| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | After removing the GST tag by TEV, samples were applied to a mono Q column (for Sub2 variants) or a mono S column (GE Healthcare, for Yra1 variants), followed by size-exclusion chromatography using a Superdex 200 column (GE Healthcare) in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.5 mM TCEP. Purified proteins were concentrated, flash frozen in liquid nitrogen, and stored at −80°C. C-box (a.a. 208–226) of Yra1 was synthesized by GenScript. | Get A Quote |
mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open ... More
