In bacteria, ribosomes stalled at the end of truncated messages are rescued by tmRNA, a bifunctional molecule that acts as both a tRNA and mRNA, and SmpB, a small protein that works in concert with tmRNA. Here we present the crystal structure at 3.2 Å resolution of a tmRNA fragment, SmpB and elongation factor Tu bound to the ribosome. The structure shows how SmpB plays the role of both the anticodon loop of tRNA and portions of mRNA to facilitate decoding in the absence of an mRNA codon in the A site of the ribosome, and explains why the tmRNASmpB system does not interfere with normal translation.Transfer-messenger RNA (tmRNA), also known as 10S RNA or SsrA, is a highly structured RNA that combines properties of tRNA and mRNA in one molecule about 350 nucleotides long (Fig. 1A) (1, 2). The tRNA-like domain (TLD) of tmRNA lacks an anticodon stem loop but contains an acceptor arm (3) that can be aminoacylated at its 3′-end by the same alanyl tRNA synthetase that aminoacylates tRNA Ala . A different region of tmRNA contains a short internal open reading frame (ORF) that acts as an mRNA template. In addition, tmRNA contains several pseudoknots and helices.Ribosomes that reach the end of prematurely truncated or defective messages are stalled because the absence of a complete codon in the A site prevents either elongation or normal termination. In bacteria, they are rescued by tmRNA in a process called trans-translation because it involves continuing translation by changing the mRNA template. In this process, EF-Tu delivers tmRNA to the A site of the stalled ribosome. The nascent polypeptide chain is transferred to the alanine on the TLD. Subsequently, translocation brings the first codon of the ORF into the A site of the ribosome and translation resumes using the ORF as the mRNA (2). The short sequence coded by the ORF thus added to the C-terminus of the partially synthesized protein acts as a degradation tag (4). Thus tmRNA acts both to rescue ribosomes as well as to target incompletely synthesized proteins for degradation.The binding of tmRNA to stalled ribosomes requires the protein SmpB (5), which can bind to tmRNA simultaneously with EF-Tu (6). Crystal structures of SmpB in complex with the TLD suggest that the protein substitutes for the missing anticodon stem inside the ribosome (7, 8), which was supported by electron microscopy studies at ~15 Å resolution (9, 10). A previous electron microscopy study found two molecules of SmpB with tmRNA in the ribosome, with the carboxy-terminus of one of them near the decoding center of the 30S ribosomal subunit (11). The observed proximity to the decoding center agrees with hydroxyl * To whom correspondence should be addressed at ramak@mrc-lmb. (14). The mechanism by which tmRNA and SmpB acting in concert can facilitate "decoding" in the absence of codon-anticodon base pairing has remained unclear.Here we present the crystal structure of the Thermus thermophilus ribosome bound to a complex consisting of a fragment of tmRNA (tmRNAΔ m ) along with SmpB and EF-Tu t...

In eubacterial translation, lack of a stop codon on the mRNA results in a defective, potentially toxic polypeptide stalled on the ribosome. Bacteria possess a specialized mRNA, called transfer messenger RNA (tmRNA), to rescue such a stalled system. tmRNA contains a transfer RNA (tRNA)-like domain (TLD), which enters the ribosome as a tRNA and places an ORF into the mRNA channel. This ORF codes for a signal marking the polypeptide for degradation and ends in a stop codon, leading to release of the faulty polypeptide and recycling of the ribosome. The binding of tmRNA to the stalled ribosome is mediated by small protein B (SmpB). By means of cryo-EM, we obtained a density map for the preaccommodated state of the tmRNA⅐SmpB⅐EF-Tu⅐70S ribosome complex with much improved definition for the tmRNA-SmpB complex, showing two SmpB molecules bound per ribosome, one toward the A site on the 30S subunit side and the other bound to the 50S subunit near the GTPase-associated center. tmRNA is strongly attached to the 30S subunit head by multiple contact sites, involving most of its pseudoknots and helices. The map clarifies that the TLD is located near helix 34 and protein S19 of the 30S subunit, rather than in the A site as tRNA for normal translation, so that the TLD is oriented toward the ORF. elongation factor Tu ͉ preaccommodated state ͉ rescue mechanism ͉ transtranslation ͉ transfer RNA-like domain

Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA-SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA-SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA-SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA-SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.

