Resolution of ribosomal stalling by EF-P and ABCF ATPases YfmR and YkpA/YbiT.

Efficiency of protein synthesis on the ribosome is strongly affected by the amino acid composition of the assembled amino acid chain. Challenging sequences include proline-rich motifs as well as highly positively and negatively charged amino acid stretches. Members of the F subfamily of ABC ATPases (ABCFs) have been long hypothesised to promote translation of such problematic motifs. In this study we have applied genetics and reporter-based assays to characterise the four housekeeping ABCF ATPases of Bacillus subtilis: YdiF, YfmM, YfmR/Uup and YkpA/YbiT. We show that YfmR cooperates with the translation factor EF-P that promotes translation of Pro-rich motifs. Simultaneous loss of both YfmR and EF-P results in a dramatic growth defect. Surprisingly, this growth defect can be largely suppressed though overexpression of an EF-P variant lacking the otherwise crucial 5-amino-pentanolylated residue K32. Using in vivo reporter assays, we show that overexpression of YfmR can alleviate ribosomal stalling on Asp-Pro motifs. Finally, we demonstrate that YkpA/YbiT promotes translation of positively and negatively charged motifs but is inactive in resolving ribosomal stalls on proline-rich stretches. Collectively, our results provide insights into the function of ABCF translation factors in modulating protein synthesis in B. subtilis.

A role for the S4-domain containing protein YlmH in ribosome-associated quality control in Bacillus subtilis.

Ribosomes trapped on mRNAs during protein synthesis need to be rescued for the cell to survive. The most ubiquitous bacterial ribosome rescue pathway is trans-translation mediated by tmRNA and SmpB. Genetic inactivation of trans-translation can be lethal, unless ribosomes are rescued by ArfA or ArfB alternative rescue factors or the ribosome-associated quality control (RQC) system, which in Bacillus subtilis involves MutS2, RqcH, RqcP and Pth. Using transposon sequencing in a trans-translation-incompetent B. subtilis strain we identify a poorly characterized S4-domain-containing protein YlmH as a novel potential RQC factor. Cryo-EM structures reveal that YlmH binds peptidyl-tRNA-50S complexes in a position analogous to that of S4-domain-containing protein RqcP, and that, similarly to RqcP, YlmH can co-habit with RqcH. Consistently, we show that YlmH can assume the role of RqcP in RQC by facilitating the addition of poly-alanine tails to truncated nascent polypeptides. While in B. subtilis the function of YlmH is redundant with RqcP, our taxonomic analysis reveals that in multiple bacterial phyla RqcP is absent, while YlmH and RqcH are present, suggesting that in these species YlmH plays a central role in the RQC.

Patchy and widespread distribution of bacterial translation arrest peptides associated with the protein localization machinery.

Regulatory arrest peptides interact with specific residues on bacterial ribosomes and arrest their own translation. Here, we analyse over 30,000 bacterial genome sequences to identify additional Sec/YidC-related arrest peptides, followed by in vivo and in vitro analyses. We find that Sec/YidC-related arrest peptides show patchy, but widespread, phylogenetic distribution throughout the bacterial domain. Several of the identified peptides contain distinct conserved sequences near the C-termini, but are still able to efficiently stall bacterial ribosomes in vitro and in vivo. In addition, we identify many arrest peptides that share an R-A-P-P-like sequence, suggesting that this sequence might serve as a common evolutionary seed to overcome ribosomal structural differences across species.

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome.

Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity.

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Identification of Bacillus subtilis YidC Substrates Using a MifM-instructed Translation Arrest-based Reporter.

YidC is a member of the YidC/Oxa1/Alb3 protein family that is crucial for membrane protein biogenesis in the bacterial plasma membrane. While YidC facilitates the folding and complex assembly of membrane proteins along with the Sec translocon, it also functions as a Sec-independent membrane protein insertase in the YidC-only pathway. However, little is known about how membrane proteins are recognized and sorted by these pathways, especially in Gram-positive bacteria, for which only a small number of YidC substrates have been identified to date. In this study, we aimed to identify Bacillus subtilis membrane proteins whose membrane insertion depends on SpoIIIJ, the primary YidC homolog in B. subtilis. We took advantage of the translation arrest sequence of MifM, which can monitor YidC-dependent membrane insertion. Our systematic screening identified eight membrane proteins as candidate SpoIIIJ substrates. Results of our genetic study also suggest that the conserved arginine in the hydrophilic groove of SpoIIIJ is crucial for the membrane insertion of the substrates identified here. However, in contrast to MifM, a previously identified YidC substrate, the importance of the negatively charged residue on the substrates for membrane insertion varied depending on the substrate. These results suggest that B. subtilis YidC uses substrate-specific interactions to facilitate membrane insertion.

