Department of Biochemistry, University of Colorado, Boulder, CO 80309-0596, USA.
Department of Biochemistry, University of Colorado, Boulder, CO 80309-0596, USA. Electronic address: https://twitter.com/Lschwanzbio.
J Mol Biol. 2023 May 15;435(10):168070. doi: 10.1016/j.jmb.2023.168070. Epub 2023 Mar 31.
The architecture and folding of complex RNAs is governed by a limited set of highly recurrent structural motifs that form long-range tertiary interactions. One of these motifs is the T-loop, which was first identified in tRNA but is broadly distributed across biological RNAs. While the T-loop has been examined in detail in different biological contexts, the various receptors that it interacts with are not as well defined. In this study, we use a cell-based genetic screen in concert with bioinformatic analysis to examine three different, but related, T-loop receptor motifs found in the flavin mononucleotide (FMN) and cobalamin (Cbl) riboswitches. As a host for different T-loop receptors, we employed the env8 class-II Cbl riboswitch, an RNA that uses two T-loop motifs for both folding and supporting the ligand binding pocket. A set of libraries was created in which select nucleotides that participate in the T-loop/T-loop receptor (TL/TLR) interaction were fully randomized. Library members were screened for their ability to support Cbl-dependent expression of a reporter gene. While T-loops appear to be variable in sequence, we find that the functional sequence space is more restricted in the Cbl riboswitch, suggesting that TL/TLR interactions are context dependent. Our data reveal clear sequence signatures for the different types of receptor motifs that align with phylogenic analysis of these motifs in the FMN and Cbl riboswitches. Finally, our data suggest the functional contribution of various nucleobase-mediated long-range interactions within the riboswitch subclass of TL/TLR interactions that are distinct from those found in other RNAs.
复杂 RNA 的结构和折叠由一组有限的高度重复的结构基序控制，这些基序形成长程三级相互作用。这些基序之一是 T 环，它最初在 tRNA 中被识别，但在广泛分布于生物 RNA 中。虽然 T 环在不同的生物背景下已经被详细研究，但它相互作用的各种受体并没有被很好地定义。在这项研究中，我们使用基于细胞的遗传筛选与生物信息学分析相结合，研究了在黄素单核苷酸 (FMN) 和钴胺素 (Cbl) 核糖开关中发现的三个不同但相关的 T 环受体基序。作为不同 T 环受体的宿主，我们采用了类 II Cbl 核糖开关，这是一种使用两个 T 环基序进行折叠和支持配体结合口袋的 RNA。创建了一组文库，其中参与 T 环/T 环受体 (TL/TLR) 相互作用的选定核苷酸完全随机化。对文库成员进行筛选，以评估它们支持 Cbl 依赖的报告基因表达的能力。虽然 T 环在序列上似乎是可变的，但我们发现 Cbl 核糖开关中的功能序列空间更受限制，这表明 TL/TLR 相互作用是上下文相关的。我们的数据揭示了不同类型的受体基序的清晰序列特征，与 FMN 和 Cbl 核糖开关中这些基序的系统发育分析一致。最后，我们的数据表明，在 TL/TLR 相互作用的核糖开关亚类中，各种碱基介导的长程相互作用的功能贡献与在其他 RNA 中发现的不同。
