Arrest peptides: cis-acting modulators of translation.

Each peptide bond of a protein is generated at the peptidyl transferase center (PTC) of the ribosome and then moves through the exit tunnel, which accommodates ever-changing segments of ≈ 40 amino acids of newly translated polypeptide. A class of proteins, called ribosome arrest peptides, contains specific sequences of amino acids (arrest sequences) that interact with distinct components of the PTC-exit tunnel region of the ribosome and arrest their own translation continuation, often in a manner regulated by environmental cues. Thus, the ribosome that has translated an arrest sequence is inactivated for peptidyl transfer, translocation, or termination. The stalled ribosome then changes the configuration or localization of mRNA, resulting in specific biological outputs, including regulation of the target gene expression and downstream events of mRNA/polypeptide maturation or localization. Living organisms thus seem to have integrated potentially harmful arrest sequences into elaborate regulatory mechanisms to express genetic information in productive directions.

Intrinsic Ribosome Destabilization Underlies Translation and Provides an Organism with a Strategy of Environmental Sensing.

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

Strolling Toward New Concepts.

For more than four decades now, I have been studying how genetic information is transformed into protein-based cellular functions. This has included investigations into the mechanisms supporting cellular localization of proteins, disulfide bond formation, quality control of membranes, and translation. I tried to extract new principles and concepts that are universal among living organisms from our observations of Escherichia coli. While I wanted to distill complex phenomena into basic principles, I also tried not to overlook any serendipitous observations. In the first part of this article, I describe personal experiences during my studies of the Sec pathway, which have centered on the SecY translocon. In the second part, I summarize my views of the recent revival of translation studies, which has given rise to the concept that nonuniform polypeptide chain elongation is relevant for the subsequent fates of newly synthesized proteins. Our studies of a class of regulatory nascent polypeptides advance this concept by showing that the dynamic behaviors of the extraribosomal part of the nascent chain affect the ongoing translation process. Vibrant and regulated molecular interactions involving the ribosome, mRNA, and nascent polypeptidyl-tRNA are based, at least partly, on their autonomously interacting properties.

Structural basis of Sec-independent membrane protein insertion by YidC.

Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane.

Multisite ribosomal stalling: a unique mode of regulatory nascent chain action revealed for MifM.

Bacillus subtilis MifM uses polypeptide-instructed ribosomal stalling to control translation of YidC2, a membrane protein biogenesis factor. In contrast to other stalling systems involving a single arrest point, our in vitro translation/toeprint experiments show that the B. subtilis ribosome stalls consecutively at multiple codons of MifM. This mode of elongation arrest depends on nascent chain residues at the middle of the ribosomal exit tunnel and a few (four for the maximum functionality) negative charges residing proximally to the arrest points. The latter element does not require exact amino acid sequence, and this feature may underlie the multisite stalling. The arrested nascent chains were not efficiently transferred to puromycin, suggesting that growing MifM nascent chains inhibit peptidyl transferase center after acquiring an acidic residue(s). Multisite stalling seems to provide a unique means for MifM to achieve a sufficient duration of ribosomal stalling required for the regulatory function.

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing.

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

Hydrophilic microenvironment required for the channel-independent insertase function of YidC protein.

The recently solved crystal structure of YidC protein suggests that it mediates membrane protein insertion by means of an intramembrane cavity rather than a transmembrane (TM) pore. This concept of protein translocation prompted us to characterize the native, membrane-integrated state of YidC with respect to the hydropathic nature of its TM region. Here, we show that the cavity-forming region of the stage III sporulation protein J (SpoIIIJ), a YidC homolog, is indeed open to the aqueous milieu of the Bacillus subtilis cells and that the overall hydrophilicity of the cavity, along with the presence of an Arg residue on several alternative sites of the cavity surface, is functionally important. We propose that YidC functions as a proteinaceous amphiphile that interacts with newly synthesized membrane proteins and reduces energetic costs of their membrane traversal.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria.

