To elucidate how T-box riboswitches sense tRNA aminoacylation state using a compact RNA structure, we screened a number of bacterial species to find a T-box construct that exhibits correct folding in vitro and that can effectively engage the tRNA 3 end. Most T-box RNA tend to form non-productive, misfolded structures in vitro, when transcribed by the non-cognate T7 RNA polymerase, thus precluding mechanistic and structural studies. We successfully identified a T-box-tRNA pair that can form stoichiometric complexes in vitro. Further we developed methods that can assemble and isolate the T-box-tRNA complex from the individual components, which permitted initial structural analysis using Small-angle X-ray Scattering (SAXS). We demonstrated that the T-box-tRNA interaction is biologically relevant as the binding is selective for the tRNA aminoacylation state of the tRNA 3 end. With the biological relevance established, we are using X-ray crystallography and single-particle Cryo-EM (in collaboration with National Center for Macromolecular Imaging) to characterize the structural mechanisms of this T-box-tRNA interaction, which functions to sense intracellular amino acid starvation in most Gram-positive bacteria. Instead of the T-boxes, eukaryotes use a highly conserved tRNA-regulated kinase (Gcn2) to sense and respond to nutrient deprivation and other types of stresses. Initially discovered by Dr. Alan Hinnebuschs lab (NICHD), Gcn2 is the sole eIF-2alpha kinase in yeast and one of the four in mammals responsible for sensing distinct stresses and shuttering global translation initiation via eIF-2alpha phosphorylation. Gcn2 senses and manages amino acid/serum starvation, UV irradiation, oxidative/osmotic/ER stress, etc, is essential for cellular survival under stress, and is a key player in several cancers and neurodegenerative diseases. Similar to T-boxes, this multi-domain protein directly engages tRNAs and evaluates their aminoacylation status to detect nutrient limitation, and couples this readout with the activation of its dormant kinase activity. To achieve these functions, Gcn2 incorporated a domain borrowed from the Histidyl-tRNA synthetase (HisRS), the enzyme responsible for charging histidine onto tRNA-his. This represents a remarkable case of enzyme repurposing and adaptation as the HisRS-like domain of Gcn2 must recognize many if not all tRNAs that carry a variety of anticodons and different 5' ends, unlike its parent enzyme specific for tRNA-his. Importantly, it is unknown how Gcn2 distinguishes uncharged from charged tRNA, a critical function of nutrient surveillance, and how the HisRS-like domain communicates with the kinase domain. In collaboration with Dr. Alan Hinnebusch (NICHD), we are investigating the structural organization of Gcn2, how Gcn2 recognizes tRNAs, reads its aminoacylation status, and couples uncharged tRNA recognition with kinase activation. In particular, structural comparison of Gcn2 with authentic HisRS enzyme would illuminate how a repurposed enzyme evolves to fit in a new functional context. Gcn2 is known to be a challenging protein to work with, whose structural elucidation has been widely awaited. Through systematic trials, we have developed a system to express and purify the HisRS-like domain and C-terminal domain of Gcn2, in biophysical quantities. We are currently performing structural analyses using X-ray crystallography and SAXS to visualize the HisRS-like domain of Gcn2.
| Stagno, J R; Liu, Y; Bhandari, Y R et al. (2017) Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541:242-246 |
| Stamatopoulou, Vassiliki; Apostolidi, Maria; Li, Shuang et al. (2017) Direct modulation of T-box riboswitch-controlled transcription by protein synthesis inhibitors. Nucleic Acids Res 45:10242-10258 |
| Zhang, Jinwei (2017) Brothers in arms: emerging roles of RNA epigenetics in DNA damage repair. Cell Biosci 7:24 |
| Zhang, Jinwei; Ferré-DAmaré, Adrian R (2016) Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers. Biomolecules 6: |
| Zhang, Jinwei; Ferré-D'Amaré, Adrian R (2016) The tRNA Elbow in Structure, Recognition and Evolution. Life (Basel) 6: |
| Zhang, Jinwei; Landick, Robert (2016) A Two-Way Street: Regulatory Interplay between RNA Polymerase and Nascent RNA Structure. Trends Biochem Sci 41:293-310 |
