Full Length Research Paper

Recent advances in the characterization of peptidyl transferase center: Zero-distance labeling of proteins at or near the catalytic site of human 80S or Escherichia coli  70S ribosomes by means of periodate-oxidized tRNA

Soria Baouz¹, Anne Woisard¹, Lila Chenoune¹, Gustave Aguié¹, Gérard Keith¹, Jean-Marie Schmitter², Jean-Pierre Le Caer³ and Codjo Hountondji^1*

¹UPMC Univ Paris 06, Equipe de Photobiologie Moléculaire, Tour 42 (5^ème Etage, couloir 42-32), Case Courrier 60, 2 place Jussieu, F-75251 Paris Cedex 05, France.

²Institut Européen de Chimie et Biologie (CNRS FRE 2247), F-33607, Pessac, France.

³CNRS-UPR 2301, Institut de Chimie des Substances Naturelles (ICSN), Avenue de la Terrasse, F-91198 Gif-Sur-Yvette, France.

*Corresponding author. E-mail: codjo.hountondji@snv.jussieu.fr.

Accepted 12 August, 2009

   Abstract

Periodate-oxidized tRNA (tRNAox), the 2’,3’-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the peptidyl transferase center (PTC), the catalytic site of the large ribosomal subunit. When human 80S or E. coli 70S ribosomes were reacted separately with tRNAox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins was observed. These proteins referred to in this work as rPox1 and rPox2 exhibited comparable physico-chemical properties including apparent molecular weights. In the case of human 80S ribosome, the protein present in the major labeled tRNA-rPox1 covalent complex was identified as the 60S ribosomal protein L36a-like (RPL36AL) by mass spectrometry. The molecular weight of the minor labeled tRNA-rPox2 covalent complex was estimated from the data of the 1-D SDS-PAGE, and a deduced molecular weight of 34,000 + 2,000 Da for the ribosomal protein referred to as rPox2 designated protein RPL5 as the candidate minor labeled protein of human 80S ribosome. Search for candidate ribosomal proteins for the tRNAox-labeled proteins rPox1 and rPox2 of 70S ribosome from E. coli designated RPL2 (M.W. 29,860 Da), the largest eubacterial rP as the tRNAox-labeled protein corresponding to the minor labeled human RPL5, and RPL15 (M.W. 14,980 Da) or RPL16 (M.W. 15,281 Da) as corresponding to the major labeled human RPL36AL.

Key words: Human 80S or E. coli 70S ribosomes/peptidyl transferase center/human RPL36AL and RPL5/P-site or A-site tRNA/periodate-oxidized tRNA.