Emerging views on tmRNA-mediated protein tagging and ribosome rescue

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.     

Trans-translation: the tmRNA-mediated surveillance mechanism for ribosome rescue, directed protein degradation, and nonstop mRNA decay

The accurate flow of genetic information from DNA to RNA to protein is essential for all living organisms. An astonishing array of quality-assurance mechanisms have evolved to ensure that high degree of fidelity is maintained at every stage of this process. One of the most fascinating quality-control mechanisms involves tmRNA, also known as SsrA or 10Sa RNA. tmRNA is a versatile and highly conserved bacterial molecule endowed with the combined structural and functional properties of both a tRNA and a mRNA. The tmRNA system orchestrates three key biological functions: (1) recognition and rescue of ribosomes stalled on aberrant mRNAs, (2) disposal of the causative defective mRNAs, and (3) addition of a degradation tag to ribosome-associated protein fragments for directed proteolysis. Although not essential in Escherichia coli, tmRNA activity is essential for bacterial survival under adverse conditions and for virulence in some, and perhaps all, pathogenic bacteria. Recent evidence suggests that in addition to its quality-control function the tmRNA system might also play a key regulatory role in certain physiological pathways. This review will focus on recent advances in our understanding of the structural properties, mechanistic details, and physiological significance of this unique RNA and its principal protein partners.     

Structure of small protein B: the protein component of the tmRNA-SmpB system for ribosome rescue

Small protein B (SmpB) is an essential component of the highly conserved tmRNA-SmpB system that has the dual function of releasing stalled ribosomes from damaged messenger RNAs and targeting incompletely synthesized protein fragments for degradation. Nuclear magnetic resonance (NMR) analysis of SmpB from Aquifex aeolicus revealed an antiparallel beta-barrel structure, with three helices packed outside the core of the barrel. While the overall structure of SmpB appears to be unique, the structure does contain an embedded oligonucleotide binding fold; in this respect SmpB has similarity to several other RNA-binding proteins that are known to be associated with translation, including IF1, ribosomal protein S17 and the N-terminal domain of aspartyl tRNA synthetase. Conserved amino acids on the protein surface that are most likely to directly interact with the tmRNA were identified. The presence of widely separated clusters of conserved amino acids suggests that SmpB could function either by stabilizing two distal regions of the tmRNA, or by facilitating an interaction between the tmRNA and another component of the translational apparatus.     

A biotin-based protein tagging system

Biotin protein ligase (BPL) mediates covalent attachment of biotin to a specific lysine residue of biotin carboxyl carrier protein (BCCP) of biotin-dependent enzymes. We recently found that the biotinylation reaction from thermophilic archaeon Sulfolobus tokodaii has a unique characteristic that the enzyme BPL forms a tight complex with the product, biotinylated BCCP (169 amino acid residues). In the current work, we attempted to apply this characteristic to a novel protein tagging system. Thus, the N terminus of S. tokodaii BCCP was truncated and the interaction of the resulting BCCP, BCCPΔ100 and BCCPΔ17 (with 69 and 152 residues, respectively), with BPL was investigated by surface plasmon resonance (SPR). It was found that the binding of BPL to the biotinylated BCCPΔ100 is extremely tight with a dissociation constant (K_D) of 1.2 nM, whereas that to the unbiotinylated counterpart was moderate with a K_D of 3.3 μM. Furthermore, chimeric proteins of glutathione S-transferase (GST) and green fluorescence protein (GFP) with BCCPΔ100 fused to their C terminus were prepared. The resulting fusion proteins were successfully biotinylated and captured on the BPL-modified SPR sensor chip or BPL-modified magnetic beads. The function of GST and GFP was hardly impaired on fusion with BCCPΔ100 and biotinylation of the latter.     

A ubiquitin-based tagging system for controlled modulation of protein stability

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (UbG76V) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of UbG76V moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized beta-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.     

Solid-phase combinatorial synthesis using MicroKan reactors, Rf tagging, and directed sorting

A modular system for high-output solid-phase combinatorial synthesis has been designed and developed. The system employs three technological innovations to achieve its high efficiency and reliability: (1) application of microreactors as the reaction units in solid-phase synthesis; (2) use of radiofrequency tagging as the non-chemical tracking method; and (3) development of the directed sorting technology for split & pool synthesis. The system has been successfully applied in the synthesis of compound libraries of several hundred to several thousand compounds in multi-milligrams per compound quantity by many organizations.     

