In spite of much progress in recent years, many of the most fundamental questions concerning the molecular mechanism of protein synthesis remain unanswered. A major challenge is to understand the molecular basis of translocation - the EF-G-catalyzed, coupled movement of mRNA and tRNA through the ribosome. Translocation requires rapid and precise large-scale molecular movements (on the order of 20 to 70 �) of tRNA from the A to P to E sites of the ribosome, which must be carried out while maintaining correct codon-anticodon pairing with the mRNA. It has long been supposed that this movement is coupled to dynamic changes in ribosome structure. The first steps were the discoveries of the involvement of hybrid-states intermediates and intersubunit rotation in the translocation process. More recently, structural and FRET studies have begun to suggest how tRNA movement is coupled to the molecular mechanics of the ribosome. Time-resolved FRET experiments allow direct observation of intersubunit movement in solution, in which rapid counter-clockwise rotation of the 30S subunit from its classical state into its hybrid (rotated) state is followed by a slower, clockwise reversal of this motion.  Parallel fluorescence-quenching experiments show that translocation of mRNA occurs during the second, clockwise rotational step. In the absence of an x-ray structure of EF-G bound to a rotated state of the ribosome, we solved the 3.3 � crystal structure of the structurally-related release factor RF3 trapped in its GTP state bound to a rotated ribosome. In this complex, the body of the 30S subunit is rotated by 7� and the 30S head by 14�. These combined rotational movements result in displacement of elements of the P-tRNA binding site by 23 �, corresponding almost exactly to the distance moved by P-tRNA in the 30S subunit during its translocation to the E site.

Some insights into the molecular mechanics of the ribosome

245th National Meeting (2013)

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