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Structure of an Archaeal Ribosome Reveals a Divergent Active Site and Hibernation Factor

ABSTRACT: Ribosomes translate mRNA into protein. Despite divergence in ribosome structure over the course of evolution, the catalytic site, known as the peptidyl transferase center (PTC), is thought to be nearly universally conserved. Here, we identify clades of archaea that have highly divergent ribosomal RNA sequences in the PTC. To understand how these PTC sequences fold, we determined cryo-EM structures of the 70S and 50S ribosomes to 2.4 Å and 2 Å, respectively, from the hyperthermophilic archaeon Pyrobaculum calidifontis. PTC sequence variation leads to the rearrangement of key base triples and differences between archaeal and bacterial ribosomal proteins enable sequence variation in archaeal PTCs. Finally, we identify a previously undescribed archaeal ribosome hibernation factor, Dri, that differs from known bacterial and eukaryotic hibernation factors and is found in multiple archaeal phyla. Overall, this work identifies factors that regulate ribosome function in archaea and reveals a larger diversity of the most ancient sequences in the ribosome

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ORGANISM(S): Pyrobaculum Calidifontis

SUBMITTER: Robert Maxwell

LAB HEAD: Jamie H. D. Cate

PROVIDER: PXD064958 | Pride | 2025-06-17

REPOSITORIES: Pride

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Project description:Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding centre can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labelling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 upregulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1 and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

			Action	DRS
	50S.mzidentml_1_2_0.xml	Mzid
	50S_1of4.mgf	Mgf
	50S_1of4.raw	Raw
	50S_2of4.mgf	Mgf
	50S_2of4.raw	Raw

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Structure of an Archaeal Ribosome Reveals a Divergent Active Site and Hibernation Factor

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