The α and γ subunits of initiation factor elF-2 can be cross-linked to 18S ribosomal RNA within the quaternary initiation complex, eIF-2-Met-tRNAfGDPCP small ribosomal subunit

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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Identification of proteins of the 40 S ribosomal subunit involved in interaction with initiation factor eIF-2 in the quaternary initiation complex by means of monospecific antibodies

FEBS Letters ◽

10.1016/0014-5793(88)81366-8 ◽

1988 ◽

Vol 233 (1) ◽

pp. 114-118 ◽

Cited By ~ 20

Author(s):

Ulrich-Axel Bommer ◽

Joachim Stahl ◽

Annemarie Henske ◽

Gudrun Lutsch ◽

Heinz Bielka

Keyword(s):

Ribosomal Subunit ◽

Initiation Factor ◽

Initiation Complex ◽

Monospecific Antibodies

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The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽

10.1261/rna.078994.121 ◽

2022 ◽

pp. rna.078994.121

Author(s):

Haina Huang ◽

Melissa D Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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rRNA Suppressor of a Eukaryotic Translation Initiation Factor 5B/Initiation Factor 2 Mutant Reveals a Binding Site for Translational GTPases on the Small Ribosomal Subunit

Molecular and Cellular Biology ◽

10.1128/mcb.00896-08 ◽

2008 ◽

Vol 29 (3) ◽

pp. 808-821 ◽

Cited By ~ 12

Author(s):

Byung-Sik Shin ◽

Joo-Ran Kim ◽

Michael G. Acker ◽

Kathryn N. Maher ◽

Jon R. Lorsch ◽

...

Keyword(s):

Translation Initiation ◽

Ribosomal Subunit ◽

The Body ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Small Ribosomal Subunit ◽

Eukaryotic Translation Initiation Factor ◽

Eukaryotic Translation ◽

Initiation Factor 2 ◽

Eukaryotic Translation Initiation

ABSTRACT The translational GTPases promote initiation, elongation, and termination of protein synthesis by interacting with the ribosome. Mutations that impair GTP hydrolysis by eukaryotic translation initiation factor 5B/initiation factor 2 (eIF5B/IF2) impair yeast cell growth due to failure to dissociate from the ribosome following subunit joining. A mutation in helix h5 of the 18S rRNA in the 40S ribosomal subunit and intragenic mutations in domain II of eIF5B suppress the toxic effects associated with expression of the eIF5B-H480I GTPase-deficient mutant in yeast by lowering the ribosome binding affinity of eIF5B. Hydroxyl radical mapping experiments reveal that the domain II suppressors interface with the body of the 40S subunit in the vicinity of helix h5. As the helix h5 mutation also impairs elongation factor function, the rRNA and eIF5B suppressor mutations provide in vivo evidence supporting a functionally important docking of domain II of the translational GTPases on the body of the small ribosomal subunit.

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Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽

10.7554/elife.45185 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 11

Author(s):

Mirjam Hunziker ◽

Jonas Barandun ◽

Olga Buzovetsky ◽

Caitlin Steckler ◽

Henrik Molina ◽

...

Keyword(s):

Ribosomal Rna ◽

Conformational Changes ◽

Ribosome Biogenesis ◽

Ribosomal Subunit ◽

Small Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Early Maturation ◽

External Transcribed Spacer ◽

Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

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