Rapid and Stable Transfer RNA Translocation through the Ribosome Ensured by Specific Contact Mechanisms

Abstract

We combined high-resolution crystal structures with cryo-EM maps of 13 intermediate states of ribosomal factorless spontaneous retro-translocation to obtain structures of the 70S ribosome in each of the 13 states in atomic detail. We then performed 100 ns all-atom, explicit-solvent molecular dynamics simulations for each of these states. Intrinsic rates for key ribosomal motions between the states were estimated from the short time fluctuations of the L1-stalk, the tRNAs and intersubunit rotations. The rates revealed rapid, sub-microsecond motions of the L1-stalk and the 30S subunit. Surprisingly, it is tRNA motions, rather than large-scale intersubunit rotations, that are rate limiting for most transitions. The interaction free energy profile of the L1-stalk with the tRNA obtained from additional umbrella sampling simulations of the L1-stalk/tRNA interactions revealed an active role of the L1-stalk in pulling the tRNA from the P to the E site. Further, by detailed analysis of the frequency of contacts between 50S ribosomal proteins L5 and L16 and the tRNAs, we identified specific residues which guide the tRNAs between the binding sites. A sequence analysis of the L1, L5 and L16 proteins revealed that the conservation score for contacting residues is significantly above average. Different types of contacts characterize the interplay of these proteins with the tRNAs and involve 1) sliding of L5 residues along the tRNA elbow 2) stepping of the tRNA between L16 contact patches 3) final pulling of the tRNA by the L1-stalk. These contact mechanisms can explain how both rapid translocation and a stable tRNA binding affinity can be achieved despite large-scale displacements.