Nascent chain-mediated translation regulation in bacteria: translation arrest and intrinsic ribosome destabilization.

Proteins that exsert physiological functions during being translated have been discovered from prokaryotes to eukaryotes. These proteins, also called regulatory nascent chains, are common in interacting co-translationally with the ribosomes to stall them. In most cases, such a translational arrest is induced or released in response to changes in the intracellular environment. Cells take advantage of such an environmental sensitivity as a sensor to feedback-regulate gene expression. Recent studies reveal that certain nascent chains could also destabilize the translating ribosomes, leading to stochastic premature translation termination. In this review, we introduce several examples of bacterial nascent chain-based mechanisms of translation regulation by which bacteria regulate cellular functions.

Search for translation arrest peptides encoded upstream of genes for components of protein localization pathways.

Regulatory nascent peptides participate in the regulation of cellular functions by the mechanisms involving regulated translation arrest. A class of them in bacteria, called monitoring substrates, feedback-regulates the expression of a specific component of protein localization machinery. Three monitoring substrates, SecM, MifM and VemP have previously been identified. Here, we attempt at identifying additional arrest peptides in bacteria. Our bioinformatic searches over more than 400 bacterial genomic sequences for proteins that have the common characteristic features shared by the known monitoring substrates and subsequent in vitro and in vivo characterization of the highlighted sequences allowed the identification of three arrest peptides termed ApcA, ApdA and ApdP. ApcA and ApdA homologs are conserved among a subset of actinobacteria, whereas ApdP has homologs in a subset of α-proteobacteria. We demonstrate that these arrest peptides, in their ribosome-tethered nascent states, inhibit peptidyl transfer. The elongation arrest occurs at a specific codon near the 3' end of the coding region, in a manner depending on the amino acid sequence of the nascent chain. Interestingly, the arrest sequences of ApcA, ApdA and ApdP share a sequence R-A-P-G/P that is essential for the elongation arrest.

Proteome-wide Capture of Co-translational Protein Dynamics in Bacillus subtilis Using TnDR, a Transposable Protein-Dynamics Reporter.

Dynamic protein maturation, such as localization, folding, and complex formation, can occur co-translationally. To what extent do nascent polypeptides engage in the co-translational dynamics to produce the functional proteome's complement? We address this question using a protein-dynamics reporter (DR) module comprising a force-sensitive arrest sequence (Bacillus subtilis MifM) followed in frame by LacZ. An engineered transposon, TnDR, carrying DR, is transposed into the B. subtilis chromosome to create translational fusions between N-terminal regions of proteins and the C-terminal DR module. By looking for LacZ+ colonies, we identify hundreds of proteins that cancel the elongation arrest, most probably reflecting their ability to initiate the maturation/localization process co-translationally. Case studies identify B. subtilis proteins that initiate assembly with a partner molecule before completion of translation. These results suggest that co-translational maturation is a frequently occurring event in protein biogenesis.

Release factor-dependent ribosome rescue by BrfA in the Gram-positive bacterium Bacillus subtilis.

Rescue of the ribosomes from dead-end translation complexes, such as those on truncated (non-stop) mRNA, is essential for the cell. Whereas bacteria use trans-translation for ribosome rescue, some Gram-negative species possess alternative and release factor (RF)-dependent rescue factors, which enable an RF to catalyze stop-codon-independent polypeptide release. We now discover that the Gram-positive Bacillus subtilis has an evolutionarily distinct ribosome rescue factor named BrfA. Genetic analysis shows that B. subtilis requires the function of either trans-translation or BrfA for growth, even in the absence of proteotoxic stresses. Biochemical and cryo-electron microscopy (cryo-EM) characterization demonstrates that BrfA binds to non-stop stalled ribosomes, recruits homologous RF2, but not RF1, and induces its transition into an open active conformation. Although BrfA is distinct from E. coli ArfA, they use convergent strategies in terms of mode of action and expression regulation, indicating that many bacteria may have evolved as yet unidentified ribosome rescue systems.

