Abstract
The translocation reaction of two tRNAs on the ribosome during elongation of the nascent peptide chain is one of the most puzzling reactions of protein biosynthesis. We show here that the ribosomal contact patterns of the two tRNAs at A and P sites, although strikingly different from each other, hardly change during the translocation reaction to the P and E sites, respectively. The results imply that the ribosomal micro-environment of the tRNAs remains the same before and after translocation and thus suggest that a movable ribosomal domain exists that tightly binds two tRNAs and carries them together with the mRNA during the translocation reaction from the A-P region to the P-E region. These findings lead to a new explanation for the translocation reaction.
Highlights
Ribosomes contain three tRNA binding sites, the A, P, and E site, viz. the A site where the decoding takes place, the P site, where the peptidyl-tRNA is located before peptide bond formation, and the E site, which is specific for deacylated tRNA [1,2,3,4,5,6]
The three basic reactions of an elongation cycle are 1) occupation of the A site by an aminoacyl-tRNA according to the corresponding codon at the A site, 2) peptidebond formation, which transfers the already synthesized peptidyl residue to the aminoacyl-tRNA so that the resulting peptidyl-tRNA, prolonged by an amino acid, resides at the A site, and 3) the translocation reaction, which moves the peptidyl-tRNA to the P site and the deacylated tRNA to the E site
The thioated deacylated tRNAPhe and AcPhe-tRNA were bound to poly(U)-programmed ribosomes to either P or A sites, respectively, and the corresponding contact patterns were analyzed before and after translocation
Summary
The sources of chemicals and plasmids are described by Dabrowski et al [10]. Preparation of Thioated tRNAPhe and AcPhe-tRNA—The thioated tRNAPhe was obtained by in vitro T7-dependent transcription [10]. Binding of deacylated thioated tRNAPhe to the PPre position was performed by taking a 50-l aliquot from the Pi binding assay and adding 60 pmol of native Ac-[14C]Phe-tRNAPhe in step two The incubation of this step was for 30 min at 37 °C. Thioated Ac-[14C]PhetRNAPhe was bound to the A position by incubating 80 pmol of 70 S ribosomes, 104 pmol of native deacylated tRNAPhe, and 50 g of poly(U) in step one and adding 104 pmol of thioated Ac-[14C]Phe-tRNAPhe in step two. Half of the aliquot obtained in step two was further processed in step three, yielding ribosomal complexes with thioated Ac-[14C]PhetRNAPhe in PPost position and a native deacylated tRNA in the E site. The result of the multiplication gives the intensity of a band of ribosomal bound AcPhe-tRNA or tRNAPhe relative to the respective control band of AcPhe-tRNA or tRNAPhe in solution, respectively (vertical comparison). If the Pi and PPre site of tRNAPhe are compared, the average deviation 0.11 is low
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