Talk abstracts

Talk on Saturday 08:40-09:00am submitted by Timea Gerczei

Mechanistic insights into RNA chaperone activities that are essential for ribosome biogenesis

Timea Gerczei (Department of Biochemistry and Molecular Biology, Rosalind Franklin University), Carl C Correll (Department of Biochemistry and Molecular Biology, Rosalind Franklin University)

Abstract:
RNA chaperone activities are essential for virtually every process containing RNA to facilitate RNA conformational changes and to assist the assembly and disassembly of RNA-RNA and RNA-protein interactions. Despite the importance of RNA chaperones, there are few mechanistic studies that further our understanding of their mode of action. To overcome these difficulties we developed a mechanistic framework to study the formation of two short hybrids between the U3 snoRNA (the RNA part of the small subunit processome) and complementary sites of the pre-rRNA. In a previous study we demonstrated that two proteins, Imp3p and Imp4p, chaperone formation of both interactions. Formation of these two hybrids is essential to initiate small ribosomal subunit biogenesis. Moreover, these hybrids represent prototypes of interactions that require a chaperone.
To further our understanding of RNA chaperone action we developed a kinetic and thermodynamic framework that enables to discriminate between common chaperone mechanisms. First, we developed FRET based assays to show that Imp3p and Imp4p assemble into a ternary complex in an ordered and RNA-dependent manner, consistent with yeast two hybrid data. Second, we measured hybridization rates and duplex affinities for both hybrids. These assays revealed that the ternary complex uses product capture mechanism to facilitate hybridization at one site, thus increasing the stability of an otherwise unstable duplex by a thousand fold. We showed that hybridization at the other site is a two-step process: first, the ternary complex unfolds the U3 stem presumably to expose the relevant bases for hybridization and second, the ternary complex increases the hybridization rate by lowering the free energy of the transition state.
Hybridization rates measured are in agreement with the timeframe these interactions are expected to form in vivo. We propose that our mechanistic framework can be used to decipher the mechanism of other RNA chaperones.

References:
Gérczei, T. and Correll, C.C. (2004) Imp3p and Imp4p mediate an essential U3-pre-rRNA hybridization, presumably to recruit the small subunit processome to the pre-rRNA. Proc Natl Acad Sci U S A 101 (43), 15301-15306

Keywords: chpaerone, folding, ribosome biogenesis