POLY(A) POLYMERASE AND FIP1 PEPTIDE COMPLEX

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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.