Regonesi Maria Elena, Briani Federica, Ghetta Andrea, Zangrossi Sandro, Ghisotti Daniela, Tortora Paolo, Dehò Gianni
Dipartimento di Biotecnologie e Bioscienze, Università degli Studi di Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy.
Nucleic Acids Res. 2004 Feb 12;32(3):1006-17. doi: 10.1093/nar/gkh268. Print 2004.
Polynucleotide phosphorylase (PNPase), a 3' to 5' exonuclease encoded by pnp, plays a key role in Escherichia coli RNA decay. The enzyme, made of three identical 711 amino acid subunits, may also be assembled in the RNA degradosome, a heteromultimeric complex involved in RNA degradation. PNPase autogenously regulates its expression by promoting the decay of pnp mRNA, supposedly by binding at the 5'-untranslated leader region of an RNase III-processed form of this transcript. The KH and S1 RNA-binding domains at the C-terminus of the protein (amino acids 552-711) are thought to be involved in pnp mRNA recognition. Here we show that a G454D substitution in E.coli PNPase impairs autogenous regulation whereas it does not affect the catalytic activities of the enzyme. Although the mutation maps outside of the KH and S1 RNA-binding domains, analysis of the mutant protein revealed a defective RNA binding, thus suggesting that other determinants may be involved in PNPase-RNA interactions. The mutation also caused a looser association with the degradosome and an abnormal electrophoretic mobility in native gels. The latter feature suggests an altered structural conformation of PNPase, which may account for the properties of the mutant protein.
多核苷酸磷酸化酶（PNPase）由pnp基因编码，是一种3'至5'核酸外切酶，在大肠杆菌RNA降解中起关键作用。该酶由三个相同的711个氨基酸亚基组成，也可能组装在RNA降解体中，这是一种参与RNA降解的异源多聚体复合物。PNPase通过促进pnp mRNA的降解来自动调节其表达，推测是通过结合在该转录本经核糖核酸酶III加工形式的5'非翻译前导区。蛋白质C末端（氨基酸552 - 711）的KH和S1 RNA结合结构域被认为参与pnp mRNA的识别。在这里，我们表明大肠杆菌PNPase中的G454D替代损害了自动调节，而不影响该酶的催化活性。尽管该突变位于KH和S1 RNA结合结构域之外，但对突变蛋白的分析显示其RNA结合存在缺陷，因此表明其他决定因素可能参与PNPase与RNA的相互作用。该突变还导致与降解体的结合更松散，并且在天然凝胶中电泳迁移率异常。后一特征表明PNPase的结构构象发生了改变，这可能解释了突变蛋白的特性。
