Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease.
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) is a powerful approach for characterizing RNA structure and dynamics at single-nucleotide resolution. However, SHAPE technology is limited, sometimes severely, because primer extension detection obscures structural information for approximately 15 nts at the 5' end and 40-60 nts at the 3' end of the RNA. Moreover, detection by primer extension is more complex than the actual structure-selective chemical interrogation step. Here we quantify covalent adducts in RNA directly by adduct-inhibited exoribonuclease degradation. RNA 2'-O-adducts block processivity of a 3'-->5' exoribonuclease, RNase R, to produce fragments that terminate three nucleotides 3' of the modification site. We analyzed the structure of the native thiamine pyrophosphate (TPP) riboswitch aptamer domain and identified large changes in local nucleotide dynamics and global RNA structure upon ligand binding. In addition to numerous changes that can be attributed to ligand recognition, we identify a single nucleotide bulge register shift, distant from the binding site, that stabilizes the ligand-bound structure. Selective 2'-hydroxyl acylation analyzed by protection from exoribonuclease (RNase-detected SHAPE) should prove broadly useful for facile structural analysis of small noncoding RNAs and for RNAs that have functionally critical structures at their 5' and 3' ends.
Steen
Kady-Ann
KA
Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, USA.
Malhotra
Arun
A
Weeks
Kevin M
KM
D000215:Acylation; D001483:Base Sequence; D020134:Catalytic Domain; D005095:Exoribonucleases / Q000737:chemistry / Q000378:metabolism*; D006878:Hydroxides / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D006878:Hydroxides; D012313:RNA; C031356:hydroxide ion; D005095:Exoribonucleases