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Genetic analysis of polynucleotide phosphorylase structure and functions

机译:多核苷酸磷酸化酶结构和功能的遗传分析

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Polynucleotide phosphorylase (PNPase) is a phosphate-dependent 3' to 5' exonuclease widely diffused among bacteria and eukaryotes. The enzyme, a homotrimer, can also be found associated with the endonuclease RNase E and other proteins in a heteromultimeric complex, the RNA degradosome. PNPase negatively controls its own gene (pnp) expression by destabilizing pnp mRNA. A current model of autoregulation maintains that PNPase and a short duplex at the 5'-end of pnp mRNA are the only determinants of mRNA stability. During the cold acclimation phase autoregulation is transiently relieved and cellular pnp mRNA abundance increases significantly. Although PNPase has been extensively studied and widely employed in molecular biology for about 50 years, several aspects of structure—function relationships of such a complex protein are still elusive. In this work, we performed a systematic PCR mutagenesis of discrete pnp regions and screened the mutants for diverse phenotypic traits affected by PNPase. Overall our results support previous proposals that both first and second core domains are involved in the catalysis of the phosphorolytic reaction, and that both phosphorolytic activity and RNA binding are required for autogenous regulation and growth in the cold, and give new insights on PNPase structure—function relationships by implicating the α-helical domain in PNPase enzymatic activity.
机译:多核苷酸磷酸化酶(PNPase)是磷酸依赖性的3'至5'核酸外切酶,广泛分布于细菌和真核生物之间。还可以在异多聚体复合物RNA降解体中发现一种酶,即同三聚体酶,与核酸内切酶RNase E和其他蛋白质相关。 PNPase通过使pnp mRNA不稳定来负控制其自身基因(pnp)的表达。当前的自动调节模型认为,PNPase和pnp mRNA 5'端的短双链体是mRNA稳定性的唯一决定因素。在冷驯化阶段,自动调节会暂时解除,并且细胞pnp mRNA的丰度会大大增加。尽管PNPase已被广泛研究并在分子生物学中广泛应用了大约50年,但这种复杂蛋白质的结构-功能关系的几个方面仍然难以捉摸。在这项工作中,我们对离散的pnp区进行了系统的PCR诱变,并筛选了受PNPase影响的多种表型性状的突变体。总体而言,我们的结果支持先前的提议,即第一和第二核心域均参与磷酸分解反应的催化,并且在低温下自发调节和生长都需要磷酸分解活性和RNA结合,并提供有关PNPase结构的新见解-通过牵涉PNPase酶活性的α-螺旋结构域的功能关系。

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