Khan Mateen A, Ma Jia, Walden William E, Merrick William C, Theil Elizabeth C, Goss Dixie J
Department of Chemistry and Biochemistry, Hunter College, City University of New York, New York, NY 10065, USA.
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612-7334, USA.
Nucleic Acids Res. 2014 Jun;42(10):6567-77. doi: 10.1093/nar/gku248. Epub 2014 Apr 11.
Metal ion binding was previously shown to destabilize IRE-RNA/IRP1 equilibria and enhanced IRE-RNA/eIF4F equilibria. In order to understand the relative importance of kinetics and stability, we now report rapid rates of protein/RNA complex assembly and dissociation for two IRE-RNAs with IRP1, and quantitatively different metal ion response kinetics that coincide with the different iron responses in vivo. kon, for FRT IRE-RNA binding to IRP1 was eight times faster than ACO2 IRE-RNA. Mn(2+) decreased kon and increased koff for IRP1 binding to both FRT and ACO2 IRE-RNA, with a larger effect for FRT IRE-RNA. In order to further understand IRE-mRNA regulation in terms of kinetics and stability, eIF4F kinetics with FRT IRE-RNA were determined. kon for eIF4F binding to FRT IRE-RNA in the absence of metal ions was 5-times slower than the IRP1 binding to FRT IRE-RNA. Mn(2+) increased the association rate for eIF4F binding to FRT IRE-RNA, so that at 50 µM Mn(2+) eIF4F bound more than 3-times faster than IRP1. IRP1/IRE-RNA complex has a much shorter life-time than the eIF4F/IRE-RNA complex, which suggests that both rate of assembly and stability of the complexes are important, and that allows this regulatory system to respond rapidly to change in cellular iron.
先前的研究表明，金属离子结合会破坏IRE-RNA/IRP1平衡，并增强IRE-RNA/eIF4F平衡。为了理解动力学和稳定性的相对重要性，我们现在报告了两种IRE-RNA与IRP1形成蛋白质/RNA复合物的组装和解离的快速速率，以及与体内不同铁反应相一致的定量不同的金属离子反应动力学。FRT IRE-RNA与IRP1结合的kon比ACO2 IRE-RNA快8倍。Mn(2+)降低了IRP1与FRT和ACO2 IRE-RNA结合的kon并增加了koff，对FRT IRE-RNA的影响更大。为了从动力学和稳定性方面进一步理解IRE-mRNA调控，我们测定了FRT IRE-RNA与eIF4F的动力学。在没有金属离子的情况下，eIF4F与FRT IRE-RNA结合的kon比IRP1与FRT IRE-RNA结合慢5倍。Mn(2+)增加了eIF4F与FRT IRE-RNA结合的缔合速率，因此在50 µM Mn(2+)时，eIF4F的结合速度比IRP1快3倍以上。IRP1/IRE-RNA复合物的寿命比eIF4F/IRE-RNA复合物短得多，这表明复合物的组装速率和稳定性都很重要，并且这使得该调节系统能够对细胞内铁的变化做出快速反应。
