Polyadenylation is essential for mRNA synthesis and its efficient translation. Regulation of this process can also affect the amount and type of mRNA derived from a gene, and thus becomes part of the cell's repertoire of responses to stimuli governing growth and differentiation. The enzyme poly(A) polymerase (PAP) is responsible for adding adenosines to the mRNA 3' end. For this activity, it needs binding sites to bring its substrates, ATP and an RNA strand, into proximity at its catalytic center. PAP must also have domains to mediate association with specificity factors which guide it to the appropriate 3' ends and modulate its activity so that it processively synthesizes tails of correct length. The mammalian PAP is further regulated by post-translational modification. The purpose of this study is to understand the molecular mechanism of PAP function, using a combination of biochemical, biophysical, and genetic approaches, and the Saccharomyces cerevisiae PAP, Pap1, as our model. First, we will use mutagenesis and kinetic analysis to explore the functional importance of motifs in Pap1 which are similar to ones found in RNases, RNA binding proteins, and other polymerases. This approach will be complemented by structural studies using X0ray diffraction analysis of crystals formed from purified Pap1 alone and in complex with ATP and RNA primer. We will also characterize the interactions of Pap1 with subunits of the PF I polyadenylation factor and explore the mechanisms by which these interactions affect Pap1 activity. Finally, we will ask what cellular factors are responsible for the novel ubiquitination of Pap1 in the G2 portion of the cell cycle and determine how this modification affects Pap1 function. Understanding the organization of important domains in a simple polymerase such as PAP can give insights into how functional motifs were combined or modified through evolution to yield the current spectrum of polymerases and nucleic acid modifying enzymes. The structural and mutational analyses will help us understand how PAP selects its substrates, how it differentiates between substrate and product, and how it advances along the single-stranded poly(A) chains as it synthesis progresses. Knowledge of the function and organization of PAP's enzymatic domains is necessary to understand the consequences of modification and protein/protein interactions which modulate PAP function. Information gained about yeast specificity factors will help us to define evolutionary relationships between polyadenylation factors in yeast and higher eukaryotes and to discern why interactions between some factors have changed while others are more highly conserved.
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| Bard, J; Zhelkovsky, A M; Helmling, S et al. (2000) Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP. Science 289:1346-9 |