Beyond ribosome rescue: tmRNA and co-translational processes

tmRNA is a unique bi-functional RNA that acts as both a tRNA and an mRNA to enter stalled ribosomes and direct the addition of a peptide tag to the C terminus of nascent polypeptides. Despite a reasonably clear understanding of tmRNA activity, the reason for its absolute conservation throughout the eubacteria is unknown. Although tmRNA plays many physiological roles in different bacterial systems, recent studies suggest a general role for trans-translation in monitoring protein folding and perhaps other co-translational processes. This review will focus on these new hypotheses and the data that support them.     

An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB''s affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.     

A stress-responsive system for mitochondrial protein degradation

We show that Ydr049 (renamed VCP/Cdc48-associated mitochondrial stress-responsive--Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress, and decreased chronological life span. Both yeast and mammalian Vms1 stably interact with Cdc48/VCP/p97, a component of the ubiquitin/proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function, and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular, and organismal viability.     

Recombineering BAC transgenes for protein tagging

Protein tagging offers many advantages for proteomic and regulomic research, particularly due to the use of generic and highly sensitive methods that can be applied with reasonable throughput. Ideally, protein tagging is equivalent to having a high affinity antibody for every chosen protein. However, these advantages are compromised if the tagged protein is overexpressed, which is usually the case from cDNA expression vectors. BAC (bacterial artificial chromosome) transgenes present a way to express a chosen protein at physiological levels with all regulatory elements in their native configurations, including cell cycle, alternative splicing and microRNA regulation. Recombineering has become the method of choice for modifying large constructs like BACs. Here, we present a method for protein tagging by recombineering BACs, transfecting cells and evaluating tagged protein expression.     

RIEDL tag: A novel pentapeptide tagging system for transmembrane protein purification

Affinity tag systems are an essential tool in biochemistry, biophysics, and molecular biology. Although several different tag systems have been developed, the epitope tag system, composed of a polypeptide “tag” and an anti-tag antibody, is especially useful for protein purification. However, almost all tag sequences, such as the FLAG tag, are added to the N- or C-termini of target proteins, as tags inserted in loops tend to disrupt the functional structure of multi-pass transmembrane proteins. In this study, we developed a novel “RIEDL tag system,” which is composed of a peptide with only five amino acids (RIEDL) and an anti-RIEDL monoclonal antibody (mAb), LpMab-7. To investigate whether the RIEDL tag system is applicable for protein purification, we conducted the purification of two kinds of RIEDL-tagged proteins using affinity column chromatography: whale podoplanin (wPDPN) with an N-terminal RIEDL tag (RIEDL-wPDPN) and human CD20 with an internal RIEDL tag insertion (CD20-₁₆₉RIEDL₁₇₀). Using an LpMab-7-Sepharose column, RIEDL-wPDPN and CD20-₁₆₉RIEDL₁₇₀ were efficiently purified in one-step purification procedures, and were strongly detected by LpMab-7 using Western blot and flow cytometry. These results show that the RIEDL tag system can be useful for the detection and one-step purification of membrane proteins when inserted at either the N-terminus or inserted in an internal loop structure of multi-pass transmembrane proteins.     

Sequence- and position-dependent tagging protects extracellular-regulated kinase 3 protein from 26S proteasome-mediated degradation

Extracellular-regulated kinase 3, an atypical member of the mitogen-activated protein kinase subfamily of extracellular-regulated kinases, was originally identified in 1991. Little is known about the biochemical properties, regulation, and biological functions of this protein kinase, partially due to the unstable nature of endogenous and low ectopical expression level of the protein. Here, we report that a single C-terminal c-myc tag increases the half-life of ectopic expressed tagged extracellular-regulated kinase 3 approximately four times compared to the reported 30 min half-life time for the endogenous protein and ectopic expressed extracellular-regulated kinase 3 deprived of its c-myc tag. These findings indicate that this C-terminal tag stabilizes the extracellular-regulated kinase 3. The stabilizing effect of the C-terminal c-myc tag is observed in all cell types tested, but is position- and tag sequence-dependent as neither N-terminal c-myc tag nor C-terminal HA tag stabilize the protein. The c-myc tag on extracellular-regulated kinase 3 did not interfere with its kinase activity, nor did it abrogate its ability to interacts with its bona fide substrate mitogen-activated protein kinase-activated protein kinase 5, indicating that tagging did not alter the known biological properties of the protein. Stabilization of the tagged extracellular-regulated kinase 3 protein probably results from reduced ubiquitination. In conclusion, position and sequence specific tagging should provide an easy and useful tool to generate a more stable protein that can functionally substitute the endogenous unstable protein. A stabilized variant may facilitate studies on the biological role of the protein.     

