret this wealth of biophysical data. This is critical, as translation is the major energy-consuming process in many cells. Leave it to Peter Moore, a wise sage of translation—deeply versed in physical chemistry—to provide an elegant solution in the manuscript highlighted here (12). First, Moore provides clear thermodynamic and theoretical support that the irreversibility of translation elongation does not arise from conformational changes per se of the ribosome and ligands—the process under normal conditions satisfies a steady-state condition and, thus, is cyclical. Moore demonstrates clearly that directionality arises from the large negative free energy from irreversible chemistries of GTP hydrolysis and peptide bond formation. Building on this foundation, Moore outlines a simple steady-state kinetic model for the flux of tRNAs through the ribosome during elongation using the underlying model of Rudorf (13) and rates for transitions between states as derived from experiments. By integrating the multiple differential equations, Moore calculates the probability of ribosomes in any state and then expands his treatment to include fluxes of near and noncognate tRNAs. His results predict in vivo elongation rates and show that error rates of translation are independent of the concentrations of ternary complexes.

Finally, Moore calculates the responsiveness to force of the translocation rate to understand whether a power stroke mechanism (where a barrier is located early in translocation (small displacement of mRNA-tRNA compared to full translocation) or a Brownian Ratchet mechanism (barrier located late in translocation with a large displacement of the mRNA-tRNA complex compared to full translocation). His calculations support the earlier conclusion of the Bustamante lab that translocation is a Brownian ratchet process while providing a firmer kinetic foundation for the overall process.

The work of Moore provides a firm theoretical model to understand bacterial translation elongation dynamics in vitro and in vivo; this model can be readily applied to eukaryotic translation as well. He freely admits to the weaknesses of the simple treatment and the existence of codon-specific and tRNA-specific effects on translation elongation rates. His work also highlights existing gaps in our knowledge of bacterial translation. We need far better measurements of elongation dynamics in vivo, as well as global and local translational fidelity as a function of different conditions and growth rates. The rapid advances of cryoelectron tomography for identifying states of ribosomes in vivo, of single-molecule methods for creating dynamic trajectories, and of computational/theoretical methods to model these systems bode well for the deep biophysical understanding of translation that Moore demands.

Editor: Mark Williams.