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Trans-translation is a quality-control process, activated upon premature termination of protein elongation, which recycles stalled ribosomes and degrades incomplete polypeptides. These functions are facilitated by transfer-messenger RNA (tmRNA, also called 10Sa RNA or SsrA RNA), a small stable RNA molecule encoded by the SsrA gene found in bacteria, chloroplasts and mitochondria. Most tmRNAs consist of a tRNA- and an mRNA-like domain connected by up to four pseudoknots. Comparative sequence analysis provided the first insight into tmRNA secondary and three-dimensional structure. Studies of the E. coli tmRNA in vitro and in vivo demonstrated that tmRNA functions as a ribonucleoprotein (RNP) complex with elongation factor Tu (EF-Tu), protein SmpB and ribosomal protein S1. The tRNA-like and mRNA-like activities of tmRNA mark prematurely terminated proteins for degradation by attaching to their C-termini peptide tags, which are recognized by numerous proteases. Studies aimed at understanding the details of the molecular mechanisms of trans-translation are ongoing. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mt), is one of the deadliest infectious diseases. The rise of multidrug-resistant strains represents a major public health threat, requiring new therapeutic options. Bacterial aminoacyl-tRNA synthetases (aaRS) have been shown to be highly promising drug targets, including for TB treatment. These enzymes play an essential role in translating the DNA gene code into protein sequence by attaching specific amino acid to their cognate tRNAs. They have multiple binding sites that can be targeted for inhibitor discovery: amino acid binding pocket, ATP binding pocket, tRNA binding site and an editing domain. Recently we reported several high-resolution structures of M. tuberculosis phenylalanyl-tRNA synthetase (MtPheRS) complexed with tRNA Phe and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. Here, using Nucleic Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) we identified fragments that bind to MtPheRS and we determined crystal structures of their complexes with MtPheRS/tRNA Phe . All the binders interact with the L-Phe amino acid binding site. The analysis of interactions of the new compounds combined with adenylate analog structure provides insights for the rational design of anti-tuberculosis drugs. The 3' arm of the tRNA Phe in all the structures was disordered with exception of one complex with D-735 compound. In this structure the 3' CCA end of the acceptor stem is observed in the editing domain of MtPheRS providing insights regarding the post-transfer editing activity of class II aaRS., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Andrzej Joachimiak reports financial support was provided by National Institutes of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Masson SAS.)

Nucleic acids are versatile materials capable of forming smart nanocarriers with highly controllable therapeutic delivery. DNA-gated release is a mechanism by which DNA oligonucleotides physically block the release of encapsulated drugs from porous nanoparticles. We extend this mechanism to be used with drugs bound to the surface of DNA-capped gold nanoparticles (AuNPs). We investigated DNA monolayers of different thicknesses and hybridization states to determine how DNA surface architecture can affect the release of a template drug bound to the gold surface. DNA layers are investigated on the planar gold surface via quartz crystal microbalance with dissipation and on AuNPs via dynamic light scattering. The resultant layer architectures were studied for their effect on the release rate of drugs. We observed that varying DNA architectures on AuNPs result in different release rates of the drug. The rate of drug release can be slowed using either folded or randomly coiled DNA sequences, which act as a physical barrier to diffusion. DNA monolayers with upright orientation release drugs more quickly. When the longer single-stranded DNA is used, the drug release is slowed even further. However, even upright DNA layers provide a barrier to drug diffusion at longer sequence lengths. We hypothesize that it is the architecture of the DNA layer, influenced by the folded or upright orientation of individual DNA molecules, that affects the free diffusion of the drug away from the AuNP surface. This mechanism may improve the biological availability of many surface-bound drugs on solid, DNA-capped nanoparticles.

