Nixon P L, Theimer C A, Giedroc D P
Department of Biochemistry and Biophysics, Center for Macromolecular Design, Texas A&M University, College Station, TX 77843-2128, USA.
Biopolymers. 1999 Oct 5;50(4):443-58. doi: 10.1002/(SICI)1097-0282(19991005)50:4<443::AID-BIP9>3.0.CO;2-C.
Equilibrium unfolding (folding) studies reveal that the autoregulatory RNA pseudoknots derived from the bacteriophage T2 and T4 gene 32 mRNAs exhibit significant stabilization by increasing concentrations of divalent metal ions in solution. In this report, the apparent affinities of exchange inert trivalent Co(NH(3))(3+)(6) have been determined, relative to divalent Mg(2+), for the folded, partially folded (K(f)), and fully unfolded (K(u)) conformations of these molecules. A general nonspecific, delocalized ion binding model was developed and applied to the analysis of the metal ion concentration dependence of individual two-state unfolding transitions. Trivalent Co(NH(3))(3+)(6) was found to associate with the fully folded and partially unfolded pseudoknotted forms of these RNAs with a K(f) of 5-8 x 10(4) M(-1) in a background of 0.10 M K(+), or 3- to 5-fold larger than the K(f) obtained for two model RNA hairpins and hairpin unfolding intermediates, and approximately 40-50-fold larger than K(f) for Mg(2+). The magnitude of K(f) was found to be strongly dependent on the monovalent salt concentration in a manner qualitatively consistent with polyelectrolyte theory, with K(f) reaching 1.2 x 10(5) M(-1) in 50 mM K(+). Two RNA hairpins were found to have affinities for Co(NH(3))(3+)(6) and Ru(NH(3))(3+)(6) of 1-2 x10(4) M(-1), or approximately 15-fold larger than the K(f) of approximately 1000 M(-1) observed for Mg(2+). Additionally, the K(u) of 4,800 M(-1) for the trivalent ligands is approximately 8-fold larger than the K(u) of 600 M(-1) observed for Mg(2+). These findings suggest that the T2 and T4 gene 32 mRNA pseudoknots possess a site(s) for Mg(2+) and Co(NH(3))(3+)(6) binding of significantly higher affinity than a "duplexlike" delocalized ion binding site that is strongly linked to the thermodynamic stability of these molecules. Imino proton perturbation nmr spectroscopy suggests that this site(s) lies near the base of the pseudoknot stem S2, near a patch of high negative electrostatic potential associated with the region where the single loop L1 adenosine crosses the major groove of stem S2.
平衡去折叠（折叠）研究表明，源自噬菌体T2和T4基因32 mRNA的自动调节RNA假结在溶液中二价金属离子浓度增加时表现出显著的稳定性。在本报告中，已确定相对于二价Mg²⁺，交换惰性三价Co(NH₃)₆³⁺对这些分子的折叠态、部分折叠态（Kf）和完全去折叠态（Ku）构象的表观亲和力。开发了一个通用的非特异性、离域离子结合模型，并将其应用于分析单个两态去折叠转变对金属离子浓度的依赖性。发现在0.10 M K⁺背景下，三价Co(NH₃)₆³⁺与这些RNA的完全折叠和部分折叠假结形式结合，Kf为5 - 8×10⁴ M⁻¹，比两个模型RNA发夹和发夹去折叠中间体获得的Kf大3至5倍，比Mg²⁺的Kf大约大40至50倍。发现Kf的大小强烈依赖于单价盐浓度，其方式在定性上与聚电解质理论一致，在50 mM K⁺中Kf达到1.2×10⁵ M⁻¹。发现两个RNA发夹对Co(NH₃)₆³⁺和Ru(NH₃)₆³⁺的亲和力为1 - 2×10⁴ M⁻¹，比观察到的Mg²⁺的Kf约1000 M⁻¹大15倍左右。此外，三价配体的Ku为4800 M⁻¹，比观察到的Mg²⁺的Ku 600 M⁻¹大约8倍。这些发现表明，T2和T4基因32 mRNA假结具有一个Mg²⁺和Co(NH₃)₆³⁺结合位点，其亲和力明显高于与这些分子的热力学稳定性密切相关的“双链样”离域离子结合位点。亚氨基质子扰动核磁共振光谱表明，该位点位于假结茎S2的基部附近，靠近与单环L1腺苷穿过茎S2大沟的区域相关的高负静电势区域。
