Structure determination of the TGE element of tra-2 messenger RNA in complex with the posttranscriptional regulatory protein GLD-1 by nuclear magnetic resonance spectroscopy
Final Report Abstract
To access the genetic information that is stored in the DNA, messenger RNAs (mRNAs) are transcribed and serve as working copies that are subsequently translated into protein. All of these steps need to be tightly controlled in order to keep a cell healthy and functional, and failure to do so results in diseases like cancer. Gene regulation at the post-transcriptional level by proteins that bind to mRNAs is one important means to achieve this goal. Visualization of these protein-RNA complexes at the atomic level helps understand their structure and function. My protein of interest is a prototypical member of the STAR protein family. These proteins regulate transcription by binding as a dimer to specific sequences in target mRNAs, but the exact mechanism of regulation is still unknown. My first aim was to determine the structure of the dimerization domain, named Qua1, of the STAR protein GLD-1 using nuclear magnetic resonance (NMR) spectroscopy. Valuable information about the secondary structure and dynamics of the protein backbone were obtained, but collecting and assigning the data for the amino acid side chains, which is necessary to determine the protein structure by NMR, was extremely challenging. However, successful crystallization of Qua1 opened the door to structure determination by X-ray crystallography. The X-ray crystal structure provides insights into how STAR proteins dimerize and, together with a homology model for the RNA-binding subdomain, allowed construction of a structural model for the STAR/RNA complex. My second aim comprised studying the structure of the RNA within the GLD-1 STAR/RNA complex by NMR. The RNA could be transcribed in sufficient quantities, but it adopted structures that prevented protein binding. Furthermore, the full protein/RNA complex proved not to be amenable for NMR studies. Attempts to crystallize the Gld-1/RNA complex together with its protein partner FOG-2 are ongoing in the Williamson lab.
