RRM
2009 Rustbelt RNA Meeting
RRM
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Talk abstracts
Abstract:
DEAD-box RNA helicases, which catalyze rearrangements of RNA in many cellular processes, are known to unwind RNA secondary structure by a mechanism distinct from canonical helicases. Rather than translocating along the substrate, DEAD-box helicases directly load on a duplex region and then pry the strands apart in an ATP-dependent fashion. This unwinding mechanism consists of multiple steps including loading of the enzyme to duplex regions, binding of ATP, strand separation, ATP hydrolysis, and product release. How these steps are coordinated in the overall kinetic framework of DEAD-box helicases is not known, and the lack of this information complicates the evaluation of structure function relationships for this important enzyme family. Here we describe the first kinetic framework for duplex unwinding by a DEAD-box RNA helicase, Ded1p from S. cerevisiae. We use a combination of complimentary kinetic approaches, including EMSA and stopped flow fluorescence spectroscopy. We find that for short duplexes, which are completely separated in a single enzyme binding event, unwinding occurs with rate constants significantly greater than the rate constant for ATP turnover, consistent with previous results showing that ATP hydrolysis is not required for strand separation. We also find that ATP binding and formation of an activated enzyme conformation are rate limiting for strand separation of short duplexes. As duplex length and stability increases, non-enzymatic strand dissociation becomes rate limiting, and multiple binding events are required for complete strand separation. Collectively, our data reveal how the stability of duplexes controls unwinding rate constants for longer and more stable duplexes. However, for short and less stable duplexes, which are thought to constitute the majority of RNA structures targeted by DEAD-box proteins, ATP binding limits the overall reaction.
Keywords: DEAD-box helicase, ATP binding, RNA metabolism
