Dial tm for rescue: tmRNA engages ribosomes stalled on defective mRNAs

Abstract

Ribosomes translate genetic information encoded by mRNAs into protein chains with high fidelity. Truncated mRNAs lacking a stop codon will cause the synthesis of incomplete peptide chains and stall translating ribosomes. In bacteria, a ribonucleoprotein complex composed of tmRNA, a molecule that combines the functions of tRNAs and mRNAs, and small protein B (SmpB) rescues stalled ribosomes. The SmpB–tmRNA complex binds to the stalled ribosome, allowing translation to resume at a short internal tmRNA open reading frame that encodes a protein degradation tag. The aberrant protein is released when the ribosome reaches the stop codon at the end of the tmRNA open reading frame and the fused peptide tag targets it for degradation by cellular proteases. The recently determined NMR structures of SmpB, the crystal structure of the SmpB–tmRNA complex and the cryo-EM structure of the SmpB–tmRNA–EF-Tu–ribosome complex have provided first detailed insights into the intricate mechanisms involved in ribosome rescue.

Introduction

Ribosomes are large ribonucleoprotein complexes of approximately 2.5 MDa that catalyze the mRNA-directed synthesis of proteins during translation. The asymmetric complex is formed by two unequal subunits with distinct functions. The small ribosomal subunit (30S) reads out the genetic information by matching up the mRNA codon with the cognate tRNA anticodon in the decoding center (DC) (Figure 1a). Subsequently, the large ribosomal subunit (50S) catalyzes peptide bond formation between the tRNA-attached amino acid and the growing polypeptide chain in the peptidyl transferase center (PTC).

The translation process starts with the formation of the initiation complex, continues with cycles of polypeptide chain elongation and is terminated by the dissociation of the ribosome–mRNA–nascent chain complex at the end of the message [1^•]. Each elongation cycle (Figure 1a) can be divided into four steps: delivery of the cognate aminoacyl-tRNA (aa-tRNA) to the ribosome; accommodation of the aa-tRNA in the ribosomal A-site; peptide bond formation in the PTC; and translocation of mRNA and tRNAs, enabling the ribosome to slide along the mRNA to the next codon (Figure 1a).

A defective mRNA missing a stop codon stalls ribosomes, because absence of the stop codon prevents proper termination of the translation process (Figure 1b). Three major problems arise for the cell: the ribosome and all ribosomes translating upstream on the same defective mRNA are ‘locked up’; the partially synthesized peptide chains cannot fold correctly and may form toxic aggregates; and the defective mRNA will continue to trap other ribosomes until it is degraded.

Almost ten years ago, Sauer and co-workers [2] discovered a rescue system that overcomes these problems. The trans-translation system, consisting of small protein B (SmpB) and transfer-messenger RNA (tmRNA), rescues stalled ribosomes by switching translation from the defective mRNA to an internal tmRNA open reading frame (ORF). The degradation tag encoded by the ORF targets the incompletely synthesized protein for degradation by cellular proteases such as ClpAP and ClpXP [3]. The stop codon at the end of the tmRNA ORF provides the missing termination signal, which causes the release of the synthesized polypeptide and the dissociation of ribosomal subunits (Figure 1b). The degradation of defective mRNAs might be facilitated by RNase R, a 3′-5′ exonuclease that associates with the SmpB–tmRNA complex [4]. Trans-translation also rescues ribosomes stalled at certain rare codons [5], possibly together with the ribonuclease RelE [6] or other still unidentified cofactors that have recently been reported to cleave mRNAs in the ribosomal A-site [7]. Phylogenic studies have shown that the trans-translation system is highly conserved among bacteria, some mitochondria and chloroplasts [8]. As trans-translation is the topic of several excellent reviews 9., 10.•, this article will focus on the most recent structural advances in the field.

The core component of the rescue system is a chimeric RNA molecule of 350–400 nucleotides, termed tmRNA, which possesses properties of both tRNAs and mRNAs. This abundant molecule, formerly known as SsrA or 10Sa RNA, is estimated to be present in approximately 10% of the molar amount of ribosomes in growing Escherichia coli cells [11] and is essential to several bacterial species. The 5′ and 3′ ends of tmRNA form a tRNA-like domain (TLD) with four extensions, including the T-arm, D-loop, the acceptor stem and the 2a stem 12., 13. (Figure 2a). Pseudo knot (pk) 1, the mRNA-like domain (MLD, which contains the internal ORF encoding the approximately 10 amino acid degradation tag) and pk2–pk4 complete the secondary structure of tmRNA 14., 15.. Replacing individual pseudo knots pk1–pk4 with single-stranded RNA demonstrated that only pk1 is essential for trans-translation [16]. A small approximately 160 amino acid protein component, SmpB, forms a tight complex with tmRNA and is essential for its biological function 17., 18..