Different aa-tRNAs Are Selected Uniformly on the Ribosome

Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208, USA.
Molecular cell (Impact Factor: 14.02). 08/2008; 31(1):114-23. DOI: 10.1016/j.molcel.2008.04.026
Source: PubMed


Ten E. coli aminoacyl-tRNAs (aa-tRNAs) were assessed for their ability to decode cognate codons on E. coli ribosomes by using three assays that evaluate the key steps in the decoding pathway. Despite a wide variety of structural features, each aa-tRNA exhibited similar kinetic and thermodynamic properties in each assay. This surprising kinetic and thermodynamic uniformity is likely to reflect the importance of ribosome conformational changes in defining the rates and affinities of the decoding process as well as the evolutionary "tuning" of each aa-tRNA sequence to modify their individual interactions with the ribosome at each step.

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    • "Although a variety of nonnatural monomer units have been incorporated by native ribosomes, it is clear that these substrates are inherently suboptimal. The tuning of polymerase machines to their natural substrates is exquisite [81]. Therefore, a major design challenge lies not only in the bottom-up aspect of incorporating nonnatural building blocks, but also in reversing the evolutionary optimization of finely tuned machinery and aligning it onto an entirely new path. "
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    • "The protocol used to make ternary complexes containing [γ-32 P]-GTP was similar to the one used in ref (Ledoux and Uhlenbeck, 2008). EFTu at 20 μM was incubated with 5 mCi/mL of [γ-32 P]-GTP and 5 μM of unlabeled GTP for 15 minutes, before the addition of an equal volume of 30 μM aa-tRNA. "
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    • "Although in vitro examinations suggest that translation proceeds uniformly [6], in vivo rates of polypeptide elongation show some heterogeneity [7] [8]. In particular, prolyl-tRNA at the P-site only inefficiently participates in termination [9] [10] or transfer to puromycin [11] [12]. "
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