Mechanism of trans-translation revealed by in vitro studies

Hyouta Himeno,Akira Muto,Daisuke Kurita

doi:10.3389/fmicb.2014.00065

Abstract

tmRNA is a bacterial small RNA having a structure resembling the upper half of tRNA and its 3′ end accepts alanine followed by binding to EF-Tu like tRNA. Instead of lacking a lower half of the cloverleaf structure including the anticodon, tmRNA has a short coding sequence for tag-peptide that serves as a target of cellular proteases. An elaborate coordination of two functions as tRNA and mRNA facilitates an irregular translation termed trans-translation: a single polypeptide is synthesized from two mRNA molecules. It allows resumption of translation stalled on a truncated mRNA, producing a chimeric polypeptide comprising the C-terminally truncated polypeptide derived from truncated mRNA and the C-terminal tag-peptide encoded by tmRNA. Trans-translation promotes recycling of the stalled ribosomes in the cell, and the resulting C-terminally tagged polypeptide is preferentially degraded by cellular proteases. Biochemical studies using in vitro trans-translation systems together with structural studies have unveiled the molecular mechanism of trans-translation, during which the upper and lower halves of tRNA are mimicked by the tRNA-like structure of tmRNA and a tmRNA-specific binding protein called SmpB, respectively. They mimic not only the tRNA structure but also its behavior perhaps at every step of the trans-translation process in the ribosome. Furthermore, the C-terminal tail of SmpB, which is unstructured in solution, occupies the mRNA path in the ribosome to play a crucial role in trans-translation, addressing how tmRNA·SmpB recognizes the ribosome stalled on a truncated mRNA.

Highlights

As mRNA acts as a messenger of the genetic information encoded by the genome, while tRNA acts as a tool for decoding, it is reasonable that they are separated molecules
The function as mRNA has been suggested by an observation that a peptide of 10-amino acid sequence encoded by tmRNA is attached to the truncated C-termini of polypeptides that are exogenously expressed in Escherichia coli with an alanine residue of unknown origin in between them (Tu et al, 1995; Keiler et al, 1996; Figure 1B)
To the tag-encoding region on tmRNA (Keiler et al, 1996). It can address the missing origin of the first alanine residue of the tag-peptide, which is derived from the alanine moiety aminoacylated to tmRNA. This model has been supported by an in vitro study showing that the tmRNA-encoded tag-peptide is synthesized using E. coli cell extract depending on the presence of poly(U) as a truncated mRNA and on the aminoacylation capacity of tmRNA (Muto et al, 1996; Himeno et al, 1997; Nameki et al, 1999a)

Summary

Introduction

As mRNA acts as a messenger of the genetic information encoded by the genome, while tRNA acts as a tool for decoding, it is reasonable that they are separated molecules. CURRENT MODEL OF TRANS -TRANSLATION Based on the structural resemblance of the complex of SmpB and TLD to a tRNA molecule, the binding sites of SmpB shared by translating tRNA, and the interaction of the C-terminal tail of SmpB with the mRNA path, a model of the trans-translation process has been proposed (Figure 3; Kurita et al, 2007, 2010).

Results

Conclusion