We have used a transgenic animal model, which constitutively develops hepatocarcinoma (Antithrombin III SV40 T large Antigen: ASV), to study the involvement of Annexin 1 (ANX1) in liver regeneration and malignant transformation. Primary hepatocytes isolated from normal mice did not express ANX1. In contrast, ANX1 was strongly expressed in hepatocytes of transgenic mice during constitutive development of hepatocarcinoma. In ASV transgenic mice, an elevated ANX1 level preceded the appearance of the tumor, indicating that it could be a good marker in the diagnosis of cancer. One-third hepatectomy in normal mice resulted in stimulation of ANX1 synthesis and phosphorylation. This upregulation correlated with increased synthesis of EGF and consequently with increased phosphorylation of the EGF receptor (EGF-R). Stable transfection of a hepatocyte cell line derived from ASV transgenic mice (mhAT2) with antisense complementary DNA for ANX1 reduced the proliferation rate as well as cytosolic phospholipase A 2 (cPLA 2 ) activity. Thus, ANX1 expression and phosphorylation could be a factor implicated in liver regeneration and tumorigenesis, either through modulation of cPLA 2 activity or EGF-R function. (HEPATOLOGY 2000;31:371-380.)Annexin 1 (ANX1) is a member of a multigene family of Ca 2ϩ /phospholipid binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase. 1 We have shown that ANX1, is transcriptionally regulated by interleukin-6 (IL-6) and phorbol myristate acetate (PMA) in human lung adenocarcinoma cell (A 549) through the induction of C/EBP␤ transcription factor. Based on these data we proposed a dual role for ANX1 in cellular differentiation 2 and as a new acute-phase protein. 3 These results prompted us to investigate the role of ANX1 in the liver. While we were carrying these experiments, ANX1 was reported to be strongly expressed in human hepatocellular (hcc) carcinoma cells, suggesting that its induction in the liver could be associated with malignant transformation. 4,5 We therefore addressed the question of the role of ANX1 in liver transformation.Recently, transgenic mice (ASV) expressing the SV40 early region coding for large T antigen under the control of the liver-specific human antithrombin III gene promoter have been created in our laboratory to study oncogenic transformation on hepatocyte function. 6 Phenotypically, the animals present a hepatocellular carcinoma, with a neoplasia at 3 to 9 weeks and later (after tumor development) a severe dysplasia, an augmentation of hepatocytes in S phases, an important cytolysis (necrosis), and an early apoptosis (2.5%-5%). Up to 25 weeks no major difference was obvious between the liver weight of transgenic (ASV) and control mice. After that, liver weight progressively increased reaching sevenfold the normal liver weight at the terminal stage of hcc. Eight old mice die for a general disorder and an important cachexia. 6 Using this model as paradigm for our study of the role of ANX1 in liver, we now report that ANX1 is strongly ...

Viruses modulate ecosystems by directly altering host metabolisms through auxiliary metabolic genes. However, viral genomes are not known to encode the core components of translation machinery, such as ribosomal proteins (RPs). Here, using reference genomes and global-scale viral metagenomic datasets, we identify 14 different RPs across viral genomes arising from cultivated viral isolates and metagenome-assembled viruses. Viruses tend to encode dynamic RPs, easily exchangeable between ribosomes, suggesting these proteins can replace cellular versions in host ribosomes. Functional assays confirm that the two most common virus-encoded RPs, bS21 and bL12, are incorporated into 70S ribosomes when expressed in Escherichia coli. Ecological distribution of virus-encoded RPs suggests some level of ecosystem adaptations as aquatic viruses and viruses of animal-associated bacteria are enriched for different subsets of RPs. Finally, RP genes are under purifying selection and thus likely retained an important function after being horizontally transferred into virus genomes.

Transfer‐messenger RNA (tmRNA), also known as SsrA or 10Sa RNA, is a bacterial ribonucleic acid that recycles 70S ribosomes stalled on problematic messenger RNAs (mRNAs) and also contributes to the degradation of incompletely synthesized peptides. tmRNA acts initially as transfer RNA (tRNA), being aminoacylated at its 3′‐end by alanyl‐tRNA synthetase, to add alanine to the stalled polypeptide chain. Resumption of translation ensues not on the mRNA on which the ribosomes were stalled but at an internal position in tmRNA. Termination soon occurs, tmRNA recruiting the appropriate termination factors allowing the release of the tagged protein that is subsequently recognized and degraded by specific cytoplasmic and periplasmic proteases, and permits ribosome recycling. Recent data suggest that tmRNA tags bacterial proteins in three other instances; when ribosomes stall at internal sites; during ‘readthrough’ of canonical termination codons; and when ribosomes are at the termination codon of intact messages. The importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is also discussed. Recent in vivo and in vitro studies have identified novel ligands of tmRNA. Based on the available experimental evidences, an updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented.

In bacteria, trans-translation is the main rescue system, freeing ribosomes stalled on defective messenger RNAs. This mechanism is driven by small protein B (SmpB) and transfer-messenger RNA (tmRNA), a hybrid RNA known to have both a tRNA-like and an mRNA-like domain. Here we present four cryo-EM structures of the ribosome during trans-translation at resolutions from 3.0 to 3.4 Å. These include the high-resolution structure of the whole pre-accommodated state, as well as structures of the accommodated state, the translocated state, and a translocation intermediate. Together, they shed light on the movements of the tmRNA-SmpB complex in the ribosome, from its delivery by the elongation factor EF-Tu to its passage through the ribosomal A and P sites after the opening of the B1 bridges. Additionally, we describe the interactions between the tmRNA-SmpB complex and the ribosome. These explain why the process does not interfere with canonical translation.