SecDF interacts with the SecYEG translocon in bacteria and enhances protein export in a proton-motive-force-dependent manner. Vibrio alginolyticus, a marine-estuarine bacterium, contains two SecDF paralogs, V.SecDF1 and V.SecDF2. Here, we show that the export-enhancing function of V.SecDF1 requires Na+ instead of H+, whereas V.SecDF2 is Na+-independent, presumably requiring H+. In accord with the cation-preference difference, V.SecDF2 was only expressed under limited Na+ concentrations whereas V.SecDF1 was constitutive. However, it is not the decreased concentration of Na+ per se that the bacterium senses to up-regulate the V.SecDF2 expression, because marked up-regulation of the V.SecDF2 synthesis was observed irrespective of Na+ concentrations under certain genetic/physiological conditions: (i) when the secDF1VA gene was deleted and (ii) whenever the Sec export machinery was inhibited. VemP (Vibrio export monitoring polypeptide), a secretory polypeptide encoded by the upstream ORF of secDF2VA, plays the primary role in this regulation by undergoing regulated translational elongation arrest, which leads to unfolding of the Shine-Dalgarno sequence for translation of secDF2VA. Genetic analysis of V. alginolyticus established that the VemP-mediated regulation of SecDF2 is essential for the survival of this marine bacterium in low-salinity environments. These results reveal that a class of marine bacteria exploits nascent-chain ribosome interactions to optimize their protein export pathways to propagate efficiently under different ionic environments that they face in their life cycles.

Structure and function of a membrane component SecDF that enhances protein export.

Protein translocation across the bacterial membrane, mediated by the secretory translocon SecYEG and the SecA ATPase, is enhanced by proton motive force and membrane-integrated SecDF, which associates with SecYEG. The role of SecDF has remained unclear, although it is proposed to function in later stages of translocation as well as in membrane protein biogenesis. Here, we determined the crystal structure of Thermus thermophilus SecDF at 3.3 Å resolution, revealing a pseudo-symmetrical, 12-helix transmembrane domain belonging to the RND superfamily and two major periplasmic domains, P1 and P4. Higher-resolution analysis of the periplasmic domains suggested that P1, which binds an unfolded protein, undergoes functionally important conformational changes. In vitro analyses identified an ATP-independent step of protein translocation that requires both SecDF and proton motive force. Electrophysiological analyses revealed that SecDF conducts protons in a manner dependent on pH and the presence of an unfolded protein, with conserved Asp and Arg residues at the transmembrane interface between SecD and SecF playing essential roles in the movements of protons and preproteins. Therefore, we propose that SecDF functions as a membrane-integrated chaperone, powered by proton motive force, to achieve ATP-independent protein translocation.

Heat shock transcription factor σ32 co-opts the signal recognition particle to regulate protein homeostasis in E. coli.

All cells must adapt to rapidly changing conditions. The heat shock response (HSR) is an intracellular signaling pathway that maintains proteostasis (protein folding homeostasis), a process critical for survival in all organisms exposed to heat stress or other conditions that alter the folding of the proteome. Yet despite decades of study, the circuitry described for responding to altered protein status in the best-studied bacterium, E. coli, does not faithfully recapitulate the range of cellular responses in response to this stress. Here, we report the discovery of the missing link. Surprisingly, we found that σ(32), the central transcription factor driving the HSR, must be localized to the membrane rather than dispersed in the cytoplasm as previously assumed. Genetic analyses indicate that σ(32) localization results from a protein targeting reaction facilitated by the signal recognition particle (SRP) and its receptor (SR), which together comprise a conserved protein targeting machine and mediate the cotranslational targeting of inner membrane proteins to the membrane. SRP interacts with σ(32) directly and transports it to the inner membrane. Our results show that σ(32) must be membrane-associated to be properly regulated in response to the protein folding status in the cell, explaining how the HSR integrates information from both the cytoplasm and bacterial cell membrane.

MifM monitors total YidC activities of Bacillus subtilis, including that of YidC2, the target of regulation.

The YidC/Oxa1/Alb3 family proteins are involved in membrane protein biogenesis in bacteria, mitochondria, and chloroplasts. Recent studies show that YidC uses a channel-independent mechanism to insert a class of membrane proteins into the membrane. Bacillus subtilis has two YidC homologs, SpoIIIJ (YidC1) and YidC2 (YqjG); the former is expressed constitutively, while the latter is induced when the SpoIIIJ activity is compromised. MifM is a substrate of SpoIIIJ, and its failure in membrane insertion is accompanied by stable ribosome stalling on the mifM-yidC2 mRNA, which ultimately facilitates yidC2 translation. While mutational inactivation of SpoIIIJ has been known to induce yidC2 expression, here, we show that the level of this induction is lower than that observed when the membrane insertion signal of MifM is defective. Moreover, this partial induction of YidC2 translation is lowered further when YidC2 is overexpressed in trans. These results suggest that YidC2 is able to insert MifM into the membrane and to release its translation arrest. Thus, under SpoIIIJ-deficient conditions, YidC2 expression is subject to MifM-mediated autogenous feedback repression. Our results show that YidC2 uses a mechanism that is virtually identical to that used by SpoIIIJ; Arg75 of YidC2 in its intramembrane yet hydrophilic cavity is functionally indispensable and requires negatively charged residues of MifM as an insertion substrate. From these results, we conclude that MifM monitors the total activities of the SpoIIIJ and the YidC2 pathways to control the synthesis of YidC2 and to maintain the cellular capability of the YidC mode of membrane protein biogenesis.

