The Open Biochemistry Journal




ISSN: 1874-091X ― Volume 13, 2019
RESEARCH ARTICLE

RpbL12 Assists Catalysis by Correctly Positioning the Incoming Aminoacyl-tRNA in the A-Site of E. coli 70S Ribosomes



Jean-Bernard Créchet1, Fulbert K. Agbo’Saga2, Soria Baouz2, Codjo Hountondj2, *
1 Ecole Polytechnique, Route de Saclay, F-91120 Palaiseau, France
2 Sorbonne Université, Campus Pierre et Marie Curie, Unité de Recherche SUUR6 “Enzymologie de l’ARN”, 7 Quai Saint-Bernard, F-75252 Paris Cedex 05, France

Abstract

Introduction:

We have recently demonstrated that Lys-65 of the 62GANK65 motif of E. coli RpbL12 was affinity labeled with a tRNA analogue, resulting in the loss of activity.

Materials and Methods:

In this report, we show that mutations operated at the position of Lys-65 led to an impairment in the activity of the mutant ribosomes, except the K65R or K65H bL12 mutants, suggesting that the only requirement of the reaction catalyzed or facilitated by RpbL12is the positive charge of the side chain of Lys-65. We also demonstrate that Lys-65 did not play any role in the peptidyl transferase reaction with respect to puromycin, but rather assisted the binding of the incoming aminoacyl-tRNA to the ribosomal A-site.

Results & Discussions

The protonated, positively charged εNH3+ form of Lys-65 is likely to participate to the binding of aa-tRNA through ionic bonds with phosphate groups, in order to insure the accurate positioning required for the nucleophilic attack of its α-amino group on the carbonyl carbone of peptidyl-tRNA.

Conclusion

This α-NH2 group is likely to be generated by the unprotonated εNH2 form of Lys-65 which is capable of withdrawing a proton from the α-NH3+ group of aa-tRNA.

Keywords: E. coli ribosomal protein bL12, GGQ-like GAN motif of bL12, Lys-65 of bL12, Site-directed mutagenesis, Mechanism of peptide bond formation, Aminoacyl-tRNA.


Article Information


Identifiers and Pagination:

Year: 2018
Volume: 12
First Page: 113
Last Page: 129
Publisher Id: TOBIOCJ-12-113
DOI: 10.2174/1874091X01812010113

Article History:

Received Date: 22/3/2018
Revision Received Date: 26/6/2018
Acceptance Date: 02/7/2018
Electronic publication date: 31/07/2018
Collection year: 2018

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© 2018 Créchet et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address correspondence to this author at the Sorbonne Université, Campus Pierre et Marie Curie, Unité de Recherche SUUR6 “Enzymologie de l’ARN”, 7 Quai Saint-Bernard, F-75252 Paris Cedex 05, France; Tel: (+33)1 44 27 40 86; E-mail: codjo.hountondji@upmc.fr


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