Chloroplast Ribosomes Interact With the Insertase Alb3 in the Thylakoid Membrane

Members of the Oxa1/YidC/Alb3 protein family are involved in the insertion, folding, and assembly of membrane proteins in mitochondria, bacteria, and chloroplasts. The thylakoid membrane protein Alb3 mediates the chloroplast signal recognition particle (cpSRP)-dependent posttranslational insertion of nuclear-encoded light harvesting chlorophyll a/b-binding proteins and participates in the biogenesis of plastid-encoded subunits of the photosynthetic complexes. These subunits are cotranslationally inserted into the thylakoid membrane, yet very little is known about the molecular mechanisms underlying docking of the ribosome-nascent chain complexes to the chloroplast SecY/Alb3 insertion machinery. Here, we show that nanodisc-embedded Alb3 interacts with ribosomes, while the homolog Alb4, also located in the thylakoid membrane, shows no ribosome binding. Alb3 contacts the ribosome with its C-terminal region and at least one additional binding site within its hydrophobic core region. Within the C-terminal region, two conserved motifs (motifs III and IV) are cooperatively required to enable the ribosome contact. Furthermore, our data suggest that the negatively charged C-terminus of the ribosomal subunit uL4c is involved in Alb3 binding. Phylogenetic analyses of uL4 demonstrate that this region newly evolved in the green lineage during the transition from aquatic to terrestrial life.

Extension of the Human Fibrinogen Database with Detailed Clinical Information—The αC-Connector Segment

Fibrinogen, an abundant plasma glycoprotein, is involved in the final stage of blood coagulation. Decreased fibrinogen levels, which may be caused by mutations, are manifested mainly in bleeding and thrombotic disorders. Clinically relevant mutations of fibrinogen are listed in the Human Fibrinogen Database. For the αC-connector (amino acids Aα240–410, nascent chain numbering), we have extended this database, with detailed descriptions of the clinical manifestations among members of reported families. This includes the specification of bleeding and thrombotic events and results of coagulation assays. Where available, the impact of a mutation on clotting and fibrinolysis is reported. The collected data show that the Human Fibrinogen Database reports considerably fewer missense and synonymous mutations than the general COSMIC and dbSNP databases. Homozygous nonsense or frameshift mutations in the αC-connector are responsible for most clinically relevant symptoms, while heterozygous mutations are often asymptomatic. Symptomatic subjects suffer from bleeding and, less frequently, from thrombotic events. Miscarriages within the first trimester and prolonged wound healing were reported in a few subjects. All mutations inducing thrombotic phenotypes are located at the identical positions within the consensus sequence of the tandem repeats.

Common sequence motifs of nascent chains engage the ribosome surface and trigger factor

In the cell, the conformations of nascent polypeptide chains during translation are modulated by both the ribosome and its associated molecular chaperone, trigger factor. The specific interactions that underlie these modulations, however, are still not known in detail. Here, we combine protein engineering, in-cell and in vitro NMR spectroscopy, and molecular dynamics simulations to explore how proteins interact with the ribosome during their biosynthesis before folding occurs. Our observations of α-synuclein nascent chains in living Escherichia coli cells reveal that ribosome surface interactions dictate the dynamics of emerging disordered polypeptides in the crowded cytosol. We show that specific basic and aromatic motifs drive such interactions and directly compete with trigger factor binding while biasing the direction of the nascent chain during its exit out of the tunnel. These results reveal a structural basis for the functional role of the ribosome as a scaffold with holdase characteristics and explain how handover of the nascent chain to specific auxiliary proteins occurs among a host of other factors in the cytosol.

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

Kinetic control of nascent protein biogenesis by peptide deformylase

Synthesis of bacterial proteins on the ribosome starts with a formylated methionine. Removal of the N-terminal formyl group is essential and is carried out by peptide deformylase (PDF). Deformylation occurs co-translationally, shortly after the nascent-chain emerges from the ribosomal exit tunnel, and is necessary to allow for further N-terminal processing. Here we describe the kinetic mechanism of deformylation by PDF of ribosome-bound nascent-chains and show that PDF binding to and dissociation from ribosomes is rapid, allowing for efficient scanning of formylated substrates in the cell. The rate-limiting step in the PDF mechanism is a conformational rearrangement of the nascent-chain that takes place after cleavage of the formyl group. Under conditions of ongoing translation, the nascent-chain is deformylated rapidly as soon as it becomes accessible to PDF. Following deformylation, the enzyme is slow in releasing the deformylated nascent-chain, thereby delaying further processing and potentially acting as an early chaperone that protects short nascent chains before they reach a length sufficient to recruit other protein biogenesis factors.

Mycolactone enhances the Ca2+ leakage from endoplasmic reticulum by trapping Sec61 translocons in a Ca2+ permeable state

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of, the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon “breathing” during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone’s stabilisation of the latter underpins the phenotype observed.

Pathway of Hsp70 interactions at the ribosome

In eukaryotes, an Hsp70 molecular chaperone triad assists folding of nascent chains emerging from the ribosome tunnel. In fungi, the triad consists of canonical Hsp70 Ssb, atypical Hsp70 Ssz1 and J-domain protein cochaperone Zuo1. Zuo1 binds the ribosome at the tunnel exit. Zuo1 also binds Ssz1, tethering it to the ribosome, while its J-domain stimulates Ssb’s ATPase activity to drive efficient nascent chain interaction. But the function of Ssz1 and how Ssb engages at the ribosome are not well understood. Employing in vivo site-specific crosslinking, we found that Ssb(ATP) heterodimerizes with Ssz1. Ssb, in a manner consistent with the ADP conformation, also crosslinks to ribosomal proteins across the tunnel exit from Zuo1. These two modes of Hsp70 Ssb interaction at the ribosome suggest a functionally efficient interaction pathway: first, Ssb(ATP) with Ssz1, allowing optimal J-domain and nascent chain engagement; then, after ATP hydrolysis, Ssb(ADP) directly with the ribosome.

A nascent polypeptide sequence modulates DnaA translation elongation in response to nutrient availability

The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes; nevertheless, the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA, we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, is critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that translation regulation by nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.