The internal ribosome entry site (IRES) RNA of bovine viral diarrhoea virus (BVDV), an economically significant Pestivirus , is required for the cap-independent translation of viral genomic RNA. Thus, it is essential for viral replication and pathogenesis. We applied a combination of high-throughput biochemical RNA structure probing (SHAPE-MaP) and in silico modelling approaches to gain insight into the secondary and tertiary structures of BVDV IRES RNA. Our study demonstrated that BVDV IRES RNA in solution forms a modular architecture composed of three distinct structural domains (I-III). Two regions within domain III are represented in tertiary interactions to form an H-type pseudoknot. Computational modelling of the pseudoknot motif provided a fine-grained picture of the tertiary structure and local arrangement of helices in the BVDV IRES. Furthermore, comparative genomics and consensus structure predictions revealed that the pseudoknot is evolutionarily conserved among many Pestivirus species. These studies provide detailed insight into the structural arrangement of BVDV IRES RNA H-type pseudoknot and encompassing motifs that likely contribute to the optimal functionality of viral cap-independent translation element.

The replication transcription complex (RTC) from the virus SARS-CoV-2 is responsible for recognizing and processing RNA for two principal purposes. The RTC copies viral RNA for propagation into new virus and for ribosomal transcription of viral proteins. To accomplish these activities, the RTC mechanism must also conform to a large number of imperatives, including RNA over DNA base recognition, basepairing, distinguishing viral and host RNA, production of mRNA that conforms to host ribosome conventions, interfacing with error checking machinery, and evading host immune responses. In addition, the RTC will discontinuously transcribe specific sections of viral RNA to amplify certain proteins over others. Central to SARS-CoV-2 viability, the RTC is therefore dynamic and sophisticated. We have conducted a systematic structural investigation of three components that make up the RTC: Nsp7, Nsp8, and Nsp12 (also known as RNA-dependent RNA polymerase). We have solved high-resolution crystal structures of the Nsp7/8 complex, providing insight into the interaction between the proteins. We have used small-angle x-ray and neutron solution scattering (SAXS and SANS) on each component individually as pairs and higher-order complexes and with and without RNA. Using size exclusion chromatography and multiangle light scattering-coupled SAXS, we defined which combination of components forms transient or stable complexes. We used contrast-matching to mask specific complex-forming components to test whether components change conformation upon complexation. Altogether, we find that individual Nsp7, Nsp8, and Nsp12 structures vary based on whether other proteins in their complex are present. Combining our crystal structure, atomic coordinates reported elsewhere, SAXS, SANS, and other biophysical techniques, we provide greater insight into the RTC assembly, mechanism, and potential avenues for disruption of the complex and its functions., (Published by Elsevier Inc.)

We have developed a novel, nanosized drug carrier with high-therapeutic payload, controllable release, and the potential for active tumor targeting. It consists of a 15 nm gold nanoparticle with dense surface loading of DNA duplexes. We utilize the natural intercalating behavior of daunomycin to load the drug between DNA base pairs. We obtained a high-therapeutic payload of >1,000 drug molecules per gold nanoparticle (AuNP), one of the highest loadings reported in literature to date. We have engineered unique DNA sequences to control release of daunomycin for over 48 hr and show higher cell death compared to equivalent concentrations of free daunomycin. We have also explored cell internalization mechanisms to identify the pathways by which our gold nanoparticles enter the cell. This nanocarrier is in the ideal size range of 16-100 nm in diameter to utilize the enhanced permeability and retention effect for passive targeting to tumors. Our AuNP platform is effective as a therapeutic drug delivery device and can easily incorporate any aptamer of choice through complementary base pairing. Our work has produced an innovative nanoscale drug-delivery platform potentially leading to personalized cancer therapies through careful selection of aptamers and an adjustable drug release profile., (© 2020 Wiley Periodicals LLC.)

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.

Intercellular communication occurring by cell-to-cell contacts and via secreted messengers trafficked through extracellular vehicles is critical for regulating biological functions of multicellular organisms. Recent research has revealed that non-coding RNAs can be found in extracellular vesicles consistent with a functional importance of these molecular vehicles in virus propagation and suggesting that these essential membrane-bound bodies can be highjacked by viruses to promote disease pathogenesis. Newly emerging evidence that coronaviruses generate non-coding RNAs and use extracellular vesicles to facilitate viral pathogenicity may have important implications for the development of effective strategies to combat COVID-19, a disease caused by infection with the novel coronavirus, SARS-CoV-2. This article provides a short overview of our current understanding of the interactions between non-coding RNAs and extracellular vesicles and highlights recent research which supports these interactions as potential therapeutic targets in the development of novel antiviral therapies.

