Communication
The Effect of Ribosome Assembly Cofactors on In Vitro 30S Subunit Reconstitution

https://doi.org/10.1016/j.jmb.2010.02.036Get rights and content

Abstract

Ribosome biogenesis is facilitated by a growing list of assembly cofactors, including helicases, GTPases, chaperones, and other proteins, but the specific functions of many of these assembly cofactors are still unclear. The effect of three assembly cofactors on 30S ribosome assembly was determined in vitro using a previously developed mass-spectrometry-based method that monitors the rRNA binding kinetics of ribosomal proteins. The essential GTPase Era caused several late-binding proteins to bind rRNA faster when included in a 30S reconstitution. RimP enabled faster binding of S9 and S19 and inhibited the binding of S12 and S13, perhaps by blocking those proteins' binding sites. RimM caused proteins S5 and S12 to bind dramatically faster. These quantitative kinetic data provide important clues about the roles of these assembly cofactors in the mechanism of 30S biogenesis.

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Acknowledgements

This work was supported by a grant from the National Institutes of Health (R37-GM-53757) to J.R.W. and by the Skaggs Institute for Chemical Biology. P.M.W. was supported by the Carl Trygger Foundation. The authors thank Prof. Gary Siuzdak and Dr. Sunia Trauger of the Scripps Center for Mass Spectrometry for advice and access to instrumentation. Gunter Stier is gratefully acknowledged for providing plasmid pETM10, Dr. Michael T. Sykes for mass spectrometry data analysis programs, and Dr. Zahra

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      In their study, RimP was only found in the fractions of 30S subunit but not others in sucrose gradient centrifugation, and primer extension studies demonstrated that RimP knockout bacteria up-regulated the level of pre-16S rRNA but down-regulated mature 16S rRNA levels (4). Recently, quantitative MS studies on E. coli suggested that RimP can increase the binding kinetics of the S5 and S12 ribosomal proteins to the 5′ domain of rRNA in vitro (7). In addition, it was reported that the relative timing of the assembly of the 3′ domain and the formation of the central pseudoknot structure in the 16S rRNA depend on the presence of the assembly factor RimP (8).

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