Conformational variation of the translocon enhancing chaperone SecDF.

The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.

Conformational transition of Sec machinery inferred from bacterial SecYE structures.

Over 30% of proteins are secreted across or integrated into membranes. Their newly synthesized forms contain either cleavable signal sequences or non-cleavable membrane anchor sequences, which direct them to the evolutionarily conserved Sec translocon (SecYEG in prokaryotes and Sec61, comprising alpha-, gamma- and beta-subunits, in eukaryotes). The translocon then functions as a protein-conducting channel. These processes of protein localization occur either at or after translation. In bacteria, the SecA ATPase drives post-translational translocation. The only high-resolution structure of a translocon available so far is that for SecYEbeta from the archaeon Methanococcus jannaschii, which lacks SecA. Here we present the 3.2-A-resolution crystal structure of the SecYE translocon from a SecA-containing organism, Thermus thermophilus. The structure, solved as a complex with an anti-SecY Fab fragment, revealed a 'pre-open' state of SecYE, in which several transmembrane helices are shifted, as compared to the previous SecYEbeta structure, to create a hydrophobic crack open to the cytoplasm. Fab and SecA bind to a common site at the tip of the cytoplasmic domain of SecY. Molecular dynamics and disulphide mapping analyses suggest that the pre-open state might represent a SecYE conformational transition that is inducible by SecA binding. Moreover, we identified a SecA-SecYE interface that comprises SecA residues originally buried inside the protein, indicating that both the channel and the motor components of the Sec machinery undergo cooperative conformational changes on formation of the functional complex.

Recruitment of a species-specific translational arrest module to monitor different cellular processes.

Nascent chain-mediated translation arrest serves as a mechanism of gene regulation. A class of regulatory nascent polypeptides undergoes elongation arrest in manners controlled by the dynamic behavior of the growing chain; Escherichia coli SecM monitors the Sec protein export pathway and Bacillus subtilis MifM monitors the YidC membrane protein integration/folding pathway. We show that MifM and SecM interact with the ribosome in a species-specific manner to stall only the ribosome from the homologous species. Despite this specificity, MifM is not exclusively designed to monitor membrane protein integration because it can be converted into a secretion monitor by replacing the N-terminal transmembrane sequence with a secretion signal sequence. These results show that a regulatory nascent chain is composed of two modular elements, one devoted to elongation arrest and another devoted to subcellular targeting, and they imply that physical pulling force generated by the latter triggers release of the arrest executed by the former. The combinatorial nature may assure common occurrence of nascent chain-mediated regulation.

Post-liberation cleavage of signal peptides is catalyzed by the site-2 protease (S2P) in bacteria.

A signal peptide (SP) is cleaved off from presecretory proteins by signal peptidase during or immediately after insertion into the membrane. In metazoan cells, the cleaved SP then receives proteolysis by signal peptide peptidase, an intramembrane-cleaving protease (I-CLiP). However, bacteria lack any signal peptide peptidase member I-CLiP, and little is known about the metabolic fate of bacterial SPs. Here we show that Escherichia coli RseP, an site-2 protease (S2P) family I-CLiP, introduces a cleavage into SPs after their signal peptidase-mediated liberation from preproteins. A Bacillus subtilis S2P protease, RasP, is also shown to be involved in SP cleavage. These results uncover a physiological role of bacterial S2P proteases and update the basic knowledge about the fate of signal peptides in bacterial cells.

ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl-tRNA hydrolysis in Escherichia coli.

