Fluorescence Triple Correlation Spectroscopy Resolves Ten Intermediates Along Different Parallel Ribosome Assembly Pathways

Abstract

Efficient self-assembly of the bacterial 30S ribosomal subunit depends on the interplay of a large number of sequential RNA folding and protein binding events. In many cases the order of these events is not rigidly defined, giving rise to an assembly landscape crossed by parallel assembly pathways. The biological and biophysical implications of parallel assembly are unclear, and seem to contradict the current understanding of the ways large RNAs avoid kinetic traps. There has been very little direct characterization of the degree of parallel assembly in ribosomes because current biophysical techniques have difficulty resolving intermediates, especially with a large macromolecule (>500kDa) that requires high concentrations (100-1000nM) to assemble.To quantitate 30S intermediates, Fluorescence Correlation Spectroscopy (FCS) was extended to create Fluorescence Triple Correlation Spectroscopy (F3CS). By correlating three signals, F3CS can study complex stoichiometric systems, kinetic processes and irreversible reactions. Theory, numerical correlation strategies, and experimental practices were established and a two-photon excitation, three-color detection microscope was built to perform the first F3CS measurements. The technique is implemented as a suite of software, Triple Correlation Toolbox, to enable triple correlation experiments with existing microscopes.F3CS simultaneously identified intermediates both on and off the ribosome assembly pathways that were predicted by classic assembly maps. A weak energetic bias favors one assembly pathway over the others, but the bias energy is not strong enough to prevent the formation of intermediates off of the favored pathway. The interaction energies of ribosomal proteins S7, S9 and S19 are initially weak, which in principle reduces the potential for kinetic traps. F3CS has broad applications for identifying the subunit composition of non-ribosomal macromolecular complexes, non-destructively and in real time, and for identifying time-reversal asymmetries.