TY - JOUR
T1 - A structural inventory of native ribosomal ABCE1-43S pre-initiation complexes
AU - Kratzat, Hanna
AU - Mackens-Kiani, Timur
AU - Ameismeier, Michael
AU - Potocnjak, Mia
AU - Cheng, Jingdong
AU - Dacheux, Estelle
AU - Namane, Abdelkader
AU - Berninghausen, Otto
AU - Herzog, Franz
AU - Fromont-Racine, Micheline
AU - Becker, Thomas
AU - Beckmann, Roland
N1 - Funding Information:
The authors thank H. Sieber, J. Musial, C. Ungewickell, and S. Rieder for technical assistance, L. Kater and K. Best for support with the pre‐processing pipeline of cryo‐EM data, R. Buschauer for assistance in model building, L. Valášek and A. Jacquier for critical reading of the manuscript, J. Wells for support during the setup of splitting assays and J. Zeman for support with the XL‐MS experiments. This work was supported by German Research Council (BE1814/15‐1 and TRR174), the Center for Integrated Protein Science Munich (CiPS‐M), the ANR‐17‐CE11‐0049‐01 and the ANR‐17‐CE12‐0024‐02 grants, the Pasteur Institute and the Centre National de la Recherche Scientifique. H.K., M.A., and M.P. are supported by a DFG fellowship through the Graduate School of Quantitative Bioscience Munich (QBM).
Funding Information:
The authors thank H. Sieber, J. Musial, C. Ungewickell, and S. Rieder for technical assistance, L. Kater and K. Best for support with the pre-processing pipeline of cryo-EM data, R. Buschauer for assistance in model building, L. Valášek and A. Jacquier for critical reading of the manuscript, J. Wells for support during the setup of splitting assays and J. Zeman for support with the XL-MS experiments. This work was supported by German Research Council (BE1814/15-1 and TRR174), the Center for Integrated Protein Science Munich (CiPS-M), the ANR-17-CE11-0049-01 and the ANR-17-CE12-0024-02 grants, the Pasteur Institute and the Centre National de la Recherche Scientifique. H.K., M.A., and M.P. are supported by a DFG fellowship through the Graduate School of Quantitative Bioscience Munich (QBM).
Publisher Copyright:
© 2020 The Authors
PY - 2021/1/4
Y1 - 2021/1/4
N2 - In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
AB - In eukaryotic translation, termination and ribosome recycling phases are linked to subsequent initiation of a new round of translation by persistence of several factors at ribosomal sub-complexes. These comprise/include the large eIF3 complex, eIF3j (Hcr1 in yeast) and the ATP-binding cassette protein ABCE1 (Rli1 in yeast). The ATPase is mainly active as a recycling factor, but it can remain bound to the dissociated 40S subunit until formation of the next 43S pre-initiation complexes. However, its functional role and native architectural context remains largely enigmatic. Here, we present an architectural inventory of native yeast and human ABCE1-containing pre-initiation complexes by cryo-EM. We found that ABCE1 was mostly associated with early 43S, but also with later 48S phases of initiation. It adopted a novel hybrid conformation of its nucleotide-binding domains, while interacting with the N-terminus of eIF3j. Further, eIF3j occupied the mRNA entry channel via its ultimate C-terminus providing a structural explanation for its antagonistic role with respect to mRNA binding. Overall, the native human samples provide a near-complete molecular picture of the architecture and sophisticated interaction network of the 43S-bound eIF3 complex and the eIF2 ternary complex containing the initiator tRNA.
KW - ABCE1
KW - cryo-EM
KW - eIF3
KW - ribosome recycling
KW - translation initiation
KW - Cell Line
KW - Humans
KW - Saccharomyces cerevisiae Proteins/metabolism
KW - Protein Binding/physiology
KW - ATP-Binding Cassette Transporters/metabolism
KW - DNA-Binding Proteins/metabolism
KW - Eukaryotic Initiation Factor-2/metabolism
KW - RNA, Messenger/metabolism
KW - Saccharomyces cerevisiae/metabolism
KW - Ribosome Subunits, Small, Eukaryotic/metabolism
KW - HEK293 Cells
KW - Protein Biosynthesis/physiology
KW - RNA, Transfer/metabolism
UR - http://www.scopus.com/inward/record.url?scp=85097283996&partnerID=8YFLogxK
U2 - 10.15252/embj.2020105179
DO - 10.15252/embj.2020105179
M3 - Article
C2 - 33289941
AN - SCOPUS:85097283996
SN - 0261-4189
VL - 40
SP - e105179
JO - EMBO Journal
JF - EMBO Journal
IS - 1
M1 - e105179
ER -