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. Two independent but complementary aims are proposed.
Aim 1 seeks to understand how a novel class of mutant tRNAs interact with the ribosome to alter the three-base genetic reading frame and suppress errors.
Aim 2 is designed to understand the structural basis of a new quality control mechanism resulting from tRNA:mRNA mismatch errors.
These aims will be accomplished through a combination of structural biology of large, functional ribosomal complexes, biochemical and biophysical methods.

Public Health Relevance

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. )

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM093278-03
Application #
8280355
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Flicker, Paula F
Project Start
2010-06-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$279,279
Indirect Cost
$99,099
Name
Emory University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Dunkle, Jack A; Vinal, Kellie; Desai, Pooja M et al. (2014) Molecular recognition and modification of the 30S ribosome by the aminoglycoside-resistance methyltransferase NpmA. Proc Natl Acad Sci U S A 111:6275-80
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
Cruz, Jonathan W; Rothenbacher, Francesca P; Maehigashi, Tatsuya et al. (2014) Doc toxin is a kinase that inactivates elongation factor Tu. J Biol Chem 289:7788-98