Translation is the process by which the cell produces new proteins on the ribosome, as directed by genetic instructions, in all living organisms. Structural studies of the ribosome have shed considerable lights on its mechanism and regulation. Cryogenic electron microscopy (cryo-EM) and single-particle reconstruction technique is one of the major approaches to studying ribosome structure. In this thesis, I report the use of cryo-EM and related new techniques to study the structure of ribosome complexes. This work is divided into three parts. First, in Chapter 3, I describe the development of a computational method in the classification of cryo-EM data. Recently developed classification methods have enabled resolving multiple structures/conformations of the molecules from cryo-EM data obtained on a heterogeneous biological sample. However, the classification methods all involve various amounts of arbitrary decisions made by researchers, which can limit the use of these methods by inexperienced users. As a step toward fully automated classification, I worked with colleagues to develop a "jumper analysis" to determine the number of distinguishable classes of 3D reconstruction, based on the statistics of cryo-EM particles. Second, in Chapter 4, I document the cryo-EM study of EttA-70S ribosome complex, which provided structural insights into the mechanism of EttA in translation regulation. Energy-dependent translation throttle A (EttA, previously named YjjK in Escherichia coli) is the most prevalent member of ATP-binding cassette F family proteins in eubacteria. Through a collaboration among the Hunt, Frank, and Gonzalez labs, we combined crystallography, biochemical, cryo-EM and single-molecule fluorescence energy transfer techniques to elucidate the function and mechanism of EttA. We demonstrated that EttA gates ribosome entry into the translation elongation cycle through a nucleotide-dependent interaction sensitive to ATP/ADP ratio. We also showed that the ATP-bound form of EttA binds to the ribosomal tRNA-exit site, and restricts the ribosome and tRNA dynamics required for translation.
Thirdly, in Chapter 5, I discuss the improvements to a new technique, time-resolved cryo-EM by mixing-spraying, and its application to ribosome studies. The mixing-spraying method can study processes involving two big biological molecules that are in the sub-second time scale. I worked with colleagues to apply this method to studying ribosome subunit association. By mixing the subunits and reacting for 60 ms and 140 ms, we were able to capture the association reaction in a pre-equilibrium state. We detected three 70S ribosome conformations in the system. Quantification of the proportions of particles assuming these conformations suggested that the 70S ribosome can undergo fast conformational changes upon formation, and reaches equilibrium among these conformations earlier than 60 ms. In addition, I present preliminary results of studying translation decoding using the mixing-spraying method. This study, performed before improving the mixing-spraying method, was inconclusive mainly due to the limited size of cryo-EM data. Now that we have demonstrated the capability of the mixing-spraying method to visualize multiple states of molecules in a sub-second reaction, the translation decoding process can be revisited and many other processes, such as translation initiation, can be studied.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/D8WW7G9J |
| Date | January 2015 |
| Creators | Chen, Bo |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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