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.

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.

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.

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.

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.