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Understanding the role of RbgA in the assembly process of the 50S subunit.

ABSTRACT: Ribosomes must be formed quickly (to support cellular growth demands) and accurately (to ensure proper translation of the genetic code). The assembly of ribosomes in vivo requires not only the constituent rRNAs and ribosomal proteins (r-proteins), but also assembly factors. Several of these factors are GTPases. However, the molecular role that these GTPases play is not understood. Hypotheses range from serving a regulatory/checkpoint role to serving a direct mechanical role in structural rearrangements (such as serving as an RNA chaperone). The objective of our research is to identify the precise function of one of these essential GTPases in the assembly of the 50S subunit and provide a clear picture for the molecular rearrangements associated with its function. This essential GTPase from Bacillus subtilis, the model organism for Gram-positive bacteria, is RbgA, which constitute a key player in the late maturation steps of the 50S ribosome subunit.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Bacillus Subtilis Subsp. Subtilis Str. 168

SUBMITTER: Marlene Oeffinger

LAB HEAD: Marlene Oeffinger

PROVIDER: PXD023644 | Pride | 2022-01-27

REPOSITORIES: Pride

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			Action	DRS
	20210112_ConditionSetup.xls	Xls
	20210112_Seffouh_et_al.sne	Other
	20210112_Seffouh_et_al_Report.xls	Xls
	20210112_Seffouh_et_al_SDRF.txt	Txt
	20210112_Seffouh_et_al_Setup.txt	Txt

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Project description:Ribosomes, macromolecular machines producing a cell´s protein content, are formed from their RNA and protein components in a dynamic process referred to as ribosome assembly. While ribosome assembly in the cell starts with the successive synthesis of ribosomal RNA (rRNA) by the RNA polymerase holoenzyme, and requires numerous assembly factors, (Kaczanowska & Ryden-Aulin, 2007; Shajani et al., 2011), the process can be accomplished in vitro, using purified ribosomal components and scalable reaction conditions (Nierhaus & Dohme, 1974; Traub & Nomura, 1968). To obtain structural and conceptual insights in the early phase of the process, we performed the in vitro assembly reaction of the bacterial 50S subunit as a time course reaction, analyzed samples by sucrose density gradient ultracentrifugation, activity assay, quantitative mass spectrometry (qMS) and cryo-electron microscopy (cryo-EM). Our structural analysis reveals that early 50 assembly initiates with 23S rRNA domain I and occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Notably, in both phases parallel pathways are utilized. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity. Our study provides new insights into fundamental principles governing ribosome assembly.

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Project description:In Escherichia coli, the heat shock protein 15 (Hsp15) is part of the cellular response to elevated temperature. Hsp15 interacts with peptidyl-tRNA-50S complexes that arise upon dissociation of translating 70S ribosomes, and is proposed to facilitate their rescue and recycling. A previous structure of E. coli Hsp15 in complex with peptidyl-tRNA-50S complex reported a binding site located at the central protuberance of the 50S subunit. By contrast, recent structures of RqcP, the Hsp15 homolog in Bacillus subtilis, in complex with peptidyl-tRNA-50S complexes have revealed a distinct site positioned between the anticodon-stem-loop (ASL) of the P-site tRNA and H69 of the 23S rRNA. Here we demonstrate that exposure of E. coli cells to heat shock leads to dissociation of 70S ribosomes and accumulation of 50S subunits, thus identifying a natural substrate for Hsp15 binding. Additionally, we have determined a cryo-EM reconstruction of the Hsp15-50S-peptidyl-tRNA complex isolated from heat shocked E. coli cells, revealing that Hsp15 binds to the 50S-peptidyl-tRNA complex analogously to its B. subtilis homolog RqcP. Collectively, our findings support a model where Hsp15 promotes access to the A-site for putative rescue factors to release the aberrant nascent polypeptide chain.

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RNA Post-transcriptional Modifications in Two Large Subunit Intermediates Populated in E.coli Cells Expressing Helicase Inactive R331A DbpA

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2022-09-15 | GSE196821 | GEO

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Project description:While the protein composition of various yeast 60S ribosomal subunit assembly intermediates has been studied in detail, little is known about ribosomal RNA (rRNA) structural rearrangements that take place during early 60S assembly steps. Using a high-throughput RNA structure probing method, we provide nucleotide resolution insights into rRNA structural rearrangements during nucleolar 60S assembly. Our results suggest that many rRNA-folding steps, such as folding of 5.8S rRNA, occur at a very specific stage of assembly, and propose that downstream nuclear assembly events can only continue once 5.8S folding has been completed. Our maps of nucleotide flexibility enable making predictions about the establishment of protein-rRNA interactions, providing intriguing insights into the temporal order of protein-rRNA as well as long-range inter-domain rRNA interactions. These data argue that many distant domains in the rRNA can assemble simultaneously during early 60S assembly and underscore the enormous complexity of 60S synthesis.Ribosome biogenesis is a dynamic process that involves the ordered assembly of ribosomal proteins and numerous RNA structural rearrangements. Here the authors apply ChemModSeq, a high-throughput RNA structure probing method, to quantitatively measure changes in RNA flexibility during the nucleolar stages of 60S assembly in yeast.

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