Making the jump: new insights into the mechanism of trans-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

Comparative 3-D Modeling of tmRNA

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

Visualizing the transfer-messenger RNA as the ribosome resumes translation

Bacterial ribosomes that are stalled on mRNAs lacking a stop codon can be rescued by a process called ‘transtranslation' that involves the ribonucleoprotein complex tmRNA–SmpB. This cryo-EM study, and the copublished study by Weis et al, reveal how translation on tmRNA is resume

The tmRDB and SRPDB resources

Maintained at the University of Texas Health Science Center at Tyler, Texas, the tmRNA database (tmRDB) is accessible at the URL with mirror sites located at Auburn University, Auburn, Alabama () and the Royal Veterinary and Agricultural University, Denmark (). The signal recognition particle database (SRPDB) at is mirrored at and the University of Goteborg (). 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

HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders

Ribosomal protein S1 influences trans-translation in vitro and in vivo

When the bacterial ribosome stalls on a truncated mRNA, transfer–messenger RNA (tmRNA) acts initially as a transfer RNA (tRNA) and then as a messenger RNA (mRNA) to rescue the ribosome and add a peptide tag to the nascent polypeptide that targets it for degradation. Ribosomal protein S1 binds tmRNA but its functional role in this process has remained elusive. In this report, we demonstrate that, in vitro, S1 is dispensable for the tRNA-like role of tmRNA but is essential for its mRNA function. Increasing or decreasing the amount of protein S1 in vivo reduces the overall amount of trans-translated proteins. Also, a truncated S1 protein impaired for ribosome binding can still trigger protein tagging, suggesting that S1 interacts with tmRNA outside the ribosome to keep it in an active state. Overall, these results demonstrate that S1 has a role in tmRNA-mediated tagging that is distinct from its role during canonical translation

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3′ ends of tRNA ligands. Here we present structures, at 3.6-Å and 3.5-Å resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer

The Caulobacter crescentus GTPase CgtA C is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels

The Obg subfamily of bacterial GTP-binding proteins are biochemically distinct from Ras-like proteins raising the possibility that they are not controlled by conventional guanine nucleotide exchange factors (GEFs) and/or guanine nucleotide activating proteins (GAPs). To test this hypothesis, we generated mutations in the Caulobacter crescentus obg gene ( cgtA C ) which, in Ras-like proteins, would result in either activating or dominant negative phenotypes. In C. crescentus , a P168V mutant is not activating in vivo , although in vitro , the P168V protein showed a modest reduction in the affinity for GDP. Neither the S173N nor N280Y mutations resulted in a dominant negative phenotype. Furthermore, the S173N was significantly impaired for GTP binding, consistent with a critical role of this residue in GTP binding. In general, conserved amino acids in the GTP-binding pocket were, however, important for function. To examine the in vivo consequences of depleting CgtA C , we generated a temperature-sensitive mutant, G80E. At the permissive temperature, G80E cells grow slowly and have reduced levels of 50S ribosomal subunits, indicating that CgtA C is important for 50S assembly and/or stability. Surprisingly, at the non-permissive temperature, G80E  cells  rapidly  lose  viability  and  yet  do not display an additional ribosome defect. Thus, the essential nature of the cgtA C gene does not appear to result from its ribosome function. G80E cells arrest as predivisional cells and stalkless cells. Flow cytometry on synchronized cells reveals a G1-S arrest. Therefore, CgtA C is necessary for DNA replication and progression through the cell cycle.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75652/1/j.1365-2958.2004.04354.x.pd

Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry

The modification of RNA with fluorophores, affinity tags and reactive moieties is of enormous utility for studying RNA localization, structure and dynamics as well as diverse biological phenomena involving RNA as an interacting partner. Here we report a labeling approach in which the RNA of interest—of either synthetic or biological origin—is modified at its 3′-end by a poly(A) polymerase with an azido-derivatized nucleotide. The azide is later on conjugated via copper-catalyzed or strain-promoted azide–alkyne click reaction. Under optimized conditions, a single modified nucleotide of choice (A, C, G, U) containing an azide at the 2′-position can be incorporated site-specifically. We have identified ligases that tolerate the presence of a 2′-azido group at the ligation site. This azide is subsequently reacted with a fluorophore alkyne. With this stepwise approach, we are able to achieve site-specific, internal backbone-labeling of de novo synthesized RNA molecules

Mycobacterium tuberculosis ribosomal protein S1 (RpsA) and variants with truncated C-terminal end show absence of interaction with pyrazinoic acid.

Pyrazinamide (PZA) is an antibiotic used in first- and second-line tuberculosis treatment regimens. Approximately 50% of multidrug-resistant tuberculosis and over 90% of extensively drug-resistant tuberculosis strains are also PZA resistant. Despite the key role played by PZA, its mechanisms of action are not yet fully understood. It has been postulated that pyrazinoic acid (POA), the hydrolyzed product of PZA, could inhibit trans-translation by binding to Ribosomal protein S1 (RpsA) and competing with tmRNA, the natural cofactor of RpsA. Subsequent data, however, indicate that these early findings resulted from experimental artifact. Hence, in this study we assess the capacity of POA to compete with tmRNA for RpsA. We evaluated RpsA wild type (WT), RpsA ∆A438, and RpsA ∆A438 variants with truncations towards the carboxy terminal end. Interactions were measured using Nuclear Magnetic Resonance spectroscopy (NMR), Isothermal Titration Calorimetry (ITC), Microscale Thermophoresis (MST), and Electrophoretic Mobility Shift Assay (EMSA). We found no measurable binding between POA and RpsA (WT or variants). This suggests that RpsA may not be involved in the mechanism of action of PZA in Mycobacterium tuberculosis, as previously thought. Interactions observed between tmRNA and RpsA WT, RpsA ∆A438, and each of the truncated variants of RpsA ∆A438, are reported