Genetic Analysis of the Structure and Function of Transfer Messenger RNA Pseudoknot 1

Jonathan D Dewey,Douglas R Tanner,Allen R Buskirk,Mickey R Miller

doi:10.1074/jbc.m600167200

Abstract

tmRNA rescues stalled ribosomes in eubacteria by forcing the ribosome to abandon its mRNA template and resume translation with tmRNA itself as a template. Pseudoknot 1 (pk1), immediately upstream of this coding region in tmRNA, is a structural element that is considered essential for tmRNA function based on the analysis of pk1 mutants in vitro. pk1 binds near the ribosomal decoding site and may make base-specific contacts with tmRNA ligands. To study pk1 structure and function in vivo, we have developed a genetic selection that ties the life of Escherichia coli cells to tmRNA activity. Mutation of pk1 at 20% per base and selection for tmRNA activity yielded sequences that retain the same pseudoknot fold. In contrast, selection of active mutants from 10(6) completely random sequences identified hairpin structures that functionally replace pk1. Rational design of a hairpin with increased stability using an unrelated sequence yielded a tmRNA mutant with nearly wild-type activity. We conclude that the role of pk1 in tmRNA function is purely structural and that it can be replaced with a variety of hairpin structures. Our results demonstrate that in the study of functional RNAs, the inactivity of a mutant designed to destroy a given structure should not be interpreted as proof that the structure is necessary for RNA function. Such mutations may only destabilize a global fold that could be formed equally well by an entirely different, stable structure.

Highlights

In the trans-translation model of tmRNA function described above, the ribosome switches templates while synthesizing a single polypeptide
Valle et al [9] speculate that pk1 is pulled toward the decoding center as tmRNA transitions from the initial binding complex visualized by cryoelectron microscopy to full accommodation in the A-site resulting in Ala transfer and tmRNA translocation to the ribosomal P-site
Analysis of the homologous Aph(3Ј)-IIIa1⁄7kanamycin co-crystal structure (Fig. 2) reveals that the C-terminal helix of 15 residues plays an important role both structurally and catalytically in binding the substrate [16]. Deletion of these residues leads to loss of function; cells expressing the kanamycin resistance protein (KanR) C-terminal deletion show no more kanamycin resistance than cells lacking the kanR gene

Summary

Introduction

In the trans-translation model of tmRNA function described above, the ribosome switches templates while synthesizing a single polypeptide. The cryoelectron microscopy structure of the 70 S ribosome bound to tmRNA, and its partner protein SmpB reveals that pk are looped around the beak of the 30 S subunit without forming extensive contacts with the ribosome, pk is bound intimately between the beak and the decoding center on the 30 S subunit [9]. Pk are dispensable for trans-translation in vitro, replacing pk with single-stranded sequence destroys tmRNA function [10]. The alteration of single-stranded loop sequences lowers activity, leading to proposals that these nucleotides form Mg2ϩ binding sites [11] or make base-specific contacts with the ribosome [12]. The authors characterized 2451 colonies by replica plating and reported high background (ϳ0.1%); this screen is not robust enough to search through large collections of mutants (libraries) of tmRNA or other components of the tagging machinery

Methods

Results

Conclusion