Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements.
Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements.
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通过高通量热力学测量剖析RNA三级结构基序内的能量架构。
DOI：
10.1073/pnas.2220485120
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发表时间：
2023-03-14
影响因子：
11.1
通讯作者：
Herschlag, Daniel
中科院分区：
文献类型：
--
作者：
Shin, John H.;Bonilla, Steve L.;Denny, Sarah K.;Greenleaf, William J.;Herschlag, Daniel
正确折叠的RNA执行基本的生物过程。这种RNA通常含有三级接触“基序”，其结构和能量特性在不同RNA中是保守的。这项研究深入探讨了一个常见的RNA基序，11核苷酸受体的能量结构。由于深入研究这个和其他RNA基序的能量特性需要对许多序列变体进行热力学测量，我们使用了一种定量的高通量方法来获得基序的所有单突变体和双突变体的结合自由能。在表征这个基序的能量结构时，我们的研究揭示了基序内的能量耦合模式，并确定了具有意想不到特性的罕见变体。结构化RNA和RNA/蛋白质复合物执行关键的细胞功能。它们通常包含结构保守的三级接触“基序”，其出现简化了RNA折叠景观。先前的研究主要集中在完整基序的构象和能量模块化。在这里，我们转向一个共同的主题，11 nt受体（11ntR）的解剖，使用定量分析的RNA上的大规模平行阵列来测量所有的单和双11ntR突变体的GAAA和GUAA四环的结合，从而探测充满活力的架构的主题。虽然11ntR表现为基序，但其协同性不是绝对的。相反，我们发现了从碱基配对和相邻残基之间的高协同性到远处残基之间的加和性的梯度。如预期的，在与GAAA四环直接接触的残基处的取代导致结合的最大降低，并且突变的能量罚分对于与替代的GUAA四环的结合基本上较小，所述替代的GUAA四环缺乏与典型的GAAA四环存在的三级接触。然而，我们发现，充满活力的后果碱基伴侣取代，在一般情况下，简单地描述了碱基对类型或等排性。我们还发现了11ntR序列变异体先前建立的稳定性-丰度关系的例外情况。这些“规则的例外”的发现突出了系统性高通量方法的力量，除了提供功能RNA的能量图之外，还可以为未来的研究发现新的变体。
Properly folded RNAs perform essential biological processes. Such RNAs often contain tertiary contact “motifs” whose structural and energetic properties are conserved throughout different RNAs. This study delves into the energetic architecture of one common RNA motif, the 11-nucleotide receptor. As deep studies into the energetic properties of this and other RNA motifs require thermodynamic measurements for many sequence variants, we used a quantitative, high-throughput method to obtain binding free energies for all single and double mutants of the motif. In characterizing the energetic architecture of this motif, our study uncovered patterns of energetic coupling within the motif and identified rare variants with unexpected properties. Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact “motifs,” whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability–abundance relationship for 11ntR sequence variants. These findings of “exceptions to the rule” highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.
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通过SMFRET探测四边形/四边形受体单价离子结合位点的动力学和热力学后果。
影响因子：
3.9
作者：
Bisaria N;Herschlag D
通讯作者：
Herschlag D
影响因子：
5.5
作者：
Brown, Reid F.;Andrews, Casey T.;Elcock, Adrian H.
通讯作者：
Elcock, Adrian H.
影响因子：
64.5
作者：
Cruz, Jose Almeida;Westhof, Eric
通讯作者：
Westhof, Eric
DOI：
10.1038/2960
发表时间：
1998-11-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Basu, S;Rambo, RP;Doudna, JA
通讯作者：
Doudna, JA
DOI：
10.1007/978-1-62703-709-9_3
发表时间：
2014-01-01
期刊：
RNA SEQUENCE, STRUCTURE, AND FUNCTION: COMPUTATIONAL AND BIOINFORMATIC METHODS
影响因子：
--
作者：
Andronescu, Mirela;Condon, Anne;Mathews, David H.
通讯作者：
Mathews, David H.