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

The Open Biochemistry Journal 31 July 2018 RESEARCH ARTICLE DOI: 10.2174/1874091X01812010113



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.


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.
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