Several essential trans-acting factors are involved in the processing of ribosomal RNAs. For example, 14 DEAD-box proteins play an essential role in rRNA folding. The function of DEAD-box proteins is often tightly coupled to the conformational changes that occur during ATP hydrolysis. Unlike most proteins, Roklp, a Saccharomyces cerevisiae DEAD-box, is capable of hydrolyzing ATP in the absence of RNA. Rok1p contains two Rec-A like domains that primarily bind ATP and RNA. In addition, N-terminal and C-terminal peripheral domains (NTD and CTD) play an important role in rRNA recruitment through the binding of Rrp5. To investigate ATP binding, especially on the dissociation rate of ATP, wt-Rok1p was recombinantly purified and biochemically analyzed. Stopped-flow spectroscopy experiments were performed by monitoring the dissociation of TNP-ATP, a fluorescent ATP analog, in the presence and absence of RNA. Dissociation rates were additionally evaluated at various temperatures ranging from 16 oC to 40 oC. TNP-ATP fluorescence quenching as a function of time resulted in a double exponential regression, signifying a two-step dissociation mechanism (kfast and kslow). The predominant slow dissociation step is consistent with a required conformational change prior to dissociation. The fast rate has a greater dependence on temperature which decreases with the addition of RNA. A comparative analysis will be performed with other DEAD-co proteins.

Kinetic analysis of ATP dissociation from wt-Rok1p

2019 Spring National Meeting

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