Heat shock transcription factor σ32 defective in membrane transport can be suppressed by transposon insertion into genes encoding a restriction enzyme subunit or a putative autotransporter in Escherichia coli.

Heat shock transcription factor σ32 of Escherichia coli plays a major role in protein homeostasis and requires membrane localization for regulation. We here report that a strongly deregulated I54N-σ32 mutant defective in association with the membrane can be phenotypically suppressed by Tn5 insertion into the mcrC or ydbA2 gene, encoding a restriction enzyme subunit or part of a putative autotransporter, respectively. The suppression is specific for mutant I54N-σ32 and reduces its activity but not its abundance or stability. Moreover, the deregulated phenotype of I54N-σ32 is effectively suppressed by a plasmid carrying the same mcrC::Tn5 mutation. In contrast, deletion of the mcrC or ydbA2 gene hardly affects I54N-σ32 activity. These results, taken together, suggest that the truncated form of McrC (and presumably also of YdbA2) protein produced by the Tn5 insertion interacts specifically with I54N-σ32 to reduce its activity without substantially affecting its amount or stability.

MifM-instructed translation arrest involves nascent chain interactions with the exterior as well as the interior of the ribosome.

Bacillus subtilis MifM is a monitoring substrate of the YidC pathways of protein integration into the membrane and controls the expression of the YidC2 (YqjG) homolog by undergoing regulated translational elongation arrest. The elongation arrest requires interactions between the MifM nascent polypeptide and the ribosomal components near the peptidyl transferase center (PTC) as well as at the constriction site of the ribosomal exit tunnel. Here, we addressed the roles played by more N-terminal regions of MifM and found that, in addition to the previously-identified arrest-provoking elements, the MifM residues 41-60 likely located at the tunnel exit and outside the ribosome contribute to the full induction of elongation arrest. Mutational effects of the cytosolically exposed part of the ribosomal protein uL23 suggested its involvement in the elongation arrest, presumably by interacting with the extra-ribosomal portion of MifM. In vitro translation with reconstituted translation components recapitulated the effects of the mutations at the 41-60 segment, reinforcing the importance of direct molecular interactions between the nascent chain and the ribosome. These results indicate that the nascent MifM polypeptide interacts extensively with the ribosome both from within and without to direct the elongation halt and consequent up-regulation of YidC2.

Photocatalytic Synthesis of CdS(core)-CdSe(shell) Quantum Dots with a Heteroepitaxial Junction on TiO2 : Photoelectrochemical Hydrogen Generation from Water.

A major challenge in chemistry for the synthesis of hetero-nanostructures is to build up atomically commensurate interfaces for smooth interfacial charge transfer. Photodeposition of CdSe on a CdS-preloaded mesoporous TiO2 nanocrystalline film yields CdS(core)-CdSe(shell) quantum dots (CdS@CdSe/mp-TiO2 ) with a heteroepitaxial nanojunction at 298 K. Two-electrode quantum-dot-sensitized photoelectrochemical (QD-SPEC) cells with the structure photoanode |0.25 M Na2 S, 0.35 M Na2 SO3 (solvent=water)| cathode were fabricated. The CdS@CdSe QD-SPEC cell affords a solar-to-current efficiency (STCE) of 0.03 % without external bias under illumination of simulated sunlight (λ>430 nm, AM 1.5, one sun). By applying 0.1 V between the electrodes, the STCE increases up to 0.048 %, far surpassing the CdS/mp-TiO2 and CdSe/mp-TiO2 photoanode cells. The CdS-CdSe interfacial analysis by high-resolution transmission electron microscopy and the band energy analysis taking the size quantization and the electrolyte effect indicate that the excellent performance of the CdS@CdSe/mp-TiO2 photoanode originates from the effective charge separation due to the cascade-like band edge alignment and the heteroepitaxial junction between CdS and CdSe QDs. In addition, high surface coverage of TiO2 with QDs can contribute to the reduction in the loss of the electron transport from TiO2 to the electron collecting electrode.

