The intrinsic apoptosis pathway occurs through the release of mitochondrial cytochrome c to the cytosol, where it promotes activation of the caspase family of proteases. The observation that tRNA binds to cytochrome c revealed a previously unexpected mode of apoptotic regulation. However, the molecular characteristics of this interaction, and its impact on each interaction partner, are not well understood. Using a novel fluorescence assay, we show here that cytochrome c binds to tRNA with an affinity comparable with other tRNA-protein binding interactions and with a molecular ratio of∼3:1. Cytochrome c recognizes the tertiary structural features of tRNA, particularly in the core region. This binding is independent of the charging state of tRNA but is regulated by the redox state of cytochrome c. Compared with reduced cytochrome c, oxidized cytochrome c binds to tRNA with a weaker affinity, which correlates with its stronger pro-apoptotic activity. tRNA binding both facilitates cytochrome c reduction and inhibits the peroxidase activity of cytochrome c, which is involved in its release from mitochondria. Together, these findings provide new insights into the cytochrome c-tRNA interaction and apoptotic regulation.
Bibliographical noteFunding Information:
This work was supported in part by National Institutes of Health Grants R01 GM081601, U01 GM108972, and R01 GM114343 (to Y. M. H.), R01 GM060911, and R21 CA178581 and United States Department of Defense Grant W81XWH-13-1-0446 (to X. Y.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We thank S. Seeholzer and H. Ding at the Children's Hospital of Philadelphia's Protein Core Facility for help with the surface plasmon resonance assay; K. Shogen and W. Ardelt for providing Onconase; Y. Mei, W. Prall, and N. Charan for technical assistance; and members of the Hou and Yang laboratories for helpful discussions.