180-kD ribosome receptor is essential for both ribosome binding and protein translocation

We have previously isolated a 180-kD ribosome receptor (p180) from mammalian rough ER that, when incorporated into liposomes, bound ribosomes with an affinity similar to intact membranes. To directly assess the contribution of p180 to ribosome binding as well as protein translocation, monoclonal antibodies were used to selectively deplete p180 from the detergent extracts of rough ER membranes used in the preparation of translocation-competent proteoliposomes. Proteoliposomes prepared from p180-depleted extracts showed a reduction in ribosome binding to the level of trypsin-inactivated controls as well as a loss in their ability to cotranslationally translocate two different secretory protein precursors. When purified p180 was added back to depleted extracts before proteoliposome formation, both ribosome binding and translocation activity were restored. In addition, the monoclonal antibodies, as well as their Fab' fragments, were able to inhibit ribosome binding and protein translocation when bound to intact rough microsomes. These data provide direct evidence that the 180-kD ribosome receptor is essential for ribosome binding and for the translocation of nascent proteins across the membrane of the rough ER.     

Ribosome rescue: tmRNA tagging activity and capacity in Escherichia coli

When protein synthesis stalls in bacteria, tmRNA acts first as a surrogate tRNA and then as an mRNA in a series of reactions that append a peptide tag to the nascent polypeptide and 'rescue' the ribosome. The peptide tag encoded by wild-type tmRNA promotes rapid degradation of rescued proteins. Using a mutant tmRNA that encodes a tag that does not lead to degradation, we demonstrate that the synthesis of approximately 0.4% of all proteins terminates with tagging and ribosome rescue during normal exponential growth of Escherichia coli. The frequency of tagging was not significantly increased in cells expressing very high levels of tmRNA and its binding protein SmpB, suggesting that recognition of 'stalled' ribosomes does not involve competition between tmRNA and other translation factors for A-sites that are unoccupied transiently during protein synthesis. When the demand for ribosome rescue was increased artificially by overproduction of a non-stop mRNA, tmRNA levels did not increase but tmRNA-mediated tagging increased substantially. Thus, the ribosome-rescue system usually operates well below capacity.     

Recombinant tagging system using ribosomal frameshifting to monitor protein expression

For rapid and accurate quantitation of recombinant proteins during expression and after purification, we introduce a new tagging strategy that expresses both target proteins and limitedly tagged target proteins together in a single cell at a constant ratio by utilizing cis‐elements of programmed ‐1 ribosomal frameshifting (‐1RFS) as an embedded device. ‐1RFS is an alternative reading mechanism that effectively controls protein expression by many viruses. When a target gene is fused to the enhanced green fluorescent protein (EGFP) gene with a ‐1RFS element implanted between them, the unfused target and the target‐GFP fusion proteins are expressed at a fixed ratio. The expression ratio between these two protein products is adjustable simply by changing ‐1RFS signals. This limited‐tagging system would be valuable for the real‐time monitoring of protein expression when optimizing expression condition for a new protein, and in monitoring large‐scale bioprocesses without a large metabolic burden on host cells. Furthermore, this strategy allows for the direct measurement of the quantity of a protein on a chip surface and easy application to proteomewide study of gene products. Biotechnol. Bioeng. 2013; 110: 898–904. © 2012 Wiley Periodicals, Inc.     

Proteasome system of protein degradation and processing

In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special “label” can get into it. A polyubiquitin chain plays the role of the “label”: degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the “labeled” molecule, it is released and labels another molecule. This process has been named “Ub-dependent protein degradation”. In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.     

The role of multiply phosphorylated S6 in ribosome degradation

Rat liver ribosomes, prepared 1–24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.