The ribosomes stalled at the end of non-stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non-productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans-translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome-tethered peptidyl-tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl-tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl-tRNAs located in the P-site of the stalled ribosome. This reaction required the GGQ (Gly-Gly-Gln) hydrolysis motif, but not the SPF (Ser-Pro-Phe) codon-recognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A-site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl-tRNA in a GGQ motif-dependent but codon-independent manner. In support of this model, the ArfA-RF2 pathway did not act on the SecM-arrested ribosome, which contains an aminoacyl-tRNA in the A-site.

Dynamic nature of disulphide bond formation catalysts revealed by crystal structures of DsbB.

In the Escherichia coli system catalysing oxidative protein folding, disulphide bonds are generated by the cooperation of DsbB and ubiquinone and transferred to substrate proteins through DsbA. The structures solved so far for different forms of DsbB lack the Cys104-Cys130 initial-state disulphide that is directly donated to DsbA. Here, we report the 3.4 A crystal structure of a DsbB-Fab complex, in which DsbB has this principal disulphide. Its comparison with the updated structure of the DsbB-DsbA complex as well as with the recently reported NMR structure of a DsbB variant having the rearranged Cys41-Cys130 disulphide illuminated conformational transitions of DsbB induced by the binding and release of DsbA. Mutational studies revealed that the membrane-parallel short alpha-helix of DsbB has a key function in physiological electron flow, presumably by controlling the positioning of the Cys130-containing loop. These findings demonstrate that DsbB has developed the elaborate conformational dynamism to oxidize DsbA for continuous protein disulphide bond formation in the cell.

SecY alterations that impair membrane protein folding and generate a membrane stress.

We report on a class of Escherichia coli SecY mutants that impair membrane protein folding. The mutants also up-regulate the Cpx/sigma(E) stress response pathways. Similar stress induction was also observed in response to a YidC defect in membrane protein biogenesis but not in response to the signal recognition particle-targeting defect or in response to a simple reduction in the abundance of the translocon. Together with the previous contention that the Cpx system senses a protein abnormality not only at periplasmic and outer membrane locations but also at the plasma membrane, abnormal states of membrane proteins are postulated to be generated in these secY mutants. In support of this notion, in vitro translation, membrane integration, and folding of LacY reveal that mutant membrane vesicles allow the insertion of LacY but not subsequent folding into a normal conformation recognizable by conformation-specific antibodies. The results demonstrate that normal SecY function is required for the folding of membrane proteins after their insertion into the translocon.

Editing disulphide bonds: error correction using redox currencies.

The disulphide bond-introducing enzyme of bacteria, DsbA, sometimes oxidizes non-native cysteine pairs. DsbC should rearrange the resulting incorrect disulphide bonds into those with correct connectivity. DsbA and DsbC receive oxidizing and reducing equivalents, respectively, from respective redox components (quinones and NADPH) of the cell. Two mechanisms of disulphide bond rearrangement have been proposed. In the redox-neutral 'shuffling' mechanism, the nucleophilic cysteine in the DsbC active site forms a mixed disulphide with a substrate and induces disulphide shuffling within the substrate part of the enzyme-substrate complex, followed by resolution into a reduced enzyme and a disulphide-rearranged substrate. In the 'reduction-oxidation' mechanism, DsbC reduces those substrates with wrong disulphides so that DsbA can oxidize them again. In this issue of Molecular Microbiology, Berkmen and his collaborators show that a disulphide reductase, TrxP, from an anaerobic bacterium can substitute for DsbC in Escherichia coli. They propose that the reduction-oxidation mechanism of disulphide rearrangement can indeed operate in vivo. An implication of this work is that correcting errors in disulphide bonds can be coupled to cellular metabolism and is conceptually similar to the proofreading processes observed with numerous synthesis and maturation reactions of biological macromolecules.

The disulfide bond formation (Dsb) system.

In oxidative folding of proteins in the bacterial periplasmic space, disulfide bonds are introduced by the oxidation system and isomerized by the reduction system. These systems utilize the oxidizing and the reducing equivalents of quinone and NADPH, respectively, that are transmitted across the cytoplasmic membrane through integral membrane components DsbB and DsbD. In both pathways, alternating interactions between a Cys-XX-Cys-containing thioredoxin domain and other regulatory domain lead to the maintenance of oxidized and reduced states of the specific terminal enzymes, DsbA that oxidizes target cysteines and DsbC that reduces an incorrect disulfide to allow its isomerization into the physiological one. Molecular details of these remarkable biochemical cascades are being rapidly unraveled by genetic, biochemical, and structural analyses in recent years.