Impact of P-Site tRNA and antibiotics on ribosome mediated protein folding: studies using the Escherichia coli ribosome.

Surojit Mondal,Chandana Barat,Sutapa Ray,Bani Kumar Pathak,Hans-Joachim Wieden

doi:10.1371/journal.pone.0101293

Surojit Mondal, Chandana Barat + Show 3 more

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https://doi.org/10.1371/journal.pone.0101293

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Journal: PloS one	Publication Date: Jul 7, 2014
Citations: 59	License type: CC BY 4.0

Affiliation: St Xavier’s College, University of Calcutta

Abstract

BackgroundThe ribosome, which acts as a platform for mRNA encoded polypeptide synthesis, is also capable of assisting in folding of polypeptide chains. The peptidyl transferase center (PTC) that catalyzes peptide bond formation resides in the domain V of the 23S rRNA of the bacterial ribosome. Proper positioning of the 3′ –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is also the center for ribosomal chaperoning activity. The unfolded polypeptide chains interact with the specific nucleotides of the PTC and are released in a folding competent form. In vitro transcribed RNA corresponding to this domain (bDV RNA) also displays chaperoning activity.ResultsThe present study explores the effects of tRNAs, antibiotics that are A- and P-site PTC substrate analogs (puromycin and blasticidin) and macrolide antibiotics (erythromycin and josamycin) on the chaperoning ability of the E. coli ribosome and bDV RNA. Our studies using mRNA programmed ribosomes show that a tRNA positioned at the P-site effectively inhibits the ribosome's chaperoning function. We also show that the antibiotic blasticidin (that mimics the interaction between 3′–CCA end of P/P-site tRNA with the PTC) is more effective in inhibiting ribosome and bDV RNA chaperoning ability than either puromycin or the macrolide antibiotics. Mutational studies of the bDV RNA could identify the nucleotides U2585 and G2252 (both of which interact with P-site tRNA) to be important for its chaperoning ability.ConclusionBoth protein synthesis and their proper folding are crucial for maintenance of a functional cellular proteome. The PTC of the ribosome is attributed with both these abilities. The silencing of the chaperoning ability of the ribosome in the presence of P-site bound tRNA might be a way to segregate these two important functions.

Highlights

The synthesis of proteins and their folding into proper three dimensional structures is of fundamental importance for the maintenance of a functional cellular proteome
We show that the antibiotic blasticidin, a P-site substrate analog [1], is more effective in inhibiting the chaperoning ability of the ribosome and bDV RNA than either puromycin or the macrolide antibiotics
In order to investigate the effect of total E. coli tRNA, we allowed it’s binding to the E. coli 70S ribosome in Buffer-P (Table S1) and the effect of ribosometRNA complex on refolding of denatured BCAII was studied

Summary

Introduction

The synthesis of proteins and their folding into proper three dimensional structures is of fundamental importance for the maintenance of a functional cellular proteome. In the highly crowded cellular milieu a majority of synthesized large polypeptide chains require the assistance of a number of molecular chaperones to either fold or be rescued from misfolding and aggregation [2]. Several studies have demonstrated that the ribosome itself is capable of assisting in folding of proteins spanning a wide range of folds and functions [3]. Proper positioning of the 39 –CCA ends of the A- and P-site tRNAs via specific interactions with the nucleotides of the PTC are crucial for peptidyl transferase activity. This RNA domain is the center for ribosomal chaperoning activity. In vitro transcribed RNA corresponding to this domain (bDV RNA) displays chaperoning activity

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