| Some classes of tRNA mimics in plant viruses, called tRNA-like structures (TLSs), are located at the 3' end of their respective genomes, driving the covalent linkage of the RNA viral genome to an amino acid species. These domains are involved in viral protein production, replication, and encapsidation, though the mechanisms of these processes remain unknown. The tertiary structures of these TLS molecules have never been fully elucidated, though models based on chemical probing have been developed. I describe here structural studies of the Turnip Yellow Mosaic Virus (TYMV) TLS, Tobacco Mosaic Virus (TMV) TLS, and Brome Mosaic Virus (BMV) TLS to determine their actual tRNA-likeness in solution, as well as assessing their individual folding characters. I found that the TYMV TLS folds into a tRNA-like architecture that is dependent on the presense of 5' upstream nucleotides. Further, I discovered that an upstream pseudoknot domain seems to pack tightly against the TLS core creating a larger functional molecule. Likewise, the TMV TLS requires 5' upstream sequences to form a solvent exclusive packing character even though one of its two pseudoknots seems to be dynamic or transiently unstable. The BMV TLS creates a tightly packed core in solution (in the absence of any upstream nucleotides), and represents a fold dissimilar to tRNA, with the replicase promoter C-stem showing both a tightly packed as well as dynamic character. My work indicates that tRNA may be far more dynamic in solution than we had previously thought, and the TYMV TLS displays a similar dynamic quality. I have also used Small Angle X-ray Scattering (SAXS) to test previous models of these structures, as well as generate low resolution solution structure surface maps. The relevance of my structural data to previous functional data is discussed, and new testable models for viral replication, translation enhancement, and TLS unfolding are presented. By studying these molecules, I have enhanced our understanding of tRNA dynamic character, tRNA mimicry, and specifically the structural regulatory role of these elements in their various viral processes. |
