New photoreactive tRNA derivatives for probing the peptidyl transferase center of the ribosome

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

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Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

10.1101/2020.08.30.274597 ◽

2020 ◽

Author(s):

Anna B. Loveland ◽

Egor Svidritskiy ◽

Denis Susorov ◽

Soojin Lee ◽

Alexander Park ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Specific Binding ◽

Structural Basis ◽

Transferase Activity ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

Ribosomal Tunnel ◽

Cellular Translation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractToxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

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Ribosome protection by antibiotic resistance ATP-binding cassette protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803313115 ◽

2018 ◽

Vol 115 (20) ◽

pp. 5157-5162 ◽

Cited By ~ 26

Author(s):

Weixin Su ◽

Veerendra Kumar ◽

Yichen Ding ◽

Rya Ero ◽

Aida Serra ◽

...

Keyword(s):

Antibiotic Resistance ◽

Conformational Changes ◽

Atp Binding ◽

Peptidyl Transferase ◽

Exit Site ◽

Peptidyl Transferase Center ◽

Biochemical Assays ◽

Exit Tunnel ◽

Insight Into ◽

Atp Binding Cassette

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

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The Peptidyl Transferase Center: a Window to the Past

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00104-21 ◽

2021 ◽

Author(s):

Madhan R. Tirumalai ◽

Mario Rivas ◽

Quyen Tran ◽

George E. Fox

Keyword(s):

Genetic Code ◽

21St Century ◽

Origins Of Life ◽

Current View ◽

Significant Progress ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

The Road ◽

The Past ◽

History Of

In his 2001 article, “Translation: in retrospect and prospect,” the late Carl Woese made a prescient observation that “our current view of translation be reformulated to become an all-embracing perspective about which 21st century Biology can develop” (RNA 7:1055–1067, 2001, https://doi:10.1017/s1355838201010615 ). The quest to decipher the origins of life and the road to the genetic code are both inextricably linked with the history of the ribosome. After over 60 years of research, significant progress in our understanding of how ribosomes work has been made.

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