Identification of phosphorylation sites in the repetitive carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The largest subunit of eukaryotic RNA polymerase II contains a carboxyl-terminal domain (CTD) which is comprised of repetitive heptapeptides with a consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We demonstrate here that the mouse CTD expressed in and purified from Escherichia coli can be phosphorylated in vitro by a p34cdc2/CDC28-containing CTD kinase from mouse ascites tumor cells. The product of this reaction, a phosphorylated form of the CTD, contains phosphoserine and phosphothreonine, but not phosphotyrosine. The same phosphoamino acid content is observed in the in vivo phosphorylated CTD from a mouse cell line. Synthetic peptides with naturally occurring non-consensus heptapeptide sequences can also be phosphorylated by CTD kinase in vitro. Phosphoamino acid analysis of these non-consensus heptapeptides together with direct sequencing of a phosphorylated heptapeptide reveals that serines (or threonines) at positions two and five are the sites phosphorylated by mouse CTD kinase. Thus, the -Ser(Thr)-Pro- motif common to p34cdc2/CDC28-containing protein kinases is the recognition site for mouse CTD kinase.     

The length, phosphorylation state, and primary structure of the RNA polymerase II carboxyl-terminal domain dictate interactions with mRNA capping enzymes

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.     

Rsp5 WW domains interact directly with the carboxyl-terminal domain of RNA polymerase II

RSP5 is an essential gene in Saccharomyces cerevisiae and was recently shown to form a physical and functional complex with RNA polymerase II (RNA pol II). The amino-terminal half of Rsp5 consists of four domains: a C2 domain, which binds membrane phospholipids; and three WW domains, which are protein interaction modules that bind proline-rich ligands. The carboxyl-terminal half of Rsp5 contains a HECT (homologous to E6-AP carboxyl terminus) domain that catalytically ligates ubiquitin to proteins and functionally classifies Rsp5 as an E3 ubiquitin-protein ligase. The C2 and WW domains are presumed to act as membrane localization and substrate recognition modules, respectively. We report that the second (and possibly third) Rsp5 WW domain mediates binding to the carboxyl-terminal domain (CTD) of the RNA pol II large subunit. The CTD comprises a heptamer (YSPTSPS) repeated 26 times and a PXY core that is critical for interaction with a specific group of WW domains. An analysis of synthetic peptides revealed a minimal CTD sequence that is sufficient to bind to the second Rsp5 WW domain (Rsp5 WW2) in vitro and in yeast two-hybrid assays. Furthermore, we found that specific "imperfect" CTD repeats can form a complex with Rsp5 WW2. In addition, we have shown that phosphorylation of this minimal CTD sequence on serine, threonine and tyrosine residues acts as a negative regulator of the Rsp5 WW2-CTD interaction. In view of the recent data pertaining to phosphorylation-driven interactions between the RNA pol II CTD and the WW domain of Ess1/Pin1, we suggest that CTD dephosphorylation may be a prerequisite for targeted RNA pol II degradation.     

Structural studies of a synthetic peptide derived from the carboxyl-terminal domain of RNA polymerase II

The conformation of the repeating heptapeptide unit of the carboxyl-terminal domain of RNA Polymerase II, Y¹S²P³T⁴S⁵P⁶S⁷ has been examined using nuclear magnetic resonance spectroscopy and circular dichroism. Nuclear Overhauser effects and CD spectra for the synthetic 56-residue peptide H₂N-(S²P³T⁴S⁵P⁶S⁷Y^l)₈-COOH in water indicate that the peptide is largely unordered. A small population of folded molecules is observed to contain β-turns located at Ser²-Pro³-Thr⁴-Ser⁵ (SPTS) and Ser⁵-Pro⁶-Ser⁷-Tyr¹ (SPSY). CD and NMR results in 90% TFE also indicate an equilibrium population of structures, but the fraction of turns is higher. Similarities of nuclear Overhauser effects in water and in 90% TFE suggest that the structures in TFE are biologically relevant. Based on these observations, the average structure of a single conformer of the heptapeptide repeat in 90% TFE was obtained by a distance geometry-simulated annealing method, using distance restraints extracted from nuclear Overhauser data. NMR spectra of the 56-mer show signals corresponding to only one repeat indicating that each repeat is in an identical environment. Thus it is possible to obtain an average structure of the heptapeptide repeat from NOE data on the 56-mer. Twenty-seven final structures were calculated and the root mean square deviations between the 27 structure and the mean coordinates was 1.52 Å for the backbone and 2.2 Å for all nonhydrogen atoms. The heptapeptide repeat consists of two overlapping β-turns which are potentially stabilized by hydrogen bonds. The hydroxyl side chains of Ser², Ser⁵, Thr⁴, and Ser⁷ all appear to be equally exposed for potential phosphorylation. The tyrosyl side chain of each repeat is folded inwards to the backbone and can potentially hydrogen bond to the carbonyl oxygen of the tyrosine in the preceding repeat. Iteration of the average structure of the heptapeptide repeat results in a model of the carboxyl-terminal domain with a regular but unusual secondary structure consisting of a series of staggered β-turns. © 1995 Wiley-Liss, Inc.     

Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II can be phosphorylated by a p34cdc2/CDC28-containing CTD kinase. Phosphorylated serine (or threonine) is located at positions 2 and 5 in the repetitive heptapeptide consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. We show here that phosphorylation of the mouse CTD retards its electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels in a way similar to that observed for the II0 form of the largest subunit of RNA polymerase II phosphorylated in vivo. At the maximum level of phosphorylation by CTD kinase in vitro, there are 15-20 phosphates evenly distributed among the 52 heptapeptide repeats that comprise the mouse CTD. Gel filtration chromatography and sucrose gradient ultracentrifugation analyses indicate that phosphorylation induces a dramatic conformational change in the CTD with the phosphorylated form adopting a far more extended structure than the unphosphorylated CTD.     

酵母RNA聚合酶II羧基端结构域激酶CTDK-I及其亚基的结构与功能

RNA聚合酶Ⅱ最大亚基Rpb1的羧基端结构域（carboxyl-terminal repeat domain,CTD）是RNA聚合酶Ⅱ发挥转录延伸功能所必需的,对其执行精确的转录调节功能至关重要。酵母细胞周期蛋白依赖性激酶CTDK-I（carboxyl-terminal repeat domain kinase,CTDK-I）由CTK1、CTK2和CTK3组成,作用于RNA聚合酶Ⅱ羧基端结构域,动态磷酸化CTD的七肽重复序列（YSPTSPS）来调控转录和翻译。酵母中的特异性蛋白CTK3与特殊的细胞周期蛋白CTK2结合形成异二聚体,再与CTDK-I的催化亚基CTK1结合以调节其活性。CTK1作为细胞周期蛋白CDK（cyclin dependent kinase,CDK）的同源蛋白,其结构与功能的研究可拓展人们对CDK蛋白家族的认识;CTK2-CTK3复合物对CTK1调控机制的研究也可为细胞周期蛋白抑制剂的研发提供新的思路。本文简述了酵母CTDK-I的功能特点及其亚基的结构与功能以及亚基间的相互作用,并展望了CTDK-I复合物的研究前景。