The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Trans-translation is catalyzed by ribonucleprotein complexes composed of SmpB protein and transfer-messenger RNA. They release stalled ribosomes from truncated mRNAs and tag defective proteins for proteolytic degradation. Comparative sequence analysis of bacterial tmRNAs provides considerable insights into their secondary structures in which a tRNA-like domain and an mRNA-like region are connected by a variable number of pseudoknots. Progress toward understanding the molecular mechanism of trans-translation is hampered by our limited knowledge about the structure of tmRNA:SmpB complexes., Results: Complexes consisting of M. tuberculosis tmRNA and E. coli SmpB tag truncated proteins poorly in E. coli. In contrast, the tagging activity of E. coli tmRNA is well supported by M. tuberculosis SmpB that is expressed in E. coli. To investigate this incompatibility, we constructed 12 chimeric tmRNA molecules composed of structural features derived from both E. coli and M. tuberculosis. Our studies demonstrate that replacing the hp5-pk2-pk3-pk4 segment of E. coli tmRNA with the equivalent segment of M. tuberculosis tmRNA has no significant effect on the tagging efficiency of chimeric tmRNAs in the presence of E. coli SmpB. Replacing either helices 2b-2d, the single-stranded part of the ORF, pk1, or residues 79-89 of E. coli tmRNA with the equivalent features of M. tuberculosis tmRNA yields chimeric tmRNAs that are tagged at 68 to 88 percent of what is observed with E. coli tmRNA. Exchanging segments composed of either pk1 and the single-stranded segment upstream of the ORF or helices 2b-2d and pk1 results in markedly impaired tagging activity., Conclusion: Our observations demonstrate the existence of functionally important but as yet uncharacterized structural constraints in the segment of tmRNA that connects its TLD to the ORF used for resuming translation. As trans-translation is important for the survival of M. tuberculosis, our work provides a new target for pharmacological intervention against multidrug-resistant tuberculosis.

RNA aptamers can fold into complex structures and bind with high affinity and selectivity to various macromolecules, viruses, and cells. They are isolated from a large pool of nucleic acids by a conceptually straightforward iterative selection process called SELEX. Aptamers have enormous potential as therapeutics due to their ability to bind to proteins and specifically inhibit their functions with minimal or no harmful side-effects. The first aptamer therapeutic was FDA approved in 2005 and a number of novel aptamer-based therapeutics are currently undergoing clinical trials for treating diseases such as macular degeneration, choroidal neovascularization, intravascular thrombus, acute coronary syndrome, von Willebrand factor related disorders, von Hippel-Lindau syndrome (VHL), angiomas, acute myeloid leukemia, renal cell carcinoma, non-small cell lung cancer, thrombotic thrombocytopenic purpura, and several others. In this review, we present aptamers in on-going, completed, and terminated clinical studies highlighting their mechanism of action as well as the inherent challenges of aptamer production and use., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We have synthesized, characterized, and optimized a novel nano drug-delivery carrier that utilizes the versatile properties of nucleic acid for programmable and on-demand drug release. The drug-delivery carrier consists of 15 nm gold nanoparticles (AuNPs) functionalized with drug binding DNA aptamers via single-stranded (ss) anchor DNA. The presence of anchor DNA makes the nanocarrier flexible to be reprogrammed with various aptamers. Under the optimum binding conditions (0.4 M NaCl and 4 μM DNA), a maximum of 101 ± 8 anchor DNA strands were conjugated per particle. On binding DNA-aptamer:drug complexes to AuNPs, a maximum of 35 neomycin molecules were bound per AuNP. Controlled and extended release of drug from the synthesized carrier was obtained by temperature and affinity modulations. Furthermore, for the first time, we demonstrated that neomycin could bind to DNA with very high affinity (K(d) = 98.101 nM). This DNA-based nanocarrier, designed using the principles of molecular biology, is expected to impact a number of treatment strategies., From the Clinical Editor: In this basic science work, the authors demonstrate the feasibility of utilizing a novel nano drug-delivery carrier with the versatile properties of nucleic acid for programmable and on-demand drug release., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Foot-and-mouth disease virus (FMDV) uses an internal ribosome entry site (IRES), a highly structured segment of its genomic RNA, to hijack the translational apparatus of an infected host. Computational analysis of 162 type II picornavirus IRES RNA sequences yielded secondary structures that included only base pairs supported by comparative or experimental evidence. The deduced helical sections provided the foundation for a hypothetical three-dimensional model of FMDV IRES RNA. The model was further constrained by incorporation of data derived from chemical modification and enzymatic probing of IRES RNAs as well as high-resolution information about IRES RNA-bound proteins.

