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bioRxiv · 10.1101/2024.05.08.593220

Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing

Sheng, K.·Dong, X.·Aiyer, S.·Lee, J.·Marquardt, S. D.·Lyumkis, D.·Williamson, J. R.
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Abstract

Investigating the intricate and rapid folding kinetics of large RNA-protein complexes (RNPs), like the bacterial ribosome, remains a formidable challenge in structural biology. Previous genetic approaches to probe assembly have focused on modulating the expression of either r-proteins or assembly factors. Here, anti-sense oligonucleotides (ASOs) were used to disrupt native RNA/RNA and RNA/protein interactions, in order to generate novel folding intermediates. In an in vitro co-transcriptional assembly assay, 8 assembly inhibitor ASOs were identified. Using cryo-electron microscopy, 38 new intermediate structures were determined resulting from the specific inhibitions. In particular a novel intermediate class provided compelling evidence of independent rRNA domain folding before proper interdomain docking. Three PNAs targeting domain-I of 23S- rRNA further subdivided the previously identified assembly core into smaller blocks representing the earliest steps in assembly. The resulting assembly graph reveals template-directed RNA foldon docking and domain consolidation, which provides a hierarchical view of the RNP assembly process.

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Sheng, K., Dong, X., Aiyer, S., Lee, J., Marquardt, S. D., Lyumkis, D., Williamson, J. R.. 2024-05-08. Domain consolidation in Bacterial 50S assembly revealed by Anti-Sense Oligonucleotide Probing. https://doi.org/10.1101/2024.05.08.593220

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