Choreography of molecular movements during ribosome progression along mRNA

Abstract

During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

The ATPase Fap7 Tests the Ability to Carry Out Translocation-like Conformational Changes and Releases Dim1 during 40S Ribosome Maturation

“…Translocation, the movement of the mRNA•tRNA pairs from the ribosomal A and P sites to the P and E sites, respectively, resets the ribosome for acceptance of a new tRNA after every round of amino acid addition. Translocation is catalyzed by the binding and subsequent GTP hydrolysis of eEF2 (EF-G in bacteria), and involves a series of concerted conformational changes largely in the small subunit (Belardinelli et al, 2016; Frank et al, 2007; Munro et al, 2010; Ratje et al, 2010; Zhou et al, 2014). First, rotation of the body of the small subunit relative to the large subunit, and swiveling of the head leads to the formation of the so-called hybrid or rotated state, which is thermodynamically stabilized by peptidyl-tRNA and binding of eEF2 (Munro et al, 2009).…”

The ribosome and its role in protein folding: looking through a magnifying glass

“…This is elegantly demonstrated by the recent advances in time-resolved cryo-electron microscopy, based on insights from single-molecule fluorescence measurements. The studies provided a detailed insight into the structure of the ribosome in real time as it transitions through different functional states during protein biosynthesis (Fischer et al, 2010;Tsai et al, 2014;Chen et al, 2015;Belardinelli et al, 2016).…”

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

“…As done in Belardinelli et al [21], we consider here that the 30S head rotations are monitored by S13-L33 FRET (30S head protein S13 labeled with the nonfluorescent acceptor and large 50S subunit protein L33 labeled with the fluorescence donor), the 30S subunit rotations are monitored by S6-L9 FRET (30S body protein S9 labeled with the nonfluorescent acceptor and 50S subunit protein L9 labeled with the fluorescence donor), the movement of mRNA is monitored with reporter Alx405 or Alx488 attached to the 3′ end of the mRNA, the deacylated tRNA dissociation is monitored by tRNA-L33 FRET, and the binding and dissociation of EF-G are monitored by L12-EF-G FRET.…”

“…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]. However, what similarities and differences are between the dynamic relations among different state transitions or processes in the translocation pathway exhibited in the smFRET data and those exhibited in the ensemble kinetic data are not clear.…”

“…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].…”