Multiperspective smFRET reveals rate-determining late intermediates of ribosomal translocation

Abstract

Directional translocation of the ribosome through the messenger RNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of transfer and messenger RNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we have tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations reveal direct evidence of structurally and kinetically distinct, late intermediates during substrate movement, whose resolution is rate-determining to the translocation mechanism. These steps involve intra-molecular events within the EFG(GDP)-bound ribosome, including exaggerated, reversible fluctuations of the small subunit head domain, which ultimately facilitate peptidyl-tRNA’s movement into its final post-translocation position.

Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome

“…Recently, a structural study has shown that the dissociation occurs upon binding of a complex of eEF2 and GTP, which stabilises the rotated conformation of the PRE ribosome that is incompatible with deacylated tRNA in the E site (46). This also agrees with a recent kinetic study, in which dissociation has been observed prior to the formation of the POST complex (44). The empty E-site allows deacylated tRNA in the P-site to adopt the hybrid P/E state.…”

“…Recent data indicate that the most rate-limiting step during translocation is the movement of the tRNA anticodon ends, associated with swivelling of the SSU head (26,44,45). Elongation factor 2 facilitates the movement in two ways.…”

Dynamic relationships between ribosomal conformational and RNA positional changes during ribosomal translocation

“…In bacteria, the translocation is catalyzed by elongation factor G (EF-G) hydrolyzing GTP. The translocation also entails multiple conformational changes of the ribosome and other positional or conformational changes of tRNAs in the ribosome such as the forward (counterclockwise) and backward (clockwise) rotations of the small 30S subunit relative to the large 50S subunit (viewed from the 30S subunit) [1, 2, 3, 4, 5, 6], forward (counterclockwise) and reverse (clockwise) rotations of the 30S head relative to the 30S body (viewed from the 30S head) [7, 8, 9, 10, 11, 12], movements of tRNAs in the 50S subunit [4, 5, 6, 13, 14], compaction of the two tRNAs [9, 15], deacylated tRNA dissociation from the E/E site [16, 17, 18, 19, 20], etc.…”

“…To this end, both single molecule fluorescence resonance energy transfer (smFRET) and ensemble kinetic (or ensemble FRET) methods were recently employed to record simultaneously the FRET data between different pairs of multiple reporters placed on both ribosomal subunits, tRNA and EF-G [15, 21]. The smFRET data revealed direct evidence of structurally distinct late intermediates during translocation such as exaggerated, reversible fluctuations of the 30S head [15].…”

“…To this end, both single molecule fluorescence resonance energy transfer (smFRET) and ensemble kinetic (or ensemble FRET) methods were recently employed to record simultaneously the FRET data between different pairs of multiple reporters placed on both ribosomal subunits, tRNA and EF-G [15, 21]. The smFRET data revealed direct evidence of structurally distinct late intermediates during translocation such as exaggerated, reversible fluctuations of the 30S head [15]. The ensemble kinetic data revealed an early forward rotation or swiveling of the 30S head taking place while the 30S body rotating in the opposite, clockwise direction, and the backward swiveling of the 30S head starting upon tRNA translocation and continuing until the posttranslocation state is reached [21].…”