Crosslinking of Translation Factor EF-G to Proteins of the Bacterial Ribosome Before and After Translocation

• Published in: Journal of molecular biology 2007-05, Vol.368 (5), p.1412-1425
• Main Authors: Nechifor, Roxana, Wilson, Kevin S.
• Format: Article
• Language: English
• Subjects: Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; bacterial ribosome; chemical crosslinking; Cross-Linking Reagents; Cysteine - metabolism; elongation factor; Escherichia coli - genetics; Escherichia coli - metabolism; Guanosine Triphosphate - metabolism; Models, Molecular; Molecular Structure; Peptide Elongation Factor G - chemistry; Peptide Elongation Factor G - metabolism; Protein Structure, Tertiary; ribosomal protein; Ribosomal Proteins - metabolism; Ribosomes - chemistry; Ribosomes - metabolism; RNA, Messenger - metabolism; RNA, Transfer - metabolism; Translocation, Genetic
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.03.009

Elongation factor G (EF-G) promotes the translocation of tRNA and mRNA in the central cavity of the ribosome following the addition of each amino acid residue to a growing polypeptide chain. tRNA/mRNA translocation is coupled to GTP hydrolysis, catalyzed by EF-G and activated by the ribosome. In this study we probed EF-G interactions with ribosomal proteins (r-proteins) of the bacterial ribosome, by using a combination of chemical crosslinking, immunoblotting and mass spectroscopy analyses. We identified three bacterial r-proteins (L7/L12, S12 and L6) crosslinked to specific residues of EF-G in three of its domains (G′, 3 and 5, respectively). EF-G crosslinks to L7/L12 and S12 were indistinguishable when EF-G was trapped on the ribosome before or after tRNA/mRNA translocation had occurred, whereas a crosslink between EF-G and L6 formed with greater efficiency before translocation had occurred. EF-G crosslinked to L7/L12 was capable of catalyzing multiple rounds of GTP hydrolysis, whereas EF-G crosslinked to S12 was inactive in GTP hydrolysis. These results imply that during the GTP hydrolytic cycle EF-G must detach from S12 within the central cavity of the ribosome, while EF-G can remain associated with L7/L12 located on one of the peripheral stalks of the ribosome. This mechanism may ensure that a single GTP molecule is hydrolyzed for each tRNA/mRNA translocation event.