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J. Biol. Chem., Vol. 276, Issue 30, 28075-28082, July 27, 2001

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The Length, Phosphorylation State, and Primary Structure of the RNA Polymerase II Carboxyl-terminal Domain Dictate Interactions with mRNA Capping Enzymes^*

Yi Pei, Stéphane Hausmann, C. Kiong Ho, Beate Schwer^§, and Stewart Shuman^¶

From the  Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021 and the ^§ Microbiology and Immunology Department, Weill Medical College of Cornell University, New York, New York 10021

The carboxyl-terminal domain (CTD) of elongating RNA polymerase II serves as a landing pad for macromolecular assemblies that regulate mRNA synthesis and processing. The capping apparatus is the first of the assemblies to act on the nascent pre-mRNA and the one for which binding of the catalytic components is most clearly dependent on CTD phosphorylation. The present study highlights a distinctive strategy of cap targeting in fission yeast whereby the triphosphatase (Pct1) and guanylyltransferase (Pce1) enzymes of the capping apparatus do not interact physically with each other (as they do in budding yeast and metazoans), but instead bind independently to the phosphorylated CTD. In vivo interactions of Pct1 and Pce1 with the CTD in a two-hybrid assay require 12 and 14 tandem repeats of the CTD heptapeptide, respectively. Pct1 and Pce1 bind in vitro to synthetic CTD peptides containing phosphoserine uniquely at position 5 or doubly at positions 2 and 5 of each of four tandem YSPTSPS repeats, but they bind weakly (Pce1) or not at all (Pct1) to a peptide containing phosphoserine at position 2. These results illustrate how remodeling of the CTD phosphorylation array might influence the recruitment and dissociation of the capping enzymes during elongation. But how does the CTD structure itself dictate interactions with the RNA processing enzymes independent of the phosphorylation state? Using CTD-Ser5 phosphopeptides containing alanine substitutions at other positions of the heptad, we define essential roles for Tyr-1 and Pro-3 (but not Thr-4 or Pro-6) in the binding of Schizosaccharomyces pombe guanylyltransferase. Tyr-1 is also essential for binding and allosteric activation of mammalian guanylyltransferase by CTD Ser5-PO₄, whereas alanine mutations of Pro-3 and Pro-6 reduce the affinity for the allosteric CTD-binding site. These are the first structure-activity relationships deduced for an effector function of the phosphorylated CTD.

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