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Theses Doctoral

Cryo-electron microscopy studies of dynamical features of ribosomes during the translation process

Sun, Ming

Cryo-electron microscopy (cryo-EM) is a structural biology technique that determines the structure of proteins and macromolecular complexes using the transmission electron microscope under cryogenic conditions. In my Ph.D. studies, I took advantage of this technique, in the study of dynamical features of ribosomes in both eukaryotes and prokaryotes.
In Chapter 2, I report my graduate research on the investigation of ribosomes from the human malaria parasite, Plasmodium falciparum, using single-particle cryo-EM. In collaboration with Dr. Jeffrey Dvorin at Harvard Medical School, we obtained five cryo-EM reconstructions of ribosomes purified from P. falciparum blood-stage schizonts, and discovered structural and dynamical features that differentiate the ribosomes of P. falciparum from those of the mammalian system. Moreover, we discovered that RACK1, a necessary ribosomal protein in eukaryotes, does not specifically co-purify with the 80S fraction in the P. falciparum schizonts stage and would mainly function in a ribosome-unbound, free state during the blood-stage. More extensive studies, using cryo-EM methodology, of translation in the parasite, will provide structural knowledge that could help in the design of effective anti-malaria drugs.
In Chapter 3, I describe the cryo-EM studies of the Saccharomyces cerevisiae ribosome in response to a carbon source switch. In collaboration with Dr. Andrew Link at Vanderbilt University, we obtained reconstructions of the 80S ribosomes at selected time points after the glucose-to-glycerol carbon source shift, and observed that a fraction of ribosomes lacked densities for r-proteins, mainly eS1 (yeast rpS1) on the 40S subunit and uL16 (yeast rpL10) on the 60S subunit. We found that the binding ratio of eS1 and uL16 to ribosomes changed as a function of time, consistent with the change in translational activities as gauged by polysome profiling. On the basis of these observations, along with previous structural and genetics studies, we propose that rapid control of translation is exerted through the dissociation of r-protein eS1/rpS1 and uL16/rpL10 from the ribosome. Our studies thus open a new venue on the exploration of S. cerevisiae’s rapid adaption to carbon source shifts at the level of translation.
In Chapter 4, I have documented a collaborative work on the development and application of a new technique, time-resolved cryo-EM, which can be used to study processes involving two reaction partners on a sub-second time scale. With my colleagues at the Frank and Gonzalez labs at Columbia University, we successfully applied this method to study the process of E. coli ribosomal subunits association. By mixing and reacting the two subunits for 60 ms and 140 ms, we captured the association reaction in a pre-equilibrium state, and detected different conformations of E. coli 70S ribosomes. With the current capability of this mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, we expect to be able to standardize this method and apply it to more challenging biological processes, such as translation recycling and initiation processes.


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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Frank, Joachim
Ph.D., Columbia University
Published Here
August 16, 2016