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

Jean-Bernard Créchet,Codjo Hountondj,Fulbert K Agbo’Saga,Soria Baouz

doi:10.2174/1874091x01812010113

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 & DiscussionsThe 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.

Highlights

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
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
We have demonstrated that Lys-53 of the 49GGQTKP54 motif of eL42 binds to the CCA-end of a tRNA at the P/E hybrid site on the human 80S ribosomes [4], suggesting that, to the GGQ motif of A-site bound eRF1, the GGQ motif of eL42 is likely to play a functional role at the Peptidyl Transferase Center (PTC)

Summary

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. The CCA-end of a tRNA in the P/P state was shown to interact in majority with a lysyl residue (Lys-197) in the neighborhood of the GGQ motif of eRF1 bound to an A-site stop codon [7] This result suggests that the CCA-arm of PtRNA binds to the GGQ motif of eRF1, as previously predicted by crystallographic data [8]. Lys-65 residue of the 62GANK65 motif of bL12 which was shown to be affinity labeled with a reactive tRNA analogue, matches with Lys-53 of the 49GGQTK53 motif of eL42 [16] which was found labeled with the same reactive tRNA analogue in a previous study [4] These results are consistent with a functional role for both residues at the tRNA-CCA binding site of these proteins on the ribosomes. For clarity, when necessary, we will preferably use the ancient system for naming ribosomal proteins, in order to distinguish between these two almost identical proteins

Methods

Results

Conclusion