Thermodynamic Analysis: The Biphasic Nature of the DEAD-box RNA helicase Rok1p

Carlisle, Robert Miller

Thermodynamic Analysis: The Biphasic Nature of the DEAD-box RNA helicase Rok1p

Persistent URL

http://hdl.handle.net/10456/15730

Author(s)

Carlisle, Robert Miller

Date Issued

May 1, 2011

Abstract

RNA must fold into distinct and dynamic structures in order to function in the cell. In many cases, proteins are recruited to aid in RNA folding and refolding. For example, DEAD-box proteins are putative RNA helicases that utilize the energy of ATP binding and hydrolysis to disrupt hydrogen bonding in RNA. A large majority of DEAD-box proteins exhibit a substantial enhancement in ATP hydrolysis activity upon RNA binding. Stimulation of ATPase activity is facilitated by the conversion of a disorder to an ordered ATP binding site. However, Rok1p, a DEAD-box protein associated with early ribosomal assembly in S. cerevisiae, has proven to be RNA independent. This observation suggests that the protein is capable of adopting an ordered conformation in the absence of RNA binding. An Erying analysis was performed by monitoring the ATPase activity over a range of 10°C to 38°C to determine the thermodynamic parameters of the transition state in an effort to understand the disordered/ordered nature of Rok1p. These experiments showed that the RNA independent activity of Rok1p dramatically decreases as the temperature increases due to the entropic component of the protein. Our analyses support the hypothesis that Rok1p takes part in a disordered-ordered equilibrium; where, at low temperatures the protein favors the ordered conformation. The data also support the hypothesis that in the presence of RNA, Rok1p exists in two distinct catalytically competent conformations which have two separate rate limiting steps. A similar analysis was performed with Dbp3p, a DEAD-box protein associated with late ribosomal assembly in S. cerevisiae, to determine if these behaviors are general characteristics of DEAD-box proteins. It appears that in the absence of RNA, Dbp3p does not take part in a disordered-ordered equilibrium like Rok1p; rather it favors the open disordered state. This suggests that in the absence of RNA, the disordered-ordered equilibrium is unique to Rok1p. Further analysis of Dbp3p in the presence of RNA is required before it can be determined if the biphasic nature of Rok1p is common to all DEAD-box proteins.

Honors

Biochemistry, 2011

First Reader(s)

Garcia, Ivelitza