Proteomics

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Depletion and reconstitution of 5S RNP complexes in early pre60S ribosomes

ABSTRACT: We purified pre60S ribosomes from yeast using Nsa1-FtpA as bait in Tandem Affinity Purification experiments. We tested the depletion of RPF2, using a Galactose-depletion system, to obtain ribosomal particles depleted of 5S RNP complexes. Sample A1: Control group (wild type strain) grown in YPGal; Sample A2: Control group (wild type strain) grown in YPGlu; Sample A3: Control group (Gal::HA-RPF2 strain) grown in YPGal; and Sample A4: Test group (Gal::HA-RPF2) shifted from YPGal to YPGlu for 6 hours to achieve 5S RNP depletion. Sample N1: Purified pre-60S Nsa1-associated with 5S RNP depleted after Sucrose gradient separation; Sample N2: Purified pre-60S Nsa1-associated with 5S RNP reconstituted after Sucrose gradient separation.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Douglas Lamont  

LAB HEAD: Professor Ed Hurt

PROVIDER: PXD040306 | Pride | 2023-10-24

REPOSITORIES: Pride

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027-2017-BZH-JBoutput.csv Csv
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027-2017-BZH-JBoutput.xlsx Xlsx
144-2017-BZH-JBoutput.csv Csv
144-2017-BZH-JBoutput.txt Txt
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Publications

Structure of nascent 5S RNPs at the crossroad between ribosome assembly and MDM2-p53 pathways.

Castillo Duque de Estrada Nestor Miguel NM   Thoms Matthias M   Flemming Dirk D   Hammaren Henrik M HM   Buschauer Robert R   Ameismeier Michael M   Baßler Jochen J   Beck Martin M   Beckmann Roland R   Hurt Ed E  

Nature structural & molecular biology 20230608 8

The 5S ribonucleoprotein (RNP) is assembled from its three components (5S rRNA, Rpl5/uL18 and Rpl11/uL5) before being incorporated into the pre-60S subunit. However, when ribosome synthesis is disturbed, a free 5S RNP can enter the MDM2-p53 pathway to regulate cell cycle and apoptotic signaling. Here we reconstitute and determine the cryo-electron microscopy structure of the conserved hexameric 5S RNP with fungal or human factors. This reveals how the nascent 5S rRNA associates with the initial  ...[more]

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