Structural distinctions between NAD+ riboswitch domains 1 and 2 determine differential folding and ligand binding

Abstract

Riboswitches are important gene regulatory elements frequently encountered in bacterial mRNAs. The recently discovered nadA riboswitch contains two similar, tandemly arrayed aptamer domains, with the first domain possessing high affinity for nicotinamide adenine dinucleotide (NAD+). The second domain which comprises the ribosomal binding site in a putative regulatory helix, however, has withdrawn from detection of ligand-induced structural modulation thus far, and therefore, the identity of the cognate ligand and the regulation mechanism have remained unclear. Here, we report crystal structures of both riboswitch domains, each bound to NAD+. Furthermore, we demonstrate that ligand binding to domain 2 requires significantly higher concentrations of NAD+ (or ADP retaining analogs) compared to domain 1. Using a fluorescence spectroscopic approach, we further shed light on the structural features which are responsible for the different ligand affinities, and describe the Mg2+-dependent, distinct folding and pre-organization of their binding pockets. Finally, we speculate about possible scenarios for nadA RNA gene regulation as a putative two-concentration sensor module for a time-controlled signal that is primed and stalled by the gene regulation machinery at low ligand concentrations (domain 1), and finally triggers repression of translation as soon as high ligand concentrations are reached in the cell (domain 2).

Impact of 3-deazapurine nucleobases on RNA properties

“…Additionally, deaza-nucleobase substitutions have identified crucial metal binding sites that are responsible for structuring of riboswitch aptamers and ribozyme active sites ( 13 , 67 ), as well as have disclosed key coordination sites of hydrated Mg 2+ ions that critically participate in proton transfer to release the 5′- O leaving group in the course of phosphodiester cleavage ( 15 , 16 , 68 , 69 ). Recent prominent examples refer to the NAD + class-I riboswitch ( 68 ) and the pistol ribozyme ( 15 , 16 ). In pistol RNA, a highly conserved guanine (G33) interacts with a Mg 2+ cation through innersphere coordination with its N7 atom.…”

“…In the NAD + class-I riboswitch, a highly conserved adenine (A10) exhibits N7-innersphere coordination to a Mg 2+ cation that additionally coordinates to the oxygen atom of a sequence-distant phosphate backbone unit and thus molds a critical part of the binding pocket. Replacement of this adenine by c 7 A abolishes binding to NAD + ( 68 ).…”

Overview of Methods for Large-Scale RNA Synthesis

“…The lack of a sequence requirement has resulted in the HDV ribozyme being widely used by structural researchers in the preparation of RNAs for crystallization. Examples include domain IV of the ribosomal 4.5S RNA from E. coli (PDB: 1DUH), the nadA riboswitch (PDB: 7D82), and the constitutive decay element (CDE1) from human 3′ untranslated region (PDB: 6XWW) [118,129,130].…”

Highlights from the ‘Noncoding RNA world: From Mechanism to Therapy’ conference, July 21-23, 2021

“…For the former, Dr Ren illustrated the mechanism of pepper activation and shed light onto its application. As regards the latter and based on the tertiary structure, Dr Ren founded the regulatory model of the NAD+1 riboswitch (17).…”