Binding of the 3' terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation.
A key event in ribosomal protein synthesis is the translocation of deacylated tRNA, peptidyl tRNA and mRNA, which is catalyzed by elongation factor G (EF-G) and requires GTP. To address the molecular mechanism of the reaction we have studied the functional role of a tRNA exit site (E site) for tRNA release during translocation. We show that modifications of the 3' end of tRNAPhe, which considerably decrease the affinity of E-site binding, lower the translocation rate up to 40-fold. Furthermore, 3'-end modifications lower or abolish the stimulation by P site-bound tRNA of the GTPase activity of EF-G on the ribosome. The results suggest that a hydrogen-bonding interaction of the 3'-terminal adenine of the leaving tRNA in the E site, most likely base-pairing with 23S rRNA, is essential for the translocation reaction. Furthermore, this interaction stimulates the GTP hydrolyzing activity of EF-G on the ribosome. We propose the following molecular model of translocation: after the binding of EF-G.GTP, the P site-bound tRNA, by a movement of the 3'-terminal single-stranded ACCA tail, establishes an interaction with 23S rRNA in the adjacent E site, thereby initiating the tRNA transfer from the P site to the E site and promoting GTP hydrolysis. The co-operative interaction between the E site and the EF-G binding site, which are distantly located on the 50S ribosomal subunit, is probably mediated by a conformational change of 23S rRNA.
Lill
R
R
Institut für Molekularbiologie, Universität Witten/Herdecke, FRG.
Robertson
J M
JM
Wintermeyer
W
W
D001483:Base Sequence; D001665:Binding Sites; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D007700:Kinetics; D010445:Peptide Elongation Factors / Q000378:metabolism; D014176:Protein Biosynthesis; D012335:RNA, Ribosomal / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000378:metabolism*; D012345:RNA, Transfer, Amino Acid-Specific / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D010445:Peptide Elongation Factors; D012335:RNA, Ribosomal; D012338:RNA, Ribosomal, 23S; D012345:RNA, Transfer, Amino Acid-Specific; D012360:RNA, Transfer, Phe; D020963:GTP Phosphohydrolase-Linked Elongation Factors