Kaushal, P. S. | bioRxiv Science

bioRxiv2024

Cryo- EM structure of ribosome from pathogenic protozoa Entamoeba histolytica, reveals unique features of its architecture

Entamoeba histolytica, an anaerobic parasite protozoan, is the causative agent of amoebiasis, the bloody diarrhea, and liver abscesses in humans. Amoebiasis is more predominant in tropical areas with poor sanitation conditions, and it remains the fourth leading cause of death due to a protozoan infection. E. histolytica life cycle spans between an infective cyst stage and an active disease-causing trophozoite stage. We have determined cryo-EM structures of E. histolytica ribosomes, large subunit (LSU), 53S ribosome at 2.8 [A] resolution and associated, 75S ribosome at 3.3 [A] resolution, isolated from its trophozoite stage. The overall core of the ribosome is conserved. However, the periphery has evolved with Entamoeba specific unique features. The most notable features are, the presence of the rRNA triple helix near the peptide exit tunnel on LSU and the co-evolution of rRNA expansion segments and extensions in r-proteins. To the best of our knowledge, this structure reports the presence of RNA triple helix in the ribosome for the first time.

biochemistry↗

bioRxiv2023

Cryo- EM structure of the mycobacterial 70S ribosome in complex with ribosome hibernation promotion factor RafH, reveals the unique mode of mycobacterial ribosome hibernation

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. Mycobacteria under hypoxia (low oxygen) stress overexpresses RafH protein regulated under DosR regulon, a critical factor 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 [A] resolution. The RafH N-terminus domain (NTD) is conserved and binds to the decoding center of the ribosomal small subunit, a similar binding site of HPFlong NTD, but additionally it also interacts with the inter subunit bridge, B2a. Contrary to the HPFlong 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 forming 100S like hibernating ribosomes.

biochemistry↗