Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of <i>Giardia intestinalis</i>

Majumdar, Soneya; Emmerich, Andrew; Krakovka, Sascha; Mandava, Chandra Sekhar; Svärd, Staffan G.; Sanyal, Suparna

doi:10.1093/nar/gkad176

Cited by 8 publications

(3 citation statements)

References 92 publications

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“…The Thr48 residue, located in the switch I region of the GTP binding pocket, is highly conserved and sequence-aligned closely to Thr56 in human eEF2. Additionally, recent time-resolved cryo-EM studies show that switch I undergoes a large flipping motion (12, 13, 26). This motion creates a Pi release channel essential for EF-G’s function on the ribosome.…”

Section: Introductionmentioning

confidence: 99%

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

Johnson,

Steele,

Lin

et al. 2024

Preprint

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While elongation factor G (EF-G) is crucial for ribosome translocation, the role of its GTP hydrolysis remains ambiguous. The indispensability of EF-G is further exemplified by the phosphorylation of human eukaryotic elongation factor 2 (eEF2) at Thr56, which inhibits protein synthesis globally, but its exact mechanism is not clear. In this study, we developed a multi-channel single-molecule FRET (smFRET) microscopy methodology to examine the conformational changes of E. coli EF-G induced by mutations that closely aligned with Thr56 residue of eEF2. We utilized Alexa 488/594 double-labeled EF-G to catalyze the translocation of fMet-Phe-tRNAPhe-Cy3 inside Cy5-L27 labeled ribosomes, allowing us to probe both processes within the same complex. Our findings indicate that in the presence of either GTP or GDPCP, wild-type EF-G undergoes a conformational extension upon binding to the ribosome to promote normal translocation. On the other hand, T48E and T48V mutations did not affect GTP/GDP binding or GTP hydrolysis, but impeded Poly(Phe) synthesis and caused EF-G to adopt a unique compact conformation, which was not observed when the mutants interact solely with the sarcin/ricin loop. This study provides new insights into EF-G's adaptability and sheds light on the modification mechanism of human eEF2.

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Section: Introductionmentioning

confidence: 99%

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

Johnson,

Steele,

Lin

et al. 2024

Preprint

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“…Most eukaryotic ribosomes, monosome, LSU, and SSU sediments, at the 80S, 60S, and 40S, respectively, and possess four rRNA species 28S, 18S, 5.8S and 5S. The high-resolution ribosome structures from other protozoans: Trypanosoma brucei (Hashem et al, 2013), Plasmodium falciparum (Wong et al, 2014), Leishmania donovani (Shalev-Benami et al, 2017), Trichomonas vaginalis and Toxoplasma gondii (Li et al, 2017), Euglena gracilis (Matzov et al, 2020) Giardia lamblia (Hiregange et al, 2022) (Majumdar et al, 2023); Eiler et al 2024) have revealed the species-specific unique features of their architecture and protein synthesis. The atomic details for E. histolytica ribosome architecture are absent as no structure is available.…”

Section: Introductionmentioning

confidence: 99%

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

Sharma,

Mishra,

Gourinath

et al. 2024

Preprint

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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. histolyticalife cycle spans between an infective 'cyst stage' and an active disease-causing 'trophozoite stage'. We have determined cryo- EM structures ofE. histolyticaribosomes, large subunit (LSU), 53S ribosome at 2.8 Å resolution and associated, 75S ribosome at 3.3 Å resolution, isolated from its trophozoite stage. The overall core of the ribosome is conserved. However, the periphery has evolved withEntamoebaspecific 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.

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“…The high-resolution ribosome structures from other protozoans: Trypanosoma brucei 19 , Plasmodium falciparum 20 , Leishmania donovani 21 , Trichomonas vaginalis and Toxoplasma gondii 22 , Euglena gracilis 23 Giardia lamblia 24,25,26 have revealed the speciesspecific unique features of their architecture and protein synthesis. The atomic details for E. histolytica ribosome architecture are absent as no structure is available.…”

Section: Introductionmentioning

confidence: 99%

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

Kaushal,

Sharma,

Mishra

et al. 2024

Preprint

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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 isolated ribosomes from the trophozoite stage and determined its single particle cryo- EM structures. The 53S, ribosome large subunit (LSU), at 2.8 Å resolution, and the 75S, associated ribosome, at 3.3 Å resolution. The E. histolytica possesses a reduced ribosome with a conserved core, and the periphery 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. These structures provide valuable insights into the evolutionary adaption of the Entamoeba translational machinery and could be explored further for amoebicidal therapeutic intervention.

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Insights into translocation mechanism and ribosome evolution from cryo-EM structures of translocation intermediates of Giardia intestinalis

Cited by 8 publications

References 92 publications

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

Multi-Channel smFRET study reveals a Compact conformation of EF-G on the Ribosome

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

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

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