Molecular dissection of methionyl-tRNA synthetase structure and function

Although the basic mechanisms of protein synthesis are established and structures of many of the components have been determined, many details remain unknown at the molecular level, particularly the mechanistic details regarding function and regulation of the aminoacyl-tRNA synthetases (AARSs). In addition to being key players in translation, the AARSs are good models for understanding allosteric interactions, in which a binding event triggers enzymatic catalysis at a distant site. Many AARSs bind to the anticodon portion of their matching (cognate) tRNA molecules, and the anticodon-binding site is often tens of Ångstroms removed from the enzyme active site, where amino acid attachment occurs.  Efficient anticodon-mediated aminoacylation therefore depends on communication between protein domains. 

Our lab is investigating the long-range communication in E. coli methionyl-tRNA synthetase (MetRS), an AARS that requires anticodon binding for efficient catalysis yet also aminoacylates a small tRNA mimic lacking an anticodon, making it a good model to study mechanisms of long-range communication in proteins. We have used computational and experimental techniques to address how long-range communication occurs in MetRS. The computational project is in collaboration with Dr. Jacque Fetrow (WFU Physics and Computer Science Departments), and has demonstrated that functional regions of the protein exhibit correlated motions and that there are low energy barriers to achieve the alternate conformations observed by x-ray crystallography. We have made mutations at residues predicted from structural comparisons and computational methods to be important in long-range communication, and preliminary results indicate that some of these mutations do impact tRNA aminoacylation efficiency.

As we investigate the contribution of protein local structure to RNA recognition and enzyme catalysis, we have swapped amino acid residues from the stem-contact fold domain of glutaminyl-tRNA synthetase (GlnRS) into the structurally similar domain of MetRS. This structural motif has been proposed to interact with the extreme inner corner of each enzyme’s cognate tRNA to globally orient the nucleic acid on the protein surface. Our hypothesis is that some portions of this structurally conserved motif can be swapped between proteins with minimal effect on tRNA binding and aminoacylation, while other residues may participate in essential unique interactions with each enzyme’s active site and substitution will not be tolerated. Analysis of the domain swapped constructs and other mutants already constructed by undergraduate and graduate students in the lab will allow continued evaluation of the network of interactions necessary for efficient catalysis. Funding for these projects is from the National Science Foundation and the National Foundation for Cancer Research.

Cellular information transfer is highly dynamic, with proteins processing along DNA during transcription and the ribosome traversing a messenger RNA during protein synthesis. At reduced temperatures, enhanced stability of RNA structure may prevent ribosome assembly or translation of a message. We are working with Dr. Pamela Jones at Winston-Salem State University to characterize the activity of E. coli CsdA, a protein that is produced by bacteria in response to low temperature growth conditions. CsdA has all the canonical sequence motifs attributed to the DEAD-box helicases, which are ATP-dependent RNA helicases. We have determined that CsdA indeed unwinds certain RNA substrates in the presence of ATP. The DEAD (Asp-Glu-Ala-Asp) sequence of CsdA is essential for this function. These results parallel those observed in vivo by Dr. Jones. The experimental groundwork for this project, including protein overexpression strategies and assay development, was established by a very talented WFU undergraduate working in my laboratory. We are continuing to investigate the catalytic activity and substrate specificity for this helicase. One question regarding ATP-dependent RNA helicases in general is how the free energy of ATP hydrolysis is mechanically coupled to RNA unwinding. Experiments will focus on the temperature dependence of ATP hydrolysis and RNA unwinding compared to the thermal stability of various RNA substrates. This project is funded by an NIH Research Infrastructure in Minority Institutions (RIMI) Grant to Winston-Salem State University.