The ABCF proteins in Escherichia coli individually cope with 'hard-to-translate' nascent peptide sequences.

Organisms possess a wide variety of proteins with diverse amino acid sequences, and their synthesis relies on the ribosome. Empirical observations have led to the misconception that ribosomes are robust protein factories, but in reality, they have several weaknesses. For instance, ribosomes stall during the translation of the proline-rich sequences, but the elongation factor EF-P assists in synthesizing proteins containing the poly-proline sequences. Thus, living organisms have evolved to expand the translation capability of ribosomes through the acquisition of translation elongation factors. In this study, we have revealed that Escherichia coli ATP-Binding Cassette family-F (ABCF) proteins, YheS, YbiT, EttA and Uup, individually cope with various problematic nascent peptide sequences within the exit tunnel. The correspondence between noncanonical translations and ABCFs was YheS for the translational arrest by nascent SecM, YbiT for poly-basic sequence-dependent stalling and poly-acidic sequence-dependent intrinsic ribosome destabilization (IRD), EttA for IRD at the early stage of elongation, and Uup for poly-proline-dependent stalling. Our results suggest that ATP hydrolysis-coupled structural rearrangement and the interdomain linker sequence are pivotal for handling 'hard-to-translate' nascent peptides. Our study highlights a new aspect of ABCF proteins to reduce the potential risks that are encoded within the nascent peptide sequences.

Escherichia coli small heat shock protein IbpA plays a role in regulating the heat shock response by controlling the translation of σ32.

Small heat shock proteins (sHsps) act as ATP-independent chaperones that prevent irreversible aggregate formation by sequestering denatured proteins. IbpA, an Escherichia coli sHsp, functions not only as a chaperone but also as a suppressor of its own expression through posttranscriptional regulation, contributing to negative feedback regulation. IbpA also regulates the expression of its paralog, IbpB, in a similar manner, but the extent to which IbpA regulates other protein expressions is unclear. We have identified that IbpA down-regulates the expression of many Hsps by repressing the translation of the heat shock transcription factor σ32. The IbpA regulation not only controls the σ32 level but also contributes to the shutoff of the heat shock response. These results revealed an unexplored role of IbpA to regulate heat shock response at a translational level, which adds an alternative layer for tightly controlled and rapid expression of σ32 on demand.

Nonspecific N-terminal tetrapeptide insertions disrupt the translation arrest induced by ribosome-arresting peptide sequences.

The nascent polypeptide chains passing through the ribosome tunnel not only serve as an intermediate of protein synthesis but also, in some cases, act as dynamic genetic information, controlling translation through interaction with the ribosome. One notable example is Escherichia coli SecM, in which translation of the ribosome arresting peptide (RAP) sequence in SecM leads to robust elongation arrest. Translation regulations, including the SecM-induced translation arrest, play regulatory roles such as gene expression control. Recent investigations have indicated that the insertion of a peptide sequence, SKIK (or MSKIK), into the adjacent N-terminus of the RAP sequence of SecM behaves as an "arrest canceler". As the study did not provide a direct assessment of the strength of translation arrest, we conducted detailed biochemical analyses. The results revealed that the effect of SKIK insertion on weakening SecM-induced translation arrest was not specific to the SKIK sequence, that is, other tetrapeptide sequences inserted just before the RAP sequence also attenuated the arrest. Our data suggest that SKIK or other tetrapeptide insertions disrupt the context of the RAP sequence rather than canceling or preventing the translation arrest.

Nascent polypeptide within the exit tunnel stabilizes the ribosome to counteract risky translation.

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, nascent polypeptide products bear an inherent risk of elongation abortion. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, weaken the translating ribosomal complex, causing certain nascent chain sequences to prematurely terminate translation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic and do not interrupt translation, due to two features of the nascent polypeptide. Firstly, the nascent polypeptide itself spans the exit tunnel, and secondly, its bulky amino acid residues occupy the tunnel entrance region, thereby serving as a bridge and protecting the large and small ribosomal subunits from dissociation. Thus, nascent polypeptide products have an inbuilt ability to ensure elongation continuity.

