Bridge helix and trigger loop perturbations generate superactive RNA polymerases.
Bridge helix and trigger loop perturbations generate superactive RNA polymerases.
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DOI：
10.1186/jbiol98
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发表时间：
2008-12-02
影响因子：
--
通讯作者：
Weinzierl RO
中科院分区：
文献类型：
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作者：
Tan L;Wiesler S;Trzaska D;Carney HC;Weinzierl RO
细胞RNA聚合酶是高度保守的酶，其经历复杂的构象变化以通过活性位点协调核酸底物的加工。特别是两个结构域，桥螺旋和触发环，通过在核苷酸添加循环的各个阶段采用不同的构象在该机制中发挥关键作用。这些结构变化的功能相关性很难从目前相对较少的静态晶体结构中评估。使用一种新的机器人的方法，我们的特点是367个定点突变体的詹氏甲烷球菌RNA聚合酶A'亚基的功能特性，揭示了广泛的体外表型。我们表明，一个令人惊讶的大量的单个氨基酸取代的桥螺旋，包括扭结诱导脯氨酸取代，增加RNA聚合酶的比活性。其他“超活化”取代位于触发环的相邻碱基螺旋中。结果支持的假设，核苷酸添加循环涉及一个扭结桥螺旋构象。RNA聚合酶的活性中心似乎受到桥螺旋和触发环之间的功能相互作用网络的限制，该网络控制RNA合成的基本参数。
Cellular RNA polymerases are highly conserved enzymes that undergo complex conformational changes to coordinate the processing of nucleic acid substrates through the active site. Two domains in particular, the bridge helix and the trigger loop, play a key role in this mechanism by adopting different conformations at various stages of the nucleotide addition cycle. The functional relevance of these structural changes has been difficult to assess from the relatively small number of static crystal structures currently available. Using a novel robotic approach we characterized the functional properties of 367 site-directed mutants of the Methanocaldococcus jannaschii RNA polymerase A' subunit, revealing a wide spectrum of in vitro phenotypes. We show that a surprisingly large number of single amino acid substitutions in the bridge helix, including a kink-inducing proline substitution, increase the specific activity of RNA polymerase. Other 'superactivating' substitutions are located in the adjacent base helices of the trigger loop. The results support the hypothesis that the nucleotide addition cycle involves a kinked bridge helix conformation. The active center of RNA polymerase seems to be constrained by a network of functional interactions between the bridge helix and trigger loop that controls fundamental parameters of RNA synthesis.