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Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit

ABSTRACT: In this work, we show that 23S rRNA lacking the SRL can be efficiently reconstituted into 50S subunits, which exhibit full peptidyl transferase (PT) activity. Using cryo-EM and proteomic methods, we also further characterize affinity-purified SRL particles isolated from cells. Collectively, these data suggest that the SRL is needed to promote the dissociation of several AFs during 50S subunit biogenesis in the cell.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli

SUBMITTER: Jenny Moon

LAB HEAD: Leonard J. Foster

PROVIDER: PXD056260 | Pride | 2025-05-07

REPOSITORIES: Pride

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			Action	DRS
	258_KF_LF7-100ng.raw	Raw
	264_KF_LF8-100ng.raw	Raw
	KF_LF7-75ng_8-19-2023_Slot1-31_7726.d.1.dar	Other
	KF_LF8-75ng_8-19-2023_Slot1-32_7724.d.1.dar	Other
	Sepideh_LFQ_txt.zip	Other

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Publications

Role of the sarcin-ricin loop of 23S rRNA in biogenesis of the 50S ribosomal subunit.

Aval Sepideh Fakhretaha SF Seffouh Amal A Moon Kyung-Mee KM Foster Leonard J LJ Ortega Joaquin J Fredrick Kurt K

RNA (New York, N.Y.) 20250318 4

The sarcin-ricin loop (SRL) is one of the most conserved segments of ribosomal RNA (rRNA). Translational GTPases (trGTPases), such as EF-G, EF-Tu, and IF2, form contacts with the SRL that are critical for GTP hydrolysis and factor function. Previous studies showed that expression of 23S rRNA lacking the SRL confers a dominant lethal phenotype in <i>Escherichia coli</i> Isolated ΔSRL particles were found to be not only inactive in protein synthesis but also incompletely assembled. In particular, ...[more]

PMID: 39875174

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Structure of Escherichia coli heat shock protein Hsp15 in complex with ribosomal 50S sununits bearing peptidyl-tRNA

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.

2022-11-15 | GSE216100 | GEO

R-proteins in modification deficient ribosomes - free 50S subunits

Project description:Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an E. coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Ribosomal protein composition of the mature 50S ribosomes and free 50S subunits was analyzed by SILAC based qMS approach. We have found that absence of 23S rRNA modifications around PTC does not affect significantly r-protein content of incompletely assembled 50S or mature 50S ribosomes.

2024-03-15 | PXD047376 | Pride

R-proteins in modification deficient ribosomes - dissociated 50S subunits from 70S

2024-03-15 | PXD047588 | Pride

Leishmania regulates host YY1: comparative proteomic analysis identifies infection modulated YY1 dependent proteinsLeishmania regulates host YY1: comparative proteomic analysis identifies infection modulated YY1 dependent proteins

Project description:The protein Yin-Yang 1 (YY1) is a ubiquitous multifunctional transcription factor. Interestingly, there are several cellular functions controlled by YY1 that could play a role in Leishmania pathogenesis. Leishmaniasis is a human disease caused by protozoan parasites of the genus Leishmania. This study examined the potential role of macrophage YY1 in promoting Leishmania intracellular survival. Knockdown of YY1 resulted in attenuated survival of Leishmania in infected macrophages, suggesting a role of YY1 in Leishmania persistence. Biochemical fractionation studies revealed Leishmania infection caused redistribution of YY1 to the cytoplasm from the nucleus where it is primarily located. Inhibition of nuclear transport by leptomycin B attenuates infection-mediated YY1 redistribution and reduces Leishmania survival. This suggests that Leishmania induces the translocation of YY1 from the nucleus to the cytoplasm of infected cells, where it may regulate host molecules to favour parasite survival. A label-free quantitative whole proteome approach showed that the expression of a large number of macrophage proteins was dependent on the YY1 level. Interestingly, several of these proteins were modulated in Leishmania-infected cells, revealing YY1-dependent host response and suggesting their potential role in Leishmania pathogenesis. Together, these findings identify YY1 as a novel and essential virulence factor by proxy that promotes Leishmania survival.

2025-03-14 | PXD059968 | Pride

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE20 is Required for Plastid 50S Ribosome Biogenesis

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OLA-phyto-23S

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Protein bL38 facilitates incorporation of uL6 during assembly of the 50S subunit in Flavobacterium johnsoniae

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