Department of Structural Biology, Van Andel Institute, Grand Rapids, MI, USA.
Department of Chemistry, The Pennsylvania State University, University Park, PA, USA.
Nat Commun. 2023 Jul 20;14(1):4396. doi: 10.1038/s41467-023-40106-2.
The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
T4 噬菌体 gp41 解旋酶和 gp61 引发酶组装成一个引发体，将 DNA 解旋与 RNA 引物合成偶联起来，以进行 DNA 复制。引发体如何组装以及引物长度如何确定尚不清楚。在这里，我们报告了一系列 T4 引发体组装中间体的 cryo-EM 结构。我们表明，gp41 本身是一个开放的螺旋，ssDNA 结合触发了大规模的剪刀样构象变化，从而驱动环闭合并激活解旋酶。解旋酶的激活暴露出一个隐藏的疏水面，以招募 gp61 引发酶。引发酶以二部分模式与解旋酶结合，其中 N 端 Zn 结合结构域和 C 端 RNA 聚合酶结构域都包含一个解旋酶相互作用基序，与单独的 gp41 N 端发夹二聚体结合，导致一个引发酶组装在解旋酶六聚体上。我们的研究揭示了 T4 引发体组装过程，并阐明了 RNA 引物合成机制。
