Deformylation of nascent peptide chains on the ribosome.

Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.

Cotranslational Biogenesis of Membrane Proteins in Bacteria.

Nascent polypeptides emerging from the ribosome during translation are rapidly scanned and processed by ribosome-associated protein biogenesis factors (RPBs). RPBs cleave the N-terminal formyl and methionine groups, assist cotranslational protein folding, and sort the proteins according to their cellular destination. Ribosomes translating inner-membrane proteins are recognized and targeted to the translocon with the help of the signal recognition particle, SRP, and SRP receptor, FtsY. The growing nascent peptide is then inserted into the phospholipid bilayer at the translocon, an inner-membrane protein complex consisting of SecY, SecE, and SecG. Folding of membrane proteins requires that transmembrane helices (TMs) attain their correct topology, the soluble domains are inserted at the correct (cytoplasmic or periplasmic) side of the membrane, and - for polytopic membrane proteins - the TMs find their interaction partner TMs in the phospholipid bilayer. This review describes the recent progress in understanding how growing nascent peptides are processed and how inner-membrane proteins are targeted to the translocon and find their correct orientation at the membrane, with the focus on biophysical approaches revealing the dynamics of the process. We describe how spontaneous fluctuations of the translocon allow diffusion of TMs into the phospholipid bilayer and argue that the ribosome orchestrates cotranslational targeting not only by providing the binding platform for the RPBs or the translocon, but also by helping the nascent chains to find their correct orientation in the membrane. Finally, we present the auxiliary role of YidC as a chaperone for inner-membrane proteins. We show how biophysical approaches provide new insights into the dynamics of membrane protein biogenesis and raise new questions as to how translation modulates protein folding.

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.

Lateral gate dynamics of the bacterial translocon during cotranslational membrane protein insertion.

During synthesis of membrane proteins, transmembrane segments (TMs) of nascent proteins emerging from the ribosome are inserted into the central pore of the translocon (SecYEG in bacteria) and access the phospholipid bilayer through the open lateral gate formed of two helices of SecY. Here we use single-molecule fluorescence resonance energy transfer to monitor lateral-gate fluctuations in SecYEG embedded in nanodiscs containing native membrane phospholipids. We find the lateral gate to be highly dynamic, sampling the whole range of conformations between open and closed even in the absence of ligands, and we suggest a statistical model-free approach to evaluate the ensemble dynamics. Lateral gate fluctuations take place on both short (submillisecond) and long (subsecond) timescales. Ribosome binding and TM insertion do not halt fluctuations but tend to increase sampling of the open state. When YidC, a constituent of the holotranslocon, is bound to SecYEG, TM insertion facilitates substantial opening of the gate, which may aid in the folding of YidC-dependent polytopic membrane proteins. Mutations in lateral gate residues showing in vivo phenotypes change the range of favored states, underscoring the biological significance of lateral gate fluctuations. The results suggest how rapid fluctuations of the lateral gate contribute to the biogenesis of inner-membrane proteins.

Co-translational insertion and topogenesis of bacterial membrane proteins monitored in real time.

Integral membrane proteins insert into the bacterial inner membrane co-translationally via the translocon. Transmembrane (TM) segments of nascent proteins adopt their native topological arrangement with the N-terminus of the first TM (TM1) oriented to the outside (type I) or the inside (type II) of the cell. Here, we study TM1 topogenesis during ongoing translation in a bacterial in vitro system, applying real-time FRET and protease protection assays. We find that TM1 of the type I protein LepB reaches the translocon immediately upon emerging from the ribosome. In contrast, the type II protein EmrD requires a longer nascent chain before TM1 reaches the translocon and adopts its topology by looping inside the ribosomal peptide exit tunnel. Looping presumably is mediated by interactions between positive charges at the N-terminus of TM1 and negative charges in the tunnel wall. Early TM1 inversion is abrogated by charge reversal at the N-terminus. Kinetic analysis also shows that co-translational membrane insertion of TM1 is intrinsically rapid and rate-limited by translation. Thus, the ribosome has an important role in membrane protein topogenesis.

Converting GTP hydrolysis into motion: versatile translational elongation factor G.

Elongation factor G (EF-G) is a translational GTPase that acts at several stages of protein synthesis. Its canonical function is to catalyze tRNA movement during translation elongation, but it also acts at the last step of translation to promote ribosome recycling. Moreover, EF-G has additional functions, such as helping the ribosome to maintain the mRNA reading frame or to slide over non-coding stretches of the mRNA. EF-G has an unconventional GTPase cycle that couples the energy of GTP hydrolysis to movement. EF-G facilitates movement in the GDP-Pi form. To convert the energy of hydrolysis to movement, it requires various ligands in the A site, such as a tRNA in translocation, an mRNA secondary structure element in ribosome sliding, or ribosome recycling factor in post-termination complex disassembly. The ligand defines the direction and timing of EF-G-facilitated motion. In this review, we summarize recent advances in understanding the mechanism of EF-G action as a remarkable force-generating GTPase.

Total synthesis of (±)-fumimycin and analogues for biological evaluation as peptide deformylase inhibitors.

A concise 7-step total synthesis of (±)-fumimycin in 11.6 % overall yield is reported. An acid-catalyzed intramolecular aza-Friedel-Crafts cyclization was developed to construct the benzofuranone skeleton of the natural product bearing an α,α-disubstituted amino acid moiety in a single step. Regioselective chlorination followed by a Suzuki-Miyaura cross-coupling rapidly enabled the preparation of a library of analogues which were evaluated against peptide deformylase for antibacterial activity.

Cotranslational protein targeting to the membrane: Nascent-chain transfer in a quaternary complex formed at the translocon.

