RNA Chaperone Activity of Ribosomal Protein L1: investigating conservation of chaperoning activity and functional implications in the context of the ribosome

The ribosome is a complex machine consisting of the ribosomal RNA scaffold and ribosomal proteins, which are crammed into the RNA. The Escherichia coli ribosome consists of two asymmetric subunits, the small or 30S subunit consisting of 16S rRNA and 21 proteins and the large or 50S subunit encompassing 2 RNA molecules (23S, 5S) and 34 proteins. A recent study showed that nearly a third of all large ribosomal subunit (lsu) proteins has RNA chaperone activity in vitro (Semrad et al. 2004). RNA chaperones are proteins, which either prevent RNA from misfolding or which resolve misfolded structures without ATP consumption. In this study it will be tested if this observed RNA chaperone activity of E.coli ribosomal proteins is conserved in other organisms. Ribosomal protein L1 of E.coli, which showed high RNA chaperone activity, has many homologues in other organisms. Therefore different L1 homologues will be cloned and purified and tested for their RNA chaperone activity in vitro. RNA misfolding occurs to an increased extend in the cold. Consequently, the hypothesis is that RNA chaperones will have higher activities at low temperatures. L1 homologues from psychrophilic, mesophilic and thermophilic organisms will be choosen for their evaluation of RNA chaperone activity and for testing if there is a correlation between growth temperature and RNA chaperone activity. Sequence alignments of ribosomal L1 homologues will then be performed and the outcome will suggest conserved aminoacids for mutation and subsequent testing in RNA chaperone assays. Most importantly, it will be tested if this RNA chaperone activity serves a function during translation: ribosomal protein L1 is located at the exit-site for the deacylated tRNA and it was suggested that L1 plays a role in E-site tRNA ejection. The hypothesis that the RNA chaperone activity of L1 could be involved in E-site tRNA ejection will be tested in vivo and in vitro. In vivo, L1 knock-out constructs have been shown to decrease growth rates two- fold (Baier et al. 1990). E.coli L1 will be replaced by homologues with or without RNA chaperone activity and growth rates will be measured at various temperatures. In vitro, I will establish an in vitro translation system and measure E-site tRNA ejection in 70S ribosomes, where E.coli L1 has either been removed or replaced by a L1 homologue.