Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
A Structural Method With Single Atom Resolution for Investigating the Secondary Structure of RNA: the Deuterium Kinetic Isotope Effect/Hydroxyl Radical Cleavage Experiment
The hydroxyl radical is widely used as a high-resolution footprinting agent for DNA (1) and RNA (2). The hydroxyl radical abstracts a hydrogen atom from the sugar-phosphate backbone, generating a carbon-based radical, which ultimately leads to a strand break. Substituting deuterium for hydrogen on the ribose results in a kinetic isotope effect if abstraction of that hydrogen atom by the hydroxyl radical leads to a strand break. The deuterium kinetic isotope effect (KIE) correlates well with the solvent accessibility of the deoxyribose hydrogen atoms in DNA (3). RNA, due to its single-stranded nature, adopts a multitude of secondary and tertiary structures. This results in a non-uniform exposure of its backbone, making it a very attractive system to study using the deuterium KIE/hydroxyl radical experiment. We applied this experiment to the sarcin-ricin loop (SRL) RNA molecule, a structurally well-defined component of the large subunit of ribosomal RNA. SRL plays a critical role in the activation of elongation factor Tu (a GTPase) leading to the hydrolysis of GTP during translation, which is speculated to be the reason for its universal conservation (4).
We utilized a one-pot enzymatic protocol to synthesize specifically deuterated ATP and GTP (5). We then use in vitro transcription to incorporate these deuterated nucleotides into the 29-mer SRL RNA molecule. We report the observation of a substantial deuterium kinetic isotope effect on hydroxyl radical cleavage of SRL RNA for dideuteration at the C5' position. This experiment provides direct evidence of abstraction by the hydroxyl radical of hydrogen atoms from the ribose C5' position in RNA. We also observe isotope effects for deuteration at the C4' position, although to a lesser extent. Interestingly, we observed different apparent KIEs for the same deuterated nucleotide at different positions in the SRL sequence, suggesting that the extent of reaction with hydroxyl radical depends on the local structural (and perhaps dynamic) environment. We conclude that the deuterium KIE/hydroxyl radical experiment provides a way to investigate the solvent exposure of the ribose backbone of RNA at the level of single hydrogen atoms.
This research is supported by NSF Award MCB-0843265.
Department of Chemistry