Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex. We then used total internal reflection fluorescence microscopy (TIRF) with alternating excitation to measure the single molecule FRET (smFRET) efficiency between these two sites in pol II-TFIIF. We found they exhibited a proximity consistent with that observed in the transcription pre-initiation complex by cryo-electron microscopy (cryo-EM). We further compared our non-covalent labeling approach with an enzyme-enabled covalent labeling method. The virtually indistinguishable results validate our smFRET approach and show that the observed proximity between the two sites represents a hallmark of the pol II-TFIIF complex. Taken together, we present a simple and versatile bio-orthogonal method derived from TAP to enable selective labeling of a protein complex. This method is suitable for analyzing dynamic relationships among proteins involved in transcription and it can be readily extended to many other biological processes.

中文翻译：

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从 TAP 中衍生出亚纳摩尔亲和肽，以实现对 RNA 聚合酶 II 复合物的 smFRET 分析

我们通过单分子成像技术对蛋白质复合物（如 RNA 聚合酶 II (pol II)）进行可视化的能力在很大程度上受到了缺乏用荧光探针进行选择性标记的简单生物正交方法的阻碍。在这里，我们修改了广泛使用的串联亲和纯化 (TAP) 标签中现有的钙调素结合肽 (CBP)，使其对钙调素 (CaM) 具有高亲和力，并使用染料-CaM 对 pol II 进行位点特异性标记. 为了证明这种方法的单分子适用性，我们用供体-CaM 标记了 pol II 的 Rpb9 亚基的 C 端，并用受体标记了 TFIIF 中的一个位点，以在 pol II 中产生 FRET（荧光共振能量转移）对-TFIIF 复合体。然后我们使用全内反射荧光显微镜 (TIRF) 和交替激发来测量 pol II-TFIIF 中这两个位点之间的单分子 FRET (smFRET) 效率。我们发现它们表现出的接近度与通过冷冻电子显微镜 (cryo-EM) 在转录预起始复合物中观察到的接近度一致。我们进一步将我们的非共价标记方法与酶促共价标记方法进行了比较。几乎无法区分的结果验证了我们的 smFRET 方法，并表明观察到的两个站点之间的接近度代表了 pol II-TFIIF 复合体的标志。总之，我们提出了一种源自 TAP 的简单而通用的生物正交方法，可以选择性标记蛋白质复合物。