A dual interface determines the recognition of RNA polymerase II by RNA capping enzyme.
RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5'-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes.
Suh
Man-Hee
MH
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Meyer
Peter A
PA
Gu
Meigang
M
Ye
Ping
P
Zhang
Mincheng
M
Kaplan
Craig D
CD
Lima
Christopher D
CD
Fu
Jianhua
J
D000409:Alanine / Q000737:chemistry; D001665:Binding Sites; D015966:Gene Expression Regulation, Fungal; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry*; D012315:RNA Caps / Q000737:chemistry*; D012319:RNA Polymerase II / Q000737:chemistry*; D012322:RNA Precursors / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014158:Transcription, Genetic; D014961:X-Ray Diffraction
D012315:RNA Caps; D012322:RNA Precursors; D012313:RNA; D012319:RNA Polymerase II; D000409:Alanine