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.

A method to enrich polypeptidyl-tRNAs to capture snapshots of translation in the cell.

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.

The mRNA binding-mediated self-regulatory function of small heat shock protein IbpA in γ-proteobacteria is conferred by a conserved arginine.

Bacterial small heat shock proteins, such as inclusion body-associated protein A (IbpA) and IbpB, coaggregate with denatured proteins and recruit other chaperones for the processing of aggregates thereby assisting in protein refolding. In addition, as a recently revealed uncommon feature, Escherichia coli IbpA self-represses its own translation through interaction with the 5'-untranslated region of the ibpA mRNA, enabling IbpA to act as a mediator of negative feedback regulation. Although IbpA also suppresses the expression of IbpB, IbpB does not have this self-repression activity despite the two Ibps being highly homologous. In this study, we demonstrate that the self-repression function of IbpA is conserved in other γ-proteobacterial IbpAs. Moreover, we show a cationic residue-rich region in the α-crystallin domain of IbpA, which is not conserved in IbpB, is critical for the self-suppression activity. Notably, we found arginine 93 (R93) located within the α-crystallin domain is an essential residue that cannot be replaced by any of the other 19 amino acids including lysine. We observed that IbpA-R93 mutants completely lost the interaction with the 5' untranslated region of the ibpA mRNA, but retained almost all chaperone activity and were able to sequester denatured proteins. Taken together, we propose the conserved Arg93-mediated translational control of IbpA through RNA binding would be beneficial for a rapid and massive supply of the chaperone on demand.

Prophage excision switches the primary ribosome rescue pathway and rescue-associated gene regulations in Escherichia coli.

Escherichia coli has multiple pathways to release nonproductive ribosome complexes stalled at the 3' end of nonstop mRNA: tmRNA (SsrA RNA)-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4-57 prophage adjacent to E. coli ssrA leads to the inactivation of tmRNA and switches the primary rescue pathway from trans-translation to ArfA/RF2. This "rescue-switching" rearranges not only the proteome landscape in E. coli but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA+ cells, we found ZntR, whose mRNA is transcribed together as the upstream part of nonstop arfA mRNA. Repression of ZntR and reconstituted model genes depends on the translation of the downstream nonstop ORFs that trigger the trans-translation-coupled exonucleolytic degradation by polynucleotide phosphorylase (PNPase). Namely, our studies provide a novel example of trans-translation-dependent regulation and re-define the physiological roles of prophage excision.

The landscape of translational stall sites in bacteria revealed by monosome and disome profiling.

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria.

Amyloid conformation-dependent disaggregation in a reconstituted yeast prion system.

Disaggregation of amyloid fibrils is a fundamental biological process required for amyloid propagation. However, due to the lack of experimental systems, the molecular mechanism of how amyloid is disaggregated by cellular factors remains poorly understood. Here, we established a robust in vitro reconstituted system of yeast prion propagation and found that heat-shock protein 104 (Hsp104), Ssa1 and Sis1 chaperones are essential for efficient disaggregation of Sup35 amyloid. Real-time imaging of single-molecule fluorescence coupled with the reconstitution system revealed that amyloid disaggregation is achieved by ordered, timely binding of the chaperones to amyloid. Remarkably, we uncovered two distinct prion strain conformation-dependent modes of disaggregation, fragmentation and dissolution. We characterized distinct chaperone dynamics in each mode and found that transient, repeated binding of Hsp104 to the same site of amyloid results in fragmentation. These findings provide a physical foundation for otherwise puzzling in vivo observations and for therapeutic development for amyloid-associated neurodegenerative diseases.

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

ATP-Responsive Nanoparticles Covered with Biomolecular Machine "Chaperonin GroEL".

Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.

Escherichia coli small heat shock protein IbpA is an aggregation-sensor that self-regulates its own expression at posttranscriptional levels.

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the σ32 heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsps expression. Oligomeric IbpA self-represses the ibpA/ibpB translation, and mediates its own mRNA degradation, but the self-repression is relieved by sequestration of IbpA into the protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.

Shotgun Proteomics Revealed Preferential Degradation of Misfolded In Vivo Obligate GroE Substrates by Lon Protease in Escherichia coli.

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE.

Proximity Histidine Labeling by Umpolung Strategy Using Singlet Oxygen.

While electrophilic reagents for histidine labeling have been developed, we report an umpolung strategy for histidine functionalization. A nucleophilic small molecule, 1-methyl-4-arylurazole, selectively labeled histidine under singlet oxygen (1O2) generation conditions. Rapid histidine labeling can be applied for instant protein labeling. Utilizing the short diffusion distance of 1O2 and a technique to localize the 1O2 generator, a photocatalyst in close proximity to the ligand-binding site, we demonstrated antibody Fc-selective labeling on magnetic beads functionalized with a ruthenium photocatalyst and Fc ligand, ApA. Three histidine residues located around the ApA binding site were identified as labeling sites by liquid chromatography-mass spectrometry analysis. This result suggests that 1O2-mediated histidine labeling can be applied to a proximity labeling reaction on the nanometer scale.

Novel self-regulation strategy of a small heat shock protein for prodigious and rapid expression on demand.

In this mini-review, we summarize the known and novel regulation mechanisms of small heat shock proteins (sHsps). sHsps belong to a well-conserved family of ATP-independent oligomeric chaperones that protect denatured proteins from forming irreversible aggregates by co-aggregation. The functions of sHsps as a first line of defense against acute stresses require the high abundance of sHsps on demand. The heat stress-induced expression of IbpA, one of the sHsps in Escherichia coli, is regulated by σ32, an RNA polymerase subunit, and the thermoresponsive mRNA structures in the 5' untranslated region, called RNA thermometers. In addition to the known mechanisms, a recent study has revealed unexpected processes by which the oligomeric IbpA self-represses the ibpA translation via the direct binding of IbpA to its own mRNA, and mediates the mRNA degradation. In summary, the role of IbpA as an aggregation-sensor, combined with other mechanisms, tightly regulates the expression level of IbpA, thus enabling the sHsp to function as a "sequestrase" upon acute aggregation stress, and provides new insights into the mechanisms of other sHsps in both bacteria and eukaryotes.

Nascent SecM chain interacts with outer ribosomal surface to stabilize translation arrest.

SecM, a bacterial secretion monitor protein, posttranscriptionally regulates downstream gene expression via translation elongation arrest. SecM contains a characteristic amino acid sequence called the arrest sequence at its C-terminus, and this sequence acts within the ribosomal exit tunnel to stop translation. It has been widely assumed that the arrest sequence within the ribosome tunnel is sufficient for translation arrest. We have previously shown that the nascent SecM chain outside the ribosomal exit tunnel stabilizes translation arrest, but the molecular mechanism is unknown. In this study, we found that residues 57-98 of the nascent SecM chain are responsible for stabilizing translation arrest. We performed alanine/serine-scanning mutagenesis of residues 57-98 to identify D79, Y80, W81, H84, R87, I90, R91, and F95 as the key residues responsible for stabilization. The residues were predicted to be located on and near an α-helix-forming segment. A striking feature of the α-helix is the presence of an arginine patch, which interacts with the negatively charged ribosomal surface. A photocross-linking experiment showed that Y80 is adjacent to the ribosomal protein L23, which is located next to the ribosomal exit tunnel when translation is arrested. Thus, the folded nascent SecM chain that emerges from the ribosome exit tunnel interacts with the outer surface of the ribosome to stabilize translation arrest.

