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Dr. Yale Goldman

University of Pennsylvania

March 29th, 2019 – Spring Seminar

Time and Location: Noon in Meyerhoff Chemistry, Room 120

Host: Dr. Minki Kyoung


“Structural Dynamics of Elongation Factors During Protein Synthesis”

The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during peptide synthesis. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal was never tested directly. We applied single-molecule total internal reflection fluorescence resonance energy transfer (FRET) microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution “GTP” to the “GDP” structures identified by x-ray crystallography. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The dynamics are altered when aa-tRNA binds to a cognate versus a near-cognate codon suggesting that EF-Tu may be more important in proof-reading than previously realized.
Elongation factor G (EF-G) facilitates translocation by one codon in the ribosome of transfer RNAs (tRNAs) and messenger RNA (mRNA). Whether energy liberated by EF-G’s GTPase activity is used mainly to drive movements of the tRNAs and mRNA or to foster EF-G dissociation from the ribosome after translocation has been a long-lasting debate. The structural dynamics of EF-G bound to the ribosome had not been described during normal translocation in the absence of inhibitors. We measured the rotational motions of EF-G domains during normal translocation by single-molecule polarized total internal reflection fluorescence (polTIRF) microscopy. EF-G exhibits a small (∼10°) global rotational motion relative to the ribosome that exerts a force which unlocks the ribosome. A larger rotation of one of its domains (III) enables comparison of EF-G to classical molecular motors.
Premature termination codons (PTCs) are present in many inherited diseases. Nonsense suppressors (NonSups) induce “readthrough”, i.e. insertion of near-cognate amino acids into nascent polypeptides at PTCs, potentially alleviating the disease. We developed a new, highly-purified in vitro assay to measure extent and mechanisms of nonsense suppressor-induced readthrough. Of 16 NonSups tested, 12 operate directly on the ribosome, and act by at least two different mechanisms. We examined the potential of single molecule FRET measurements to elucidate mechanisms of NonSup-induced readthrough.