The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of three enzymes: RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-7) methyltransferase. Our goal is to understand the mechanisms and structures of the cap-forming enzymes and the means by which capping is coupled to transcription. We have shown that the guanylyltransferase (GTase) component of the capping apparatus is directed to nascent pre-mRNAs by binding to the phosphorylated carboxyl-terminal domain (CTD) of the largest subunit of Pol I1. The RNA triphosphatase (RTPase) component may either be chaperoned to the CTD-PO4by physical association with the GTase (as in metazoans and S. cerevisiae) or RTPase may bind to CTD-PO4 directly (as in the fission yeast S. pombe). The capping enzymes discriminate different CTD phosphorylation arrays, suggesting that remodeling of the CTD array by protein kinases and phosphatases can be a means to regulate cotranscriptional mRNA processing. Mammalian and S. pombe capping enzymes also bind to the Pol II elongation factor Spt5, which, in conjunction with other factors, elicits an elongation arrest that is overcome by the protein kinase activity of P-TEFb (Cdk9/cyclinT). HIV Tat protein, which recruits and activates Cdk9/cyclinT during HIV transcription, interacts with the mammalian capping enzyme and stimulates the capping of nascent HIV pre-mRNA. We find that the S. pombe Cdk9 ortholog interacts with the RTPase component of the S. pombe capping apparatus. These observations suggest a transcriptional checkpoint that ensures a temporal window for capping of nascent mRNAs before committing Pol II to processive elongation. This proposal focuses on the interactions of mammalian and S. pombe capping enzymes with their binding partners (CTD-PO4, Spt5, Tat, Cdk9), the mechanism of CTD remodeling by the protein phosphatase Fcpl, and the structural basis for the cap methylation reaction. Insights gained from the proposed studies can be exploited to develop new approaches to antifungal and anti-HIV therapies designed to block capping of the pathogen's mRNAs.
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