The internal ribosomal entry site (IRES) RNA of bovine viral diarrhea virus (BVDV) has been implicated in virus propagation. To gain insight into the structure and potential function of the BVDV IRES RNA, we collected and aligned 663 of its sequences. Compensatory Watson-Crick and wobble G·U pairs were investigated to establish phylogenetically supported secondary structures for each of the BVDV IRES RNA sequences. The extensively folded BVDV IRES RNAs were composed of helices 2, 3 and 4. Helix 2 consisted of five helical sections. Helix 3 contained sections 3a to 3j as well as six helical insertions 3.1-3.6. Sections 3a and 3b together with helices 3.6 and 4 formed an RNA pseudoknot. Two highly variable regions corresponded to hairpins 3j and 3.4. Three-dimensional modeling of the BVDV-1b strain Osloss IRES RNA predicted an elongated structure with approximate dimensions of 170 Å by 65 Å by 90 Å. The model of the IRES RNA-ribosome complex suggested proximity between helix 2 of the BVDV IRES and ribosomal proteins S5 and S25., (Published by Elsevier B.V.)

Trans-translation is a universal quality-control process eubacteria use to degrade incompletely synthesized proteins and rescue ribosome stalled on defective mRNAs. This process is facilitated by a ribonucleoprotein complex composed of transfer-messenger RNA (tmRNA)-a chimera made of a tRNA-like molecule and a short open reading frame (ORF) -and small protein B (SmpB). Determination of the structure of tmRNA and SmpB in complex with the ribosome, at the stage when translation has resumed on tmRNA, has provided an increased understanding of the structure of tmRNA as it transits the ribosome, and unique insights into the complex mechanism of template switching on the ribosome and SmpB-driven selection of the correct reading frame on tmRNA's ORF.

Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable 'arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.

tmRNA employs both tRNA-like and mRNA-like properties as it rescues stalled bacterial ribosomes, while targeting the defective mRNA and incomplete nascent protein for degradation. We describe variation of the tmRNA gene (ssrA) and how it informs tmRNA structure and function. Endosymbiont tmRNAs tend to lose secondary structure and length in the mRNA-like region as nucleotide composition drifts with that of the whole genome. A dramatic gene structure variation is circular permutation, which produces two-piece tmRNAs in three bacterial lineages; new sequences blur these lineages. We present evidence that Sinorhizobium two-piece tmRNA retains the 5'-triphosphate of transcriptional initiation and predict a new structure at the 5' end of cyanobacterial two-piece tmRNA precursor. ssrA is a target for some mobile DNAs and a passenger on others. It has been found interrupted (but not functionally disrupted) by mobile elements such as group I introns, genomic islands and palindromic elements. The alphaproteobacterial permuted genes are significantly less frequently interrupted by genomic islands than are their standard counterparts, yet are a hotspot for insertion or swapping of rickettsial palindromic elements, in contrast to other rickettsial loci that show steady decay of a single ancestral element. Bacteriophages, plasmids and genomic islands can carry tmRNA genes; we describe a native bacterial ssrA disrupted by insertion of a genomic island that carries its own ssrA, a genome encoding both one- and two-piece tmRNA, and a phage encoding a tmRNA variant lacking the mRNA-like function, which may counteract host tmRNA during infection.

