Poster abstracts
Poster number 53 submitted by Toby Bomser
Kinetic studies to elucidate the activation of an archaeal RNase P RNA by L7Ae, a ribosomal protein
Toby Bomser (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Vaishnavi Sidharthan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Alex Baumgardner (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Rachel Robertson (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Emily Wong (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Walter Zahurancik, Venkat Gopalan (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
RNase P is an essential ribonucleoprotein (RNP) that removes the 5′ leader from precursor tRNAs (pre-tRNAs) to generate mature tRNAs used in translation. Archaeal RNase P consists of the RNase P RNA (RPR) ribozyme and five accessory RNase P proteins (RPPs) that facilitate catalysis. The roles of four of these RPPs (RPP21, RPP29, POP5, RPP30) are better understood compared to the fifth protein, L7Ae. We previously determined that L7Ae, a ribosomal protein, binds to kink-turns (KTs) in the RPR and at a stoichiometry proportional to the number of KTs.1 Additionally, kinetic studies showed that L7Ae increases the catalytic efficiency (kcat/KM) of the holoenzyme and decreases the [Mg2+] required for maximal activity. These results support the hypothesis that L7Ae scaffolds multi-RPP holoenzyme assembly.1,2 However, we recently observed that L7Ae alone increases the activity of the RPR from Methanobrevibacter smithii (Msm), a mesophilic archaeon. Here, we investigated how L7Ae enhances the activity of the Msm RPR and which KTs must be bound by L7Ae to elicit this enhanced activity. To disrupt L7Ae binding, we generated RPR variants with the KTs mutated either alone or in combination. We then performed Michaelis–Menten kinetic analyses to examine the effects of L7Ae on kcat and KM during pre-tRNA cleavage by the WT or KT mutants ± L7Ae. Additionally, because of the proximity of KT1 to the P6 pseudoknot, a tertiary interaction essential for RPR folding and activity, we are now also testing the idea that formation of the P6 pseudoknot depends on KT1 and its stabilization by L7Ae. Overall, this work aims to further our understanding of the diverse roles of L7Ae across RNPs and provide insight into how RNA structure has been shaped through co-evolution with L7Ae.
References:
1.Lai, LB, Tanimoto A, Lai SM, Chen W-Y, Marathe IA, Westhof E, Wysocki VH, Gopalan V. (2017) A novel double kink-turn module in euryarchaeal RNase P RNAs. Nucleic Acids Res 45, 7432–7440.
2.Cho I-M, Lai LB, Susanti D, Mukhopadhyay B, Gopalan V. (2010) Ribosomal protein L7Ae is a subunit of archaeal RNase P. Proc Natl Acad Sci (USA)107, 14573–14578.
Keywords: RNase P, L7Ae, RNA-protein interactions