Molecular Basis for the Cooperative Action of the Reverse Gyrase Helicase and Topoisomerase Domains in Positive DNA Supercoiling (B13)

反向旋转酶是一种DNA拓扑异构酶，由一个解旋酶和一个拓扑异构酶结构域组成。这些结构域的复杂协作产生了依赖于ATP的正DNA超螺旋的新功能。在tRNA乙酰化酶TMCA中，解旋酶和转乙酰基酶结构域在tRNA乙酰化过程中相互作用。我们将通过交联/质谱学和X射线结晶学分析DNA与反向旋转酶结合所涉及的协作性，并通过单分子FRET剖析反向旋转酶整个核苷酸周期中协同构象变化的时间协调性。此外，我们将解决TMCA在tRNA乙酰化过程中与配体结合的协同性和构象变化。目标是揭开解旋酶结构域和其他酶结构域之间功能相互作用的分子基础。

Reverse gyrase is a DNA topoisomerase that consists of a helicase and a topoisomerase domain. The intricate cooperation of these domains generates the novel function of ATP-dependent positive DNA supercoiling. In the tRNA acetylase TmcA, a helicase and a transacetylase domain cooperate in tRNA acetylation. We will analyze the cooperativity involved in DNA binding to reverse gyrase by cross-linking/mass-spectrometry and X-ray crystallography, and dissect the temporal coordination of cooperative conformational changes throughout the nucleotide cycle of reverse gyrase by single-molecule FRET. Further, we will address cooperativity in ligand binding by and conformational changes of TmcA during tRNA acetylation. The goal is to unravel the molecular basis for the functional interplay between helicase domains and other enzymatic domains.