Electron Filtering by an Intervening ZnS Thin Film in the Gold Nanoparticle-Loaded CdS Plasmonic Photocatalyst.

In the gold nanoparticle (Au NP)-loaded CdS film on fluorine-doped tin oxide electrode (Au/CdS/FTO), the localized plasmonic resonance excitation-induced electron injection from Au NP to CdS has been proven by photoelectrochemical measurements. Formation of ZnS thin films between the Au NP and CdS film leads to a drastic increase of the photocurrent under visible-light irradiation (λ > 610 nm) in a 0.1 M NaClO4 aqueous electrolyte solution due to the electron filtering effect. The photocurrent strongly depends on the thickness of the ZnS film, and the maximum value is obtained at a thickness as thin as 2.1 nm. Furthermore, the ZnS overlayer significantly stabilizes the photocurrent of the CdS/FTO electrode in a polysulfide/sulfide electrolyte solution even under the excitation of CdS (λ > 430 nm). This work presents important information about the design for new plasmonic photocatalysts consisting of plasmonic metal NPs and chalcogenide semiconductors with high conduction band edge.

Stable accumulation of seed storage proteins containing vaccine peptides in transgenic soybean seeds.

There has been a significant increase in the use of transgenic plants for the large-scale production of pharmaceuticals and industrial proteins. Here, we report the stable accumulation of seed storage proteins containing disease vaccine peptides in transgenic soybean seeds. To synthesize vaccine peptides in soybean seeds, we used seed storage proteins as a carrier and a soybean breeding line lacking major seed storage proteins as a host. Vaccine peptides were inserted into the flexible disordered regions in the A1aB1b subunit three-dimensional structure. The A1aB1b subunit containing vaccine peptides in the disordered regions were sorted to the protein storage vacuoles where vaccine peptides are partially cleaved by proteases. In contrast, the endoplasmic reticulum (ER)-retention type of the A1aB1b subunit containing vaccine peptides accumulated in compartments that originated from the ER as an intact pro-form. These results indicate that the ER may be an organelle suitable for the stable accumulation of bioactive peptides using seed storage proteins as carriers.

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α' subunit of soybean ß-conglycinin.

Soybean 7S globulin, known as ß-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of ß-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of ß-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the ß-conglycinin α' subunit.

Differential expression and elution behavior of basic 7S globulin among cultivars under hot water treatment of soybean seeds.

Basic 7S globulin (Bg7S), which accumulates in mature soybean (Glycine max) seeds, is an extracellular matrix protein. A large amount of Bg7S is synthesized de novo and is eluted from soybean seeds when immersed in 50-60°C water (hot water treatment, HWT). However, the Bg7S elution mechanism remains unclear. Under HWT, the seeds probably undergo heat stress and flooding stress. To obtain fundamental knowledge related to how Bg7S is eluted from hot-water-treated seeds, this study compared Bg7S elution among soybean cultivars having different flooding tolerance during pre-germination. The amounts of Bg7S eluted from seeds varied significantly among cultivars. Elution was suppressed by seed coats regarded as preventing the leakage of seed contents by rapid water imbibition. Furthermore, Bg7S expression levels differed among cultivars, although the difference did not result from any variation in Bg7S promoter sequences. However, the expression levels of Bg7S under HWT were not associated with the flooding tolerance level. Immunoelectron microscopy revealed that the Bg7S accumulated in the intercellular space of hot-water-treated seeds. Plasma membrane shrinkage was observed. The main proteins eluted from seeds under HWT were located in the extracellular space. This study clarified the mechanism of Bg7S elution from seeds under HWT.

Soybean basic 7S globulin: subunit heterogeneity and molecular evolution.

Basic 7S globulin, a cysteine-rich protein from soybean seeds, consists of subunits containing 27 kD and 16 kD chains linked by disulfide bonding. Three differently sized subunits of the basic 7S globulin were detected and partially separated by SP Sepharose chromatography. The basic 7S globulin was characterized as a member of a superfamily of structurally related but functionally distinct proteins descended from a specific group of plant aspartic proteinases.