Mechanism of Protein Synthesis
- Dr. Peter Cornish University of Missouri
- Dr. Gloria Culver
- Dr. Nikolaus Grigorieff Brandeis University
- Dr. Andrei Korostelev University of Massachusetts
- Dr. P. Spiegel Western Washington University
Prism-type total internal reflection microscope setup assembled in our laboratory for single molecule FRET measurements.
All ribosomes have a ribonucleoprotein structure made up of two unequal subunits, both of which contain three binding sites for tRNA: the A (aminoacyl), P (peptidyl) and E (exit) sites. Sequential movement of tRNAs from the A site to the P site to the E site is coupled with movement of their associated codons in the mRNA. Following peptide bond formation, the acceptor ends of the tRNAs first move on the large subunit, resulting in the A/P and P/E hybrid binding states, in which the anticodon ends of the peptidyl-tRNA and deacylated tRNA remain bound to the A and P sites, respectively, of the small subunit, while their acceptor ends move into the P and E sites of the large subunit.
Subsequently, the tRNAs move from the A/P and P/E states to the P/P and E/E states, in a step catalyzed by elongation factor EF-G (or EF-2, in eukaryotes) and GTP. In cryo-EM reconstructions of ribosome complexes containing bound EF-G, it was observed that the small subunit was rotated in a counter-clockwise direction compared with its usual orientation relative to the large subunit. Using Förster resonance energy transfer (FRET), we found that ribosomes undergo spontaneous intersubunit rotational movement in the absence of ribosomal factors and GTP, fluctuating between two conformations corresponding to the classical and hybrid states of the tRNA translocational cycle (Ermolenko et al, JMB, 2007; Ermolenko et al, NSMB, 2007 & Cornish et al, 2008). In view of the high structural and sequence conservation of the ribosome throughout all branches of life, the intersubunit movement observed in our FRET experiments is most likely a universal feature of translation that accounts for conservation of the two-subunit organization of ribosomes in all organisms.
Positions of FRET pairs in the 70S ribosome that are used to monitor intersubunit movements during ribosomal translocation. View from the solvent side of the small subunit of the 70S ribosome crystal structure showing positions of the fluorescent donor-acceptor pairs on ribosomal proteins S6, S11 and L9 (Ermolenko et al, JMB 2007).
We are currently addressing following questions:
- How does the catalyst of ribosomal translocation, elongation factor G, rectify spontaneous intersubunit movement into unidirectional translocation of the ribosome?
- What is the mechanism of programmed ribosome frame-shifting and bypassing segments of mRNA?
- How do elongation factor G and ribosome recycling factor (RRF) disassemble ribosome into subunits after termination of protein synthesis?