LeCuyer K A, Behlen L S, Uhlenbeck O C
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1995 Aug 22;34(33):10600-6. doi: 10.1021/bi00033a035.
An RNA binding assay measuring cooperative protein binding has been used to evaluate the effects of mutations in the MS2 phage coat protein expected to disrupt capsid assembly. By using the crystal structure of the virus as a guide, six different mutations in the FG loop structure were selected in which hydrophobic residues were replaced with charged residues. Most of these proteins form capsids in Escherichia coli, but not in an in vitro assembly assay, suggesting that interdimer interactions are weaker than wild type. These mutant proteins reduce the free energy of cooperative protein binding to a double-hairpin RNA from its wild-type value of -1.9 kcal/mol. Several of the variants that have large effects on cooperativity have no effect on RNA affinity, suggesting that protein-RNA interactions can be affected independently of dimer-dimer interactions. The V75E;A81G protein, which shows no measurable cooperativity, binds operator RNA equally well as the wild-type protein under a variety of buffer conditions. Because this protein also exhibits similar specificity for variant RNA sequences, it will be useful for studying RNA binding properties independent of capsid assembly.
一种用于测量协同蛋白结合的RNA结合试验已被用于评估预期会破坏衣壳组装的MS2噬菌体外壳蛋白中的突变效应。以病毒的晶体结构为指导，在FG环结构中选择了六个不同的突变，其中疏水残基被带电残基取代。这些蛋白中的大多数在大肠杆菌中形成衣壳，但在体外组装试验中则不然，这表明二聚体间相互作用比野生型弱。这些突变蛋白将协同蛋白与双发夹RNA结合的自由能从其野生型值-1.9千卡/摩尔降低。几个对协同性有很大影响的变体对RNA亲和力没有影响，这表明蛋白质-RNA相互作用可以独立于二聚体-二聚体相互作用而受到影响。V75E;A81G蛋白没有可测量的协同性，在各种缓冲条件下与操纵子RNA的结合能力与野生型蛋白相当。由于该蛋白对变异RNA序列也表现出类似的特异性，因此它将有助于独立于衣壳组装来研究RNA结合特性。