Biohybrid platforms such as synthetic polymer networks engineered from artificial and natural materials hold immense potential as drug and gene delivery vehicles. Here, we report the synthesis and characterization of novel polymer networks that release oligonucleotide sequences via enzymatic and physical triggers. Chemical monomers and acrylated oligonucleotides were copolymerized into networks, and phosphoimaging revealed that 70% of the oligonucleotides were incorporated into the networks. We observed that the immobilized oligonucleotides were readily cleaved when the networks were incubated with the type II restriction enzyme BamHI. The diffusion of the cleaved fragments through the macromolecular chains resulted in relatively constant release profiles very close to zero-order. To our knowledge, this is the first study which harnesses the sequence-specificity of restriction endonucleases as triggering agents for the cleavage and release of oligonucleotide sequences from a synthetic polymer network. The polymer networks exhibited an oligonucleotide diffusion coefficient of 5.6 x 10(-8) cm(2)/s and a diffusional exponent of 0.92. Sigmoidal temperature responsive characteristics of the networks matched the theoretical melting temperature of the oligonucleotides and indicated a cooperative melting transition of the oligonucleotides. The networks were also triggered to release a RNA-cleaving deoxyribozyme, which degraded a HIV-1 mRNA transcript in vitro. To tailor release profiles of the oligonucleotides, we controlled the structure of the macromolecular architecture of the networks by varying their cross-linking content. When incubated with DNase I, networks of cross-linking content 0.15%, 0.22%, and 0.45% exhibited oligonucleotide diffusion coefficients of 1.67 x 10(-8), 7.65 x 10(-9), and 2.7 x 10(-9) cm(2)/s, and diffusional exponents of 0.55, 0.8, and 0.8, respectively. The modular nature of our platform promises to open new avenues for the creation and optimization of a rich toolbox of novel drug and gene delivery platforms. We anticipate further inquiry into nucleic acid based programmable on-demand switches and modulatory devices of exquisite sensitivity and control.

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3' end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA- and mRNA-like domains that are usually connected by four pseudoknots (pk1-pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Deltah4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.

The transfer-messenger ribonucleoprotein (tmRNP), which is composed of RNA and a small protein, small protein B (SmpB), recycles ribosomes that are stalled on broken mRNAs lacking stop codons and tags the partially translated proteins for degradation. Although it is not yet understood how the ribosome gets from the 3' end of the truncated message onto the messenger portion of the tmRNA to add the tag, a recent study in BMC Biology has shed some light on this astonishing feat.

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL http://psyche.uthct.edu/dbs/tmRDB/tmRDB.html with mirror sites located at Auburn University, Auburn, Alabama (http://www.ag.auburn.edu/mirror/tmRDB/) and the Royal Veterinary and Agricultural University, Denmark (http://tmrdb.kvl.dk/). The signal recognition particle database (SRPDB) at http://psyche.uthct.edu/dbs/SRPDB/SRPDB.html is mirrored at http://srpdb.kvl.dk/ and the University of Goteborg (http://bio.lundberg.gu.se/dbs/SRPDB/SRPDB.html). The databases assist in investigations of the tmRNP (a ribonucleoprotein complex which liberates stalled bacterial ribosomes) and the SRP (a particle which recognizes signal sequences and directs secretory proteins to cell membranes). The curated tmRNA and SRP RNA alignments consider base pairs supported by comparative sequence analysis. Also shown are alignments of the tmRNA-associated proteins SmpB, ribosomal protein S1, alanyl-tRNA synthetase and Elongation Factor Tu, as well as the SRP proteins SRP9, SRP14, SRP19, SRP21, SRP54 (Ffh), SRP68, SRP72, cpSRP43, Flhf, SRP receptor (alpha) and SRP receptor (beta). All alignments can be easily examined using a new exploratory browser. The databases provide links to high-resolution structures and serve as depositories for structures obtained by molecular modeling.

