The cellular environment stabilizes adenine riboswitch RNA structure.
There are large differences between the intracellular environment and the conditions widely used to study RNA structure and function in vitro. To assess the effects of the crowded cellular environment on RNA, we examined the structure and ligand binding function of the adenine riboswitch aptamer domain in healthy, growing Escherichia coli cells at single-nucleotide resolution on the minute time scale using SHAPE (selective 2'-hydroxyl acylation analyzed by primer extension). The ligand-bound aptamer structure is essentially the same in cells and in buffer at 1 mM Mg(2+), the approximate Mg(2+) concentration we measured in cells. In contrast, the in-cell conformation of the ligand-free aptamer is much more similar to the fully folded ligand-bound state. Even adding high Mg(2+) concentrations to the buffer used for in vitro analyses did not yield the conformation observed for the free aptamer in cells. The cellular environment thus stabilizes the aptamer significantly more than does Mg(2+) alone. Our results show that the intracellular environment has a large effect on RNA structure that ultimately favors highly organized conformations.
Department of Chemistry, ‡Department of Biochemistry and Biophysics, and §Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599-3290, United States.
Jillian
J
Tyrrell
Jennifer L
JL
McGinnis
Kevin M
KM
Weeks
Gary J
GJ
Pielak
D000227:Adenine Nucleotides / Q000737:chemistry*; D052157:Aptamers, Nucleotide / Q000737:chemistry; D001483:Base Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D042541:Intracellular Space / Q000737:chemistry / Q000502:physiology*; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009994:Osmolar Concentration; D020871:RNA Stability / Q000187:drug effects / Q000502:physiology*; D012329:RNA, Bacterial / Q000737:chemistry*; D058928:Riboswitch / Q000187:drug effects / Q000502:physiology*
D000227:Adenine Nucleotides; D052157:Aptamers, Nucleotide; D012329:RNA, Bacterial; D058928:Riboswitch; D008274:Magnesium