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New features of the ribosome and ribosomal inhibitors: non-enzymatic recycling, misreading and back-translocation.

Abstract：

:We describe the optimization of a poly(Phe) synthesis system, the conditions of which have been applied for efficient translation of heteropolymeric mRNAs. Here we identify two parameters that are essential to obtain translation at efficiency and accuracy levels equivalent to those in vivo, viz., the fine-tuning of the energy-rich components with an acetyl-phosphate substrate for energy regeneration, as well as the ionic conditions. Applying this system revealed a number of new features: (i) 70S ribosomes are able to recycle within 300 s in a non-enzymatic fashion in the absence of tmRNA. This observation might explain the fact that a knockout of the tmRNA gene ssrA is not lethal for Escherichia coli cells in contrast to other bacterial strains, such as Bacillus subtilis. (ii) The high efficiency of the system was exploited to analyze the misincorporation of various amino acids (resolution limit=1:15,000). No misreading was observed at the middle codon position and only marginal effects were observed at the first one (even when misreading was artificially stimulated 20- to 30-fold), yielding an improved definition of the near-cognate and non-cognate aminoacyl-tRNAs. (iii) Aminoglycosides increase Phe and Lys incorporation about 2-fold in the presence of poly(U) or poly(UUC) and poly(A), respectively, and induce a back-translocation (except hygromycin B) exclusively in the absence of EF-G*GTP, as do the non-related drugs viomycin and edeine.

journal_name

J Mol Biol

journal_title

Journal of molecular biology

authors

Szaflarski W,Vesper O,Teraoka Y,Plitta B,Wilson DN,Nierhaus KH

doi

10.1016/j.jmb.2008.04.060

subject

Has Abstract

pub_date

2008-06-27 00:00:00

pages

193-205

issue

1

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(08)00518-4

journal_volume

380

pub_type

杂志文章

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