POLY(A) POLYMERASE AND FIP1 PEPTIDE COMPLEX

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 在真核生物中，被称为poly（A）尾的非编码mRNA延伸作为分子标记， 柄，与核输出、翻译和mRNA降解机制相互作用， 并强烈影响mRNA的稳定性和翻译效率。这些尾部由多蛋白复合物（酵母中约14种蛋白质）形成，该复合物识别新生转录物的3'非翻译区中的信号序列，并在由这些信号决定的位点切割前体RNA。这种裂解的前mRNA作为引物，通过3 '末端加工复合物的催化亚基，聚（A）-聚合酶（酵母中的Pap 1）添加聚（A）尾。虽然分离的Pap 1酶在体外保留野生型活性水平，但当与其在细胞内组成性连接的复合物的其余部分分离时，它不保留对其RNA靶的特异性或进行动力学。 Fip 1是一种由327个氨基酸组成的酸性蛋白质，被认为是一种支架蛋白，酵母3'切割/聚腺苷酸化复合物的许多组分通过该支架蛋白与聚腺苷酸聚合酶相互作用。它是酵母裂解/聚腺苷酸化复合物中唯一被证明与聚合酶直接相互作用的组分。Fip 1以纳摩尔亲和力与Pap 1结合，但当进行凝胶过滤色谱时运行异常大，并且被认为在Pap 1上和关闭时都非常延伸。Fip 1的缺失研究表明，80和105之间的区域是酵母活力所必需的。为了更好地理解这些蛋白质之间的相互作用，我们已经形成了Pap 1和Fip 1区域之间的复合物晶体。 通过凝胶电泳和质谱法证实了复合物的形成。 我们预计，综合体的结构将允许我们开始定位其余的 相对于聚合酶的复杂性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. In eukaryotes, the non-coding mRNA extensions known as poly(A) tails serve as molecular handles, which interact with nuclear export, translation and mRNA degradation machinery, and strongly effect mRNA stability and translational efficiency. These tails are formed by a multiprotein complex (~14 proteins in yeast) which recognizes signal sequences in the 3' untranslated region of a nascent transcript and cleaves the precursor RNA at a site determined by these signals. This cleaved pre-mRNA serves as a primer for addition of the poly(A) tail by the catalytic subunit of the 3'-end-processing complex, poly(A)-polymerase (Pap1 in yeast). Though isolated Pap1 enzyme retains wild type levels of activity in vitro, it does not retain specificity for its RNA target or processive kinetics when separated from the rest of the complex to which it is constitutively tethered within the cell. Fip1, an acidic protein of 327 amino acids, is thought to be a scaffolding protein through which many of the components of the yeast 3' cleavage/polyadenylation complex interact with poly(A) polymerase. It is the only component of the yeast cleavage/polyadenylation complex which has been shown to interact directly with the polymerase. Fip1 binds with nanomolar affinity to Pap1, but runs abnormally large when subjected to gel filtration chromatography and is thought to be very extended both on and off Pap1. Deletion studies of Fip1 have shown that the region between 80 and 105 is necessary for yeast viability. In an effort to better understand the interaction of these proteins we've formed crystals of the complex between Pap1 and this region of Fip1. Complex formation has been confirmed by gel electrophoresis and by mass spectrometry. We anticipate that the structure of the complex will allow us to begin to position the rest of the complex with respect to the polymerase.