【作者】 王维维；

【导师】 施蕴渝； 吴季辉；

【作者基本信息】 中国科学技术大学 ， 结构生物学， 2012， 博士

【摘要】 Hfq是细菌里重要的转录后调节因子。它能够帮助非编码sRNA与目标mRNA的配对。Hfq蛋白对于DsrA介导的rpoS mRNA在低温下的翻译激活是必须的。rpoS编码的稳态sigma因子σS是大肠杆菌中一般应激响应的核心控制因子。大肠杆菌内的Hfq蛋白是一个102个残基（11.2kDa）的Sm家族RNA分子伴侣蛋白。N端的1⁶5号残基（Hfq65）组成保守的Sm结构域而C端的部分（66¹02号残基）则是一个没有固定结构但是却在功能上十分重要的部分。Sm结构域形成同六聚体圆盘状结构，Hfq65六聚体分子量约43kDa，Hfq全长六聚体分子量约67kDa。圆盘的两个面分别构成能与U-rich单链RNA序列结合的近端和能与A-rich单链RNA序列结合的远端。然而，Hfq蛋白与DsrA相互作用的结构信息还未知。尽管有报道称Hfq有水解ATP的能力，但是ATP结合位点和ATP水解的生物学意义都不清楚。没有严整结构的HfqCTD的折叠属性和发挥功能的机制也是一个未解之谜。我们对Hfq65和HfqCTD进行了核磁共振（NMR）谱的主链归属，并且获得了Hfq65和Hfq全长（HfqFL）蛋白异亮氨酸，亮氨酸和颉氨酸侧链甲基的选择性标记样品的甲基HMQC谱。我们发现，Hfq65和HfqFL的1H-15NHSQC以及¹H-¹³C甲基HMQC都有显著的不同。HfqCTD和Hfq65的相互NMR滴定实验表明这两者之间没有显著的相互作用。我们用Gd（DPA）3作为顺磁标签，在PRE实验中获得了初步的结果显示在HfqFL中，HfqCTD可能与Hfq65之间有瞬时的相互作用。我们还获得了大肠杆菌Hfq与DsrA上面的主要Hfq识别区域AU₆A以及ADP的三元复合物晶体结构，以及Hfq与ADP的二元复合物晶体结构。AU₆A结合到一个Hfq的近端和另一个Hfq的远端。ADP结合到远端的嘌呤选择性结合位点并且有一些ADP用磷酸基团与另一个Hfq上近端的保守精氨酸或是谷氨酰胺侧链相互作用。这样的ADP结合模式与之前人们推测的不同。我们在ATP酶活实验中验证了这些与ADP磷酸基团接触的保守的极性残基对于ATP酶活性的重要性。我们发现ATP酶活性会降低Hfq与U-rich序列的结合亲和力。在RNA浓度远高于Hfq浓度的情况下，ATP的存在可以增强Hfq的分子伴侣活性。我们用Hfq和DsrA的片段在溶液核磁共振以及荧光偏振实验中获得的结果验证了多个Hfq六聚体在与DsrA结合时的合作现象。用Hfq全长和DsrA全长进行的荧光共振能量转移实验也支持Hfq全长在与DsrA全长相互作用时需要多个六聚体的相互合作。我们基于以上的结果，提出一个Hfq通过多个六聚体之间的合作来完成其功能的猜想。更多还原

【Abstract】 Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA andtarget mRNA. Hfq mediates DsrA dependent translational activation of rpoS mRNA at lowtemperatures. rpoS encodes the stationary phase sigma factorσSwhich is the central regulatorin general stress response. E.coli Hfq is a 102 amino acid residue (11.2kDa) bacterial SmRNA chaperon protein. The N-terminal 1~65 amino acid residues of Hfq protein (Hfq65)comprise a conserved Sm domain and the C-terminal part 66~102aa (HfqCTD) isnon-structured but functional essential. The Sm domain forms homo-hexameric ring structure.The molecular weights for Hfq65 and Hfq full length hexamers are 43kDa and 67kDarespectively. Distinct RNA binding sites with specificity for A-rich and U-rich ssRNArespectively has been identified on either side of the ring. However, structural information onHfq-DsrA interaction is not yet available. Though Hfq is reported to hydrolyze ATP, neitherthe ATP binding site nor the biological significance of ATP hydrolysis is known. Moreover,the structure and function of the flexible tail of HfqCTD remains an enigma. We haveassigned backbone resonances for Hfq65 and HfqCTD and acquired I, L, V methyl selectivelylabeled Hfq65 and Hfq full length (HfqFL) HMQC spectra. We found that Hfq65 and HfqFLbehaves very differently on both1H-15N HSQC and1H-13C methyl HMQC. Mutual NMRtitrations of HfqCTD with Hfq65 indicate non-prominent interaction between these twoconstructs. We have used Gd(DPA)3as paramagnetic probe and acquired preliminaryparamagnetic relaxation enhancement (PRE) results suggesting transient interactions betweenHfq65 and HfqCTD in HfqFL protein. We have also acquired ternary crystal complexstructure of E.coli Hfq bound to a major Hfq recognition region on DsrA, AU6A, togetherwith ADP and crystal complex structure of Hfq bound to ADP. AU6A binds to proximal anddistal sides of two Hfq hexamers. ADP bind to purine selective site on distal side and contactconserved arginine or glutamine residues on proximal side of another hexamer. This bindingmode is different from previously postulated. In ATPase activity assays, we confirmed thatthe residues that make contact with ADP is involved in the ATPase activity. We found thatATPase activity decreases the binding affinity of Hfq to U-rich sequence. In cases when Hfqconcentration is much lower than RNA concentration, the presence of ATP can increase thechaperoning ability of Hfq. The cooperation of two different Hfq hexamers upon nucleic acidbinding in solution is verified by fluorescence polarization and solution NMR experimentsusing fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with fulllength Hfq and DsrA also support cooperation of Hfq hexamers upon DsrA binding. Weproposed a plausible model for Hfq mechanism in which cooperation between multiple Hfqhexamers is required.更多还原