Key result
The cryo-EM structure of the mycobacterial 70S ribosome in complex with RafH at 2.8 Å resolution reveals a unique mode of ribosome hibernation that prevents 100S disome formation.
This structural biology study reveals the unique mechanism by which the RafH protein promotes ribosome hibernation in mycobacteria, providing insights into their survival under stress.
May inform ribosome-targeted TB therapies; leaves open clinical translation pending validation.
Ribosome hibernation is a key survival strategy bacteria adopt under environmental stress, where a protein, hibernation promotion factor (HPF), transitorily inactivates the ribosome and slows down its overall protein synthesis. The mechanism is well studied in enteric bacteria, which mainly hibernate its ribosome in 100S disome form through a dual domain, long HPF (HPFlong) or a single domain, short HPF (HPFshort) in concert with another ribosome modulation factor. Mycobacterium tuberculosis encounters hypoxia (low oxygen) as a major stress in the host macrophages, and it overexpresses RafH protein, which is critical for its survival. The RafH, a dual domain HPF, an orthologue of bacterial HPFlong, hibernates ribosome in 70S monosome form only. Here we report the cryo- EM structure of Mycobacterium smegmatis, a close homologue of M. tuberculosis, 70S ribosome in complex with the RafH factor at an overall 2.8 Å resolution. The RafH N- terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding for HPFlong NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPFlong C- terminus domain (CTD), the RafH CTD, which is larger, binds to a unique site at the platform binding center of the ribosomal small subunit and sandwiches between bS1 and uS11 ribosomal proteins. The two domain connecting linker regions, which remain mostly disordered in earlier reported HPFlong structures, interacts mainly with the anti-Shine Dalgarno sequence of the 16S rRNA. The helix H54a of 23S rRNA, unique to the mycobacterial ribosome, adopts a different conformation and come close to RafH CTD, suggesting its role in ribosome hibernation. RafH inhibits in-vitro protein synthesis in a concentration dependent manner. Further, the modeling studies provided the structural basis for the incompatibility of mycobacterial ribosomes to form 100S like disome architecture.
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Kaushal et al. (2023) studied Latent Tuberculosis Infection (Ribosome Hibernation). RafH protein was evaluated on Cryo-EM structure of 70S ribosome RafH complex. The cryo-EM structure of the mycobacterial 70S ribosome in complex with RafH at 2.8 Å resolution reveals a unique mode of ribosome hibernation that prevents 100S disome formation.
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