Molecularly Engineered "Janus GroEL": Application to Supramolecular Copolymerization with a Higher Level of Sequence Control.

Herein we report the synthesis and isolation of a shape-persistent Janus protein nanoparticle derived from the biomolecular machine chaperonin GroEL (AGroELB) and its application to DNA-mediated ternary supramolecular copolymerization. To synthesize AGroELB with two different DNA strands A and B at its opposite apical domains, we utilized the unique biological property of GroEL, i.e., Mg2+/ATP-mediated ring exchange between AGroELA and BGroELB with their hollow cylindrical double-decker architectures. This exchange event was reported more than 24 years ago but has never been utilized for molecular engineering of GroEL. We leveraged DNA nanotechnology to purely isolate Janus AGroELB and succeeded in its precision ternary supramolecular copolymerization with two DNA comonomers, A** and B*, that are partially complementary to A and B in AGroELB, respectively, and programmed to self-dimerize on the other side. Transmission electron microscopy allowed us to confirm the formation of the expected dual-periodic copolymer sequence -(B*/BGroELA/A**/A**/AGroELB/B*)- in the form of a laterally connected lamellar assembly rather than a single-chain copolymer.

Electrostatic interactions between middle domain motif-1 and the AAA1 module of the bacterial ClpB chaperone are essential for protein disaggregation.

ClpB, a bacterial homologue of heat shock protein 104 (Hsp104), can disentangle aggregated proteins with the help of the DnaK, a bacterial Hsp70, and its co-factors. As a member of the expanded superfamily of ATPases associated with diverse cellular activities (AAA+), ClpB forms a hexameric ring structure, with each protomer containing two AAA+ modules, AAA1 and AAA2. A long coiled-coil middle domain (MD) is present in the C-terminal region of the AAA1 and surrounds the main body of the ring. The MD is subdivided into two oppositely directed short coiled-coils, called motif-1 and motif-2. The MD represses the ATPase activity of ClpB, and this repression is reversed by the binding of DnaK to motif-2. To better understand how the MD regulates ClpB activity, here we investigated the roles of motif-1 in ClpB from Thermus thermophilus (TClpB). Using systematic alanine substitution of the conserved charged residues, we identified functionally important residues in motif-1, and using a photoreactive cross-linker and LC-MS/MS analysis, we further explored potential interacting residues. Moreover, we constructed TClpB mutants in which functionally important residues in motif-1 and in other candidate regions were substituted by oppositely charged residues. These analyses revealed that the intra-subunit pair Glu-401-Arg-532 and the inter-subunit pair Asp-404-Arg-180 are functionally important, electrostatically interacting pairs. Considering these structural findings, we conclude that the Glu-401-Arg-532 interaction shifts the equilibrium of the MD conformation to stabilize the activated form and that the Arg-180-Asp-404 interaction contributes to intersubunit signal transduction, essential for ClpB chaperone activities.

Disruption of the Gene trx-m1 Impedes the Growth of Anabaena sp. PCC 7120 under Nitrogen Starvation.

Cyanobacteria possess a sophisticated photosynthesis-based metabolism with admirable plasticity. This plasticity is possible via the deep regulation network, the thiol-redox regulations operated by thioredoxin (hereafter, Trx). In this context, we characterized the Trx-m1-deficient mutant strain of Anabaena sp., PCC 7120 (shortly named A.7120), cultivated under nitrogen limitation. Trx-m1 appears to coordinate the nitrogen response and its absence induces large changes in the proteome. Our data clearly indicate that Trx-m1 is crucial for the diazotrophic growth of A.7120. The lack of Trx-m1 resulted in a large differentiation of heterocysts (>20% of total cells), which were barely functional probably due to a weak expression of nitrogenase. In addition, heterocysts of the mutant strain did not display the usual cellular structure of nitrogen-fixative cells. This unveiled why the mutant strain was not able to grow under nitrogen starvation.