Liu C, Tolić L P, Hofstadler S A, Harms A C, Smith R D, Kang C, Sinha N
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington, 99352, USA.
Anal Biochem. 1998 Aug 15;262(1):67-76. doi: 10.1006/abio.1998.2753.
The interactions of bacteriophage T4 regA protein, a unique translational regulator, with RNAs of various size and sequence were studied using electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry. Using very gentle interface conditions, regA/RNA complexes with a 1:1 binding stoichiometry were observed for all four target RNAs studied, consistent with solution binding studies. Competitive binding of target RNAs and their degradation products with regA demonstrated that the loss of a single nucleotide resulted in a dramatic change in binding affinity in some cases. Competitive binding of regA with four target RNAs revealed similar relative binding affinity order to that suggested by previous in vitro repression experiments. The use of sustained off-resonance irradiation for collisionally induced dissociation of a regA/RNA complex suggested the potential for directly obtaining information regarding the regA binding domain.
利用电喷雾电离-傅里叶变换离子回旋共振质谱法研究了独特的翻译调节因子噬菌体T4 RegA蛋白与各种大小和序列的RNA之间的相互作用。在非常温和的接口条件下，对于所研究的所有四种靶RNA，均观察到具有1:1结合化学计量比的RegA/RNA复合物，这与溶液结合研究一致。靶RNA及其降解产物与RegA的竞争性结合表明，在某些情况下，单个核苷酸的缺失会导致结合亲和力发生显著变化。RegA与四种靶RNA的竞争性结合揭示了与先前体外抑制实验所暗示的相似的相对结合亲和力顺序。使用持续非共振辐照对RegA/RNA复合物进行碰撞诱导解离，表明有可能直接获得有关RegA结合域的信息。