Background: Trans-translation releases stalled ribosomes from truncated mRNAs and tags defective proteins for proteolytic degradation using transfer-messenger RNA (tmRNA). This small stable RNA represents a hybrid of tRNA- and mRNA-like domains connected by a variable number of pseudoknots. Comparative sequence analysis of tmRNAs found in bacteria, plastids, and mitochondria provides considerable insights into their secondary structures. Progress toward understanding the molecular mechanism of template switching, which constitutes an essential step in trans-translation, is hampered by our limited knowledge about the three-dimensional folding of tmRNA., Results: To facilitate experimental testing of the molecular intricacies of trans-translation, which often require appropriately modified tmRNA derivatives, we developed a procedure for building three-dimensional models of tmRNA. Using comparative sequence analysis, phylogenetically-supported 2-D structures were obtained to serve as input for the program ERNA-3D. Motifs containing loops and turns were extracted from the known structures of other RNAs and used to improve the tmRNA models. Biologically feasible 3-D models for the entire tmRNA molecule could be obtained. The models were characterized by a functionally significant close proximity between the tRNA-like domain and the resume codon. Potential conformational changes which might lead to a more open structure of tmRNA upon binding to the ribosome are discussed. The method, described in detail for the tmRNAs of Escherichia coli, Bacillus anthracis, and Caulobacter crescentus, is applicable to every tmRNA., Conclusion: Improved molecular models of biological significance were obtained. These models will guide in the design of experiments and provide a better understanding of trans-translation. The comparative procedure described here for tmRNA is easily adopted for the modeling the members of other RNA families.

In bacteria, translation of mRNAs lacking stop codons produces truncated polypeptides and traps ribosomes in unproductive complexes. Potentially harmful truncated proteins are tagged with short peptides encoded by the mRNA-like domain of tmRNA and targeted for digestion by housekeeping proteases. We show that altered Escherichia coli transfer-messenger RNAs (tmRNAs) produce in vivo fusion proteins with peptide tags that extend far beyond the conventional termination signal of the wild-type tmRNA. Regions of tmRNA capable of serving as templates for protein synthesis include helix 5, as well as pseudoknots 2, 3, and 4. The removal of all six in-frame stop codons negatively affects tmRNA processing, thereby preventing translation of the 3' portion of the tRNA-like domain. These findings provide evidence that trans-translation can be accompanied by the unfolding of significant portions of the tmRNA molecule. Many of these conformational changes are likely to be required during trans-translation to maintain the ribosomal subunits in close proximity to the tmRNA for monitoring its transit.

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.

Escherichia coli ribosomal protein S1 is composed of six repeating homologous oligonucleotide/oligosaccharide-binding fold (OB folds). In trans-translation, S1 plays a role in delivering transfer-messenger RNA (tmRNA) to stalled ribosomes. The second OB fold of S1 was found to be protected from tryptic digestion in the presence of tmRNA. Truncated S1 mutant Delta2, in which the first and second OB folds were deleted, showed significantly decreased tmRNA-binding activity. Furthermore, the E. coli S1 homolog (BS1) from Bacillus subtilis, which corresponds to the four C-terminal OB folds of E. coli S1, showed no interaction with E. coli tmRNA, as judged by the results of a gel shift assay. Surface plasmon resonance analysis revealed that mutant Delta2 and BS1 had decreased association rate constants (ka, 0.59 x 10(3) M(-1).S(-1); and ka, 1.89 x 10(3) M(-1).S(-1)), while they retained the respective dissociation rate constants (kd, 0.67 x 10(-3) S(-1); and kd, 0.53 x 10(-3) S(-1)), in comparison with wild-type protein S1 (ka, 3.32 x 10(3) M(-1).S(-1); and kd, 0.56 x 10(-3) S(-1)). These results suggest that the second OB fold in protein S1 is essential for the recognition of tmRNA, while the four C-terminal OB folds play a role in stabilizing the S1-tmRNA complex.