Srivastava Yoshita, Bonn-Breach Rachel, Chavali Sai Shashank, Lippa Geoffrey M, Jenkins Jermaine L, Wedekind Joseph E
Department of Biochemistry & Biophysics and Center for RNA Biology, University of Rochester School of Medicine & Dentistry, Rochester, NY 14642, USA.
Present address: Division of Biology, Alfred University, Alfred, NY 14802, USA.
Crystals (Basel). 2021 Mar;11(3). doi: 10.3390/cryst11030273. Epub 2021 Mar 10.
RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII ( of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop ( of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community.
RNA在所有生物体中都起着核心作用，并且可以折叠成复杂的结构来协调功能。此类结构的可视化通常需要结晶，这可能是结构确定过程中的一个瓶颈。为了促进结晶，U1A剪接体蛋白的一个RNA识别基序（RRM）已被用作结晶模块。具体而言，被U1A识别的U1-snRNA发夹II（hpII）单链环可以移植到RNA靶标中，以促进晶体接触，并通过使用硒代甲硫氨酸的分子置换或反常衍射方法获得相位信息。在此，我们制备了U1A的F37M/F77M突变体，以增强这个强大的结晶模块的相位确定能力。硒代甲硫氨酸取代的U1A(F37M/F77M)对hpII保持高亲和力（解离常数为59.7±11.4 nM）。分辨率为2.20 Å的晶体结构表明，突变的侧链在疏水核心中形成了新的S-π相互作用，并且可用于单波长反常衍射。还获得了与细菌前Q-II核糖开关结合的U1A(F37M/F77M)晶体。类似地，F34M/F37M/F77M突变体被引入到一个实验室进化的U1A变体（TBP6.9）中，该变体识别HIV-1 TAR RNA的内部凸起环。我们设想，这个短RNA序列可以放置在非必需的双链区域中，以促进靶标RNA的结晶和相位确定。我们表明，硒代甲硫氨酸取代的TBP6.9(F34M/F37M/F77M)与一个TAR变体结合，其中顶端环被一个GNRA四环取代（解离常数为69.8±2.9 nM），为将TBP6.9(F34M/F37M/F77M)用作结晶模块奠定了基础。这些新工具可供研究界使用。
