Low energy CD of RNA hairpin unveils a loop conformation required for λ N antitermination activity
Low energy CD of RNA hairpin unveils a loop conformation required for λ N antitermination activity
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DOI：
10.1074/jbc.m504619200
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发表时间：
2005-09-16
影响因子：
4.8
通讯作者：
von Hippel, PH
中科院分区：
文献类型：
--
作者：
Johnson, NP;Baase, WA;von Hippel, PH
由λ噬菌体编码的N蛋白是一种转录因子，其通过特异性地结合在称为boxB的茎环结构处的新生RNA转录物来刺激大肠杆菌RNA聚合酶的抗终止活性。我们使用一种新的生物物理技术，涉及监测的低能量圆二色光谱的2-氨基嘌呤残基位点特异性地放置在boxB RNA环，调查这种结合的相互作用。这些2-氨基嘌呤探针的低能CD光谱反映了与相邻核苷酸碱基的特异性不对称相互作用。因此，这些CD光谱提供了有关这些发色团处RNA链的详细且特定的构象信息，而这些信息无法从这些探针的相关荧光信号的变化中获得。在N肽与boxB RNA结合时观察到CD变化，其对应于先前通过NMR观察到的结构变化，从而验证了我们的实验方法。然后使用低能量CD方法来量化自由发夹环的有序和无序状态，并显示boxB环的显著部分在不存在蛋白质的情况下呈现产物样结构。鉴定了具有完整茎和降低浓度的有序环的boxB衍生物，并用于显示蛋白质和boxB之间的反应程度取决于RNA反应物群体中结构化环的浓度。这一结果对RNA-蛋白质相互作用的构象特异性具有普遍意义。
N protein coded by phage lambda is a transcription factor that stimulates the antitermination activity of Escherichia coli RNA polymerase by binding specifically to the nascent RNA transcript at a stem-loop structure called boxB. We use a new biophysical technique, involving the monitoring of the low energy circular dichroism spectra of 2-aminopurine residues site-specifically placed in the boxB RNA loop, to investigate this binding interaction. The low energy CDspectra of these 2-aminopurine probes reflect specific asymmetric interactions with adjacent nucleotide bases. Consequently, these CD spectra provide detailed and specific conformational information about the RNA chain at these chromophores that cannot be obtained from changes in the related fluorescence signals of these probes. CD changes were observed on binding the N peptide to boxB RNA that correspond to structural changes that had been previously seen by NMR, thus validating our experimental approach. The low energy CD method was then used to quantify the ordered and disordered states of the free hairpin loop and to show that a significant fraction of the boxB loop assumes a product-like structure in the absence of protein. A boxB derivative with an intact stem and a reduced concentration of ordered loop was identified and used to show that the extent of the reaction between protein and boxB depends on the concentration of structured loop in the RNA reactant population. This result has general implications for the conformational specificity of RNA- protein interactions.