Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity

• Published in: Nucleic acids research Vol. 43; no. 5; pp. 2968 - 2979
• Main Authors: Yates, Luke A, Durrant, Benjamin P, Fleurdépine, Sophie, Harlos, Karl, Norbury, Chris J, Gilbert, Robert J C
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 11.03.2015
• Subjects: Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Models, Molecular; Mutation; Nucleotidyltransferases - chemistry; Nucleotidyltransferases - genetics; Nucleotidyltransferases - metabolism; Protein Structure, Tertiary; RNA Nucleotidyltransferases - chemistry; RNA Nucleotidyltransferases - genetics; RNA Nucleotidyltransferases - metabolism; RNA, Fungal - genetics; RNA, Fungal - metabolism; Rotation; Schizosaccharomyces - enzymology; Schizosaccharomyces - genetics; Schizosaccharomyces pombe; Schizosaccharomyces pombe Proteins - chemistry; Schizosaccharomyces pombe Proteins - genetics; Schizosaccharomyces pombe Proteins - metabolism; Structural Biology; Uridine Triphosphate - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkv122

Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.74 Å) crystal structure of Cid1 that provides detailed evidence for uracil selection via the dynamic flipping of a single histidine residue. We also describe a novel closed conformation of the enzyme that may represent an intermediate stage in a proposed product ejection mechanism. The structural insights gained, combined with normal mode analysis and biochemical studies, demonstrate that the plasticity of Cid1, particularly about a hinge region (N164-N165), is essential for catalytic activity, and provide an explanation for its distributive uridylyl transferase activity. We propose a model clarifying observed differences between the in vitro apparently processive activity and in vivo distributive monouridylylation activity of Cid1. We suggest that modulating the flexibility of such enzymes-for example by the binding of protein co-factors-may allow them alternatively to add single or multiple uridyl residues to the 3' termini of RNA molecules.