Ribosomes are the complex, cellular machinery responsible for the production of all proteins in every living organism. This 2.5 million Dalton enzyme contains three large RNAs and more than 50 proteins that form two asymmetric subunits and promote mRNA-directed translation of the genetic code. Accurate translation requires the precise synchronization of regulatory factors, messenger RNAs and transfer RNAs to produce a mature protein. Errors associated with translation are detrimental to gene expression and hence cellular function. Furthermore, consistent with the critical importance of error- free protein synthesis for proper cellular function, there are numerous examples where human disease is linked to alterations in this macromolecular machinery that monitors the accuracy of these events. The major question that underlies translational regulation is how the ribosome is able to distinguish errors from non-canonical three-base decoding and tRNA misreading from normal function. Our long-term goal is to understand how this large macromolecular machine on a molecular level identifies such errors and how this process impacts human disease. This long-term goal will be addressed here by testing the hypothesis that mRNA and tRNA interactions with the ribosome cause conformational changes that prevent errors either through suppression of the mRNA mutation or via a new and novel proofreading mechanism for quality control purposes. Three independent but complementary aims are proposed. Experiments in Aim 1 will test if the +1 shift into the new frame is promoted by interactions with elongation factor G in the A site or by ribosomal components that structurally obstruct the tRNA path between the A and P site.
In Aim 2 we will structurally characterize how fs tRNASufA6 interacts with the ribosomal P site and biochemically test whether mutations of important tRNA nts affect fs tRNAs affinity for the A site and/or how it is recognized by EF-G and moved from the A to the P site. These experiments build upon our new model for +1 frameshifting we established in the prior funding period.
In Aim 3 we will investigate a second, possibly linked phenomenon: how mismatched P-site mRNA-tRNA interactions on the ribosome arising from tRNA selection errors lead to premature termination of protein synthesis.
These aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes using both X-ray crystallography and single particle cryo-electron microscopy (in collaboration with Dr. Skiniotis) approaches and complementary biochemical methods.
The goal of this project is to understand how the genetic code is regulated during translation from RNA to protein. We are interested in how cells have evolved ways to either prevent genomic errors or use a newly discovered quality control mechanism to avoid various disease states that can arise.
| Zhang, Yan; Hong, Samuel; Ruangprasert, Ajchareeya et al. (2018) Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel. Structure 26:437-445.e3 |
| Hoffer, Eric D; Maehigashi, Tatsuya; Fredrick, Kurt et al. (2018) Ribosomal ambiguity (ram) mutations promote the open (off) to closed (on) transition and thereby increase miscoding. Nucleic Acids Res : |
| Hong, Samuel; Sunita, S; Maehigashi, Tatsuya et al. (2018) Mechanism of tRNA-mediated +1 ribosomal frameshifting. Proc Natl Acad Sci U S A 115:11226-11231 |
| Nguyen, Ha An; Dunham, Christine M (2017) Genome mining: Digging the tunnel for chemical space. Nat Chem Biol 13:1061-1062 |
| Pierson, William E; Hoffer, Eric D; Keedy, Hannah E et al. (2016) Uniformity of Peptide Release Is Maintained by Methylation of Release Factors. Cell Rep 17:11-18 |
| Schureck, Marc A; Repack, Adrienne; Miles, Stacey J et al. (2016) Mechanism of endonuclease cleavage by the HigB toxin. Nucleic Acids Res 44:7944-53 |
| Schureck, Marc A; Maehigashi, Tatsuya; Miles, Stacey J et al. (2016) mRNA bound to the 30S subunit is a HigB toxin substrate. RNA 22:1261-70 |
| Dunkle, Jack A; Dunham, Christine M (2015) Mechanisms of mRNA frame maintenance and its subversion during translation of the genetic code. Biochimie 114:90-6 |
| Cruz, Jonathan W; Sharp, Jared D; Hoffer, Eric D et al. (2015) Growth-regulating Mycobacterium tuberculosis VapC-mt4 toxin is an isoacceptor-specific tRNase. Nat Commun 6:7480 |
| Maehigashi, Tatsuya; Dunkle, Jack A; Miles, Stacey J et al. (2014) Structural insights into +1 frameshifting promoted by expanded or modification-deficient anticodon stem loops. Proc Natl Acad Sci U S A 111:12740-5 |
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