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Nucleic Acids Res. 2016 Nov 16;44(20):9918-9932. Epub 2016 Jul 5.

Binding properties of YjeQ (RsgA), RbfA, RimM and Era to assembly intermediates of the 30S subunit.

Author information

1
Department of Biochemistry and Biomedical Sciences and M.G. DeGroote Institute for Infectious Diseases Research, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S4K1, Canada.
2
Department of Integrative Computational and Structural Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
3
Department of Biochemistry, University of Toronto, 1 King's College Circle, Toronto, Ontario M5S1A8, Canada.
4
Department of Biochemistry and Biomedical Sciences and M.G. DeGroote Institute for Infectious Diseases Research, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S4K1, Canada ortegaj@mcmaster.ca.

Abstract

Our understanding regarding the function of YjeQ (also called RsgA), RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in null strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that these immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells. We measured by microscale thermophoresis that YjeQ and Era binds to the mature 30S subunit with high affinity. However, the binding affinity of these factors to the immature particles and of RimM and RbfA to mature or immature particles was weak, suggesting that binding is not occurring at physiological concentrations. These results suggest that in the absence of these factors, the immature particles evolve into a thermodynamically stable intermediate that exhibits low affinity for the assembly factors. These results imply that the true substrates of YjeQ, RbfA, RimM and Era are immature particles that precede the ribosomal particles accumulating in the knockouts strains.

PMID:
27382067
PMCID:
PMC5175332
DOI:
10.1093/nar/gkw613
[Indexed for MEDLINE]
Free PMC Article

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