Riboswitch structure: an internal residue mimicking the purine ligand

Abstract

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.

Multiple conformational states of riboswitches fine-tune gene regulation

“…Most prominent changes are restricted to the ligand-binding pocket. In the crystallized apo-state it is often occupied by internal residues, a behavior that is also reported for mutants of the purine riboswitches, where an remote nucleotide is able to mimic the purine ligand in the ligand-free state [32].…”

Comparison of a PreQ1 Riboswitch Aptamer in Metabolite-bound and Free States with Implications for Gene Regulation

“…2D versus supplemental Fig. S3B) also has been observed for free-state crystal structures of the lysine riboswitch (9, 10) and the U65C mutant of the pbuE adenine riboswitch (62). Because such conformations are ill suited to ligand binding, it is most plausible that they are in equilibrium with open states in solution (11).…”

Molecular insights into the ligand-controlled organization of the SAM-I riboswitch

“…SHAPE, 2AP fluorescence and in vitro transcription assays indicate that the presence of the P4 stem is associated with a different organization of the SAM-I aptamer that performs more efficiently at the levels of ligand binding and transcription regulation. Thus, as previously suggested for key nucleotides of purine riboswitches 39,40 , the presence of the P4 stem may have been selected to restrain the ligand-free aptamer in a conformation compatible with ligand binding. Notably, preliminary experiments show that a similar situation exists in the lysine riboswitch in which the highly variable P5 stem influences the riboswitch activity 41 .…”