1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA
2. Department of Molecular, Cellular and Developmental Biology and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520, USA

Correspondence to: Alexander Serganov¹Dinshaw J. Patel¹ Correspondence and requests for materials should be addressed to A.S. (Email: serganoa@mskcc.org) or D.J.P. (Email: pateld@mskcc.org).Coordinates of the X-ray structure of the TPP-bound riboswitch have been deposited in the RCSB Protein Data Bank under accession code 2GDI.

Abstract

Riboswitches are metabolite-sensing RNAs, typically located in the non-coding portions of messenger RNAs, that control the synthesis of metabolite-related proteins^{1, 2, 3}. Here we describe a 2.05 Å crystal structure of a riboswitch domain from the Escherichia coli thiM mRNA⁴ that responds to the coenzyme thiamine pyrophosphate (TPP). TPP is an active form of vitamin B₁, an essential participant in many protein-catalysed reactions⁵. Organisms from all three domains of life^{6, 7, 8, 9}, including bacteria, plants and fungi, use TPP-sensing riboswitches to control genes responsible for importing or synthesizing thiamine and its phosphorylated derivatives, making this riboswitch class the most widely distributed member of the metabolite-sensing RNA regulatory system. The structure reveals a complex folded RNA in which one subdomain forms an intercalation pocket for the 4-amino-5-hydroxymethyl-2-methylpyrimidine moiety of TPP, whereas another subdomain forms a wider pocket that uses bivalent metal ions and water molecules to make bridging contacts to the pyrophosphate moiety of the ligand. The two pockets are positioned to function as a molecular measuring device that recognizes TPP in an extended conformation. The central thiazole moiety is not recognized by the RNA, which explains why the antimicrobial compound pyrithiamine pyrophosphate targets this riboswitch and downregulates the expression of thiamine metabolic genes. Both the natural ligand and its drug-like analogue stabilize secondary and tertiary structure elements that are harnessed by the riboswitch to modulate the synthesis of the proteins coded by the mRNA. In addition, this structure provides insight into how folded RNAs can form precision binding pockets that rival those formed by protein genetic factors.

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