大腸桿菌 PriB 蛋白分子之結晶學結構研究

1. 大腸桿菌PriB蛋白分子之結晶學結構研究 • 細菌染色體去氧核醣核酸(DNA)的複製，需要先合成一個引物合成體(primosome)以合成引物(primer)，並打開雙鏈以利染色體在複製叉(Replication fork)進行DNA複製。在大腸桿菌(E.coli)，至少有兩種引物合成體已經被確認。一種結合在oriC 位置並需DnaA 蛋白來啟動﹔另一種則結合在n’-pas 序列並靠PriA蛋白來進行啟動。這兩種primosomes 分別被命名為DnaA-primosome 及PriA-primosomes。在大腸桿菌中當複製叉功能已經受損，還能再重新啟動複製的功能是非常必需且重要地。在重新啟動的過程中PriA-primosomes包含 PriA、 PriB、 PriC、 DnaT 、DnaC和 DnaB 扮演非常重要地角色。在本研究中我們可以得到大量且純度高的PriB 蛋白並嘗試利用各種結晶方式的到三種PriB 蛋白的結晶。第一種為PriB (Native) 屬於四方晶系(primitive tetragonal)， 此晶系所得之晶胞參數為a=b=50.86 Å c=139.10 Å α=β=γ=90° 所屬空間群為P41212 or P43212。第二種為 PriB (Se-Met)，其屬於三方晶系(trigonal) 所得之晶胞參數為a=b=57.78 Å c=83.10 Å α=β=90° γ=120° 所屬空間群為P3121。第三種為 PriB (Hg)是屬於正交晶系(orthorhombic) ，所得之晶胞參數為a=39.14 Å b=58.21 Å c=100.24 Å α=β=γ=90° 所屬空間群為P212121。此外更進一步利用MAD方法解出PriB (Hg) 蛋白的結構。此外利用電泳遷移率變動分析法(EMSA)，我們更進一步證實，PriB 蛋白和single stranded DNA (ssDNA)之間有相互作用力存在，會形成PriB-ssDNA 複合體(complex)。

2. Crystallization and Preliminary X-ray Crystallographic Analysis of Escherichia coli PriB Protein • Bacterical chromosomal DNA replication requires assembly of a primosome responsible for primer synthesis and continuous unwinding of double-stranded DNA at the replication fork. In E coli, there are at least two types of distinct primosomes that have been identified in vitro. One that is assembled at oriC is dependent on DnaA protein, and the other that depends on PriA protein is assembled at n’-pas sequence (primosome assembly site). These two primosomes, although independent of each other, are co-required for the viability of cells. PriB is one of the primosomal proteins (PriA, PriB, PriC, DnaT, DnaC and DnaB), and it plays an essential role in both PriA- and DnaA-dependent pathways. In this study, the Escherichia coli PriB protein has been overexpressed and purified. There different crystal forms of PriB have been obtained by using various types of crystallization methods. The crystals of PriB (Native) belong to the tetragonal space group P43212 or P41212 with cell dimension a=b=53.348 Å , c=139.664 Å , α=β=γ=90°. The crystal of PriB (Se-Met) belong to the trigonal space group P3121 with cell dimension a=b=57.78 Å , c=83.10 Å , α=β=90° γ=120°。The crystal of PriB (Hg) belong to the orthorhombic space group P212121 with cell dimension a=39.14 Å , b=58.21 Å , c=100.24 Å , α=β=γ=90°。 These crystals can be diffracted to at least 2.5 Å, appropriate for high-resolution structure determination. We solved the phase problem by using MAD method from PriB (Hg) crystal. The structure of PriB is similar to that of single-stranded DNA binding protein (SSB), even though they share only 19% amino acid sequence identity. The gel-mobility shift assay also shows PriB could bind to 35mer dT (ssDNA). transcription of these receptors.