Talk on Saturday 08:30-08:42am submitted by Sanjaya Abeysirigunawardena
Ribosomal proteins influence RNA dynamics during 30S assembly
Sanjaya Abeysirigunawardena (T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore MD ), Hajin Kim (Department of Physics and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL ), Taekjip Ha (Department of Physics and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, Urbana, IL ), Sarah Woodson (T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore MD )
Binding of ribosomal proteins with the rRNA has been shown to stabilize productive assembly intermediates that lead to the formation of active ribosomes. Recently our single-molecule FRET experiments showed binding of ribosomal protein S4 to the 5’-domain of 16S rRNA, modulate the dynamics of rRNA during early stages of 30S assembly. Initial highly dynamic S4-rRNA complexes go through a stable non-native intermediate complex before forming the native complex. Furthermore, fluctuations between non-native intermediate complex in which h3 flipped (F), and fully folded native complex with h3 docked under the base of h18 are observed at equilibrium. Ribosomal proteins S17, S16 and S20 bind to the core region of the 5’-domain RNA. These proteins stabilize certain RNA-RNA and RNA-Protein contacts hence can preferentially stabilize different S4-rRNA complexes. In this research S4 titrations in bulk and single molecule ALEX-FRET (Alternative Excitation Foster Resonance Energy Transfer) experiments are used to understand the effects of 5’-domain binding proteins on equilibrium between native and intermediate S4-RNA complexes. Surprisingly, protein S17 stabilizes the non-native intermediate complex, which is in agreement with the hydroxyl radical footprinting experiments. Protein S20 binds farther away from h3 and shows less influence on the equilibrium between native and non-native S4-RNA complexes. Interestingly however, S4 titrations in bulk showed that presence of proteins S20 and S16 together strongly favored the native S4-RNA complex. Neither protein S20 nor S16 showed significant perturbation to the equilibrium when added individually. Our 3-color single molecule ALEX-FRET experiments showed that native S4-rRNA complex becomes is highly preferred in complexes that are bound with protein S16. However, addition of protein S20 to RNA-S4-S16 complex did not increase the preference to native complex. Nevertheless, addition of protein S20 increased the number of stable RNA-S4-S16 complexes. Real-time S16Cy3 binding experiments showed that S16 binds preferably to native S4-RNA complex. Interestingly, binding of S16 protein slows down the motions of helix3 and stabilizes native complex that is required for the addition of more proteins during the ribosomal assembly.
Keywords: RNA-protein interactions, Ribosome assembly, smFRET