Contributions of Pseudoknots and Protein SmpB to the Structure and Function of tmRNA in trans-Translation

Iwona K Wower,Christian Zwieb,Jacek Wower

doi:10.1074/jbc.m410488200

Iwona K Wower, Christian Zwieb + Show 1 more

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https://doi.org/10.1074/jbc.m410488200

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Abstract

Bacteria contain transfer-messenger RNA (tmRNA), a molecule that during trans-translation tags incompletely translated proteins with a small peptide to signal the proteolytic destruction of defective polypeptides. TmRNA is composed of tRNA- and mRNA-like domains connected by several pseudoknots. Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli tmRNA in trans-translation. Site-directed mutagenesis experiments showed that pseudoknot 2 and the abutting helix 5 were particularly important for the binding of ribosomal protein S1 to tmRNA. Pseudoknot 4 not only facilitated tmRNA maturation but also promoted tagging. In addition, the three pseudoknots (pk2 to pk4) were shown to play a significant role in the proper folding of the tRNA-like domain. Protein SmpB enhanced tmRNA processing, suggesting a new role for SmpB in trans-translation. Taken together, these results provide unanticipated insights into the functions of the pseudoknots and protein SmpB during tmRNA folding, maturation, and protein synthesis.

Highlights

An interruption of the elongation step of protein synthesis results in the production of truncated proteins and leaves ribosomes stalled at the 3Ј end of mRNA templates lacking a stop codon(s)
Using truncated ribosomal protein L27 as a reporter for tagging in vitro and in vivo, we have developed exceptionally sensitive assays to study the role of Escherichia coli transfer-messenger RNA (tmRNA) in trans-translation
A truncated L27 encoded by mRNA lacking a stop codon(s) was expected to be suitable for studying tmRNA-dependent tagging in cell lysates by detection of polypeptides using SDS-PAGE and staining with Coomassie Blue

Summary

Introduction

An interruption of the elongation step of protein synthesis results in the production of truncated proteins and leaves ribosomes stalled at the 3Ј end of mRNA templates lacking a stop codon(s). The entry of tmRNA into a stalled E. coli ribosome has been visualized by cryo-electron microscopy [7] At this particular step of trans-translation, the TLD, pk, and the MLD contact the ribosome, whereas the pk to pk segment forms an arc that remains outside the ribosome. Previous in vitro experiments suggested that pk is essential for tmRNA folding and protein tagging, whereas the three remaining pseudoknots, pk through pk, are interchangeable and replaceable with stretches of single-stranded RNA (14 –16) These data were derived exclusively using an insensitive assay in which poly(U)-programmed ribosomes produce hydrophobic polypeptides (polyphenylalanine) that are inefficiently tagged and because of their heterogeneity are difficult to analyze qualitatively

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