Membrane proteins in bacteria are cotranslationally inserted into the plasma membrane through the SecYEG translocon. Ribosomes exposing the signal-anchor sequence (SAS) of a membrane protein are targeted to the translocon by the signal recognition particle (SRP) pathway. SRP scans translating ribosomes and forms high-affinity targeting complexes with those exposing a SAS. Recognition of the SAS activates SRP for binding to its receptor, FtsY, which, in turn, is primed for SRP binding by complex formation with SecYEG, resulting in a quaternary targeting complex. Here we examine the effect of SecYEG docking to ribosome-nascent-chain complexes (RNCs) on SRP binding and SAS transfer, using SecYEG embedded in phospholipid-containing nanodiscs and monitoring FRET between fluorescence-labeled constituents of the targeting complex. SecYEG-FtsY binding to RNC-SRP complexes lowers the affinity of SRP to both ribosome and FtsY, indicating a general weakening of the complex due to partial binding competition near the ribosomal peptide exit. The rearrangement of the quaternary targeting complex to the pre-transfer complex requires an at least partially exposed SAS. The presence of SecYEG-bound FtsY and the length of the nascent chain strongly influence nascent-chain transfer from SRP to the translocon and repositioning of SRP in the post-transfer complex.

Signal recognition particle binds to translating ribosomes before emergence of a signal anchor sequence.

The bacterial signal recognition particle (SRP) is part of the machinery that targets ribosomes synthesizing membrane proteins to membrane-embedded translocons co-translationally. Recognition of nascent membrane proteins occurs by virtue of a hydrophobic signal-anchor sequence (SAS) contained in the nascent chain, usually at the N terminus. Here we use fluorescence-based stopped-flow to monitor SRP-ribosome interactions with actively translating ribosomes while an SRP substrate is synthesized and emerges from the peptide exit tunnel. The kinetic analysis reveals that, at cellular concentrations of ribosomes and SRP, SRP rapidly binds to translating ribosomes prior to the emergence of an SAS and forms an initial complex that rapidly rearranges to a more stable engaged complex. When the growing peptide reaches a length of ∼50 amino acids and the SAS is partially exposed, SRP undergoes another conformational change which further stabilizes the complex and initiates targeting of the translating ribosome to the translocon. These results provide a reconciled view on the timing of high-affinity targeting complex formation, while emphasizing the existence of preceding SRP recruitment steps under conditions of ongoing translation.

Signal recognition particle prevents N-terminal processing of bacterial membrane proteins.

Bacterial proteins are synthesized with an N-formylated amino-terminal methionine, and N-formylated peptides elicit innate-immunity responses against bacterial infections. However, the source of these formylated peptides is not clear, as most bacterial proteins are co-translationally deformylated by peptide deformylase. Here we develop a deformylation assay with translating ribosomes as substrates, to show that the binding of the signal recognition particle (SRP) to signal sequences in nascent proteins on the ribosome prevents deformylation, whereas deformylation of nascent proteins without signal sequence is not affected. Deformylation and its inhibition by SRP are not influenced by trigger factor, a chaperone that interacts with nascent chains on the ribosome. We propose that bacterial inner-membrane proteins, in particular those with N-out topology, can retain their N-terminal formyl group during cotranslational membrane insertion and supply formylated peptides during bacterial infections.

Translocation as continuous movement through the ribosome.

In each round of translation elongation, tRNAs and mRNA move within the ribosome by one codon at a time. tRNA-mRNA translocation is promoted by elongation factor G (EF-G) at the cost of GTP hydrolysis. The key questions for understanding translocation are how and when the tRNAs move and how EF-G coordinates motions of the ribosomal subunits with tRNA movement. Here we present 2 recent papers which describe the choreography of movements over the whole trajectory of translocation. We present the view that EF-G accelerates translocation by promoting the steps that lead to GTPase-dependent ribosome unlocking. EF-G facilitates the formation of the rotated state of the ribosome and uncouples the backward motions of the ribosomal subunits, forming an open conformation in which the tRNAs can rapidly move. Ribosome dynamics are important not only in translocation, but also in recoding events, such as frameshifting and bypassing, and mediate sensitivity to antibiotics.

The bacterial SRP receptor, FtsY, is activated on binding to the translocon.

Proteins are inserted into the bacterial plasma membrane cotranslationally after translating ribosomes are targeted to the translocon in the membrane via the signal recognition particle (SRP) pathway. The targeting pathway involves an interaction between SRP and the SRP receptor, FtsY. Here we focus on the role of FtsY and its interaction with the translocon in controlling targeting. We show that in unbound FtsY the NG and A domains interact with one another. The interaction involves the membrane-targeting region at the junction between A and N domain. The closed form of FtsY is impaired in binding to SRP. Upon binding to the phospholipid-embedded translocon the domains of FtsY move apart. This enhances the docking of the FtsY NG domain to the homologous NG domain of the SRP protein Ffh. Thus, FtsY binding to the translocon has a central role in orchestrating the formation of a quaternary transfer complex in which the nascent peptide is transferred to the translocon. We propose that FtsY activation at the translocon ensures that ribosome-SRP complexes are directed to available translocons. This way sequestering SRP in futile complexes with unbound FtsY can be avoided and efficient targeting to the translocon achieved.

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY.

Integral membrane proteins in bacteria are co-translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N-terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C-terminal membrane targeting sequence. The central A domain binds to the translocon non-specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome-bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.

Protein Elongation, Co-translational Folding and Targeting.

The elongation phase of protein synthesis defines the overall speed and fidelity of protein synthesis and affects protein folding and targeting. The mechanisms of reactions taking place during translation elongation remain important questions in understanding ribosome function. The ribosome-guided by signals in the mRNA-can recode the genetic information, resulting in alternative protein products. Co-translational protein folding and interaction of ribosomes and emerging polypeptides with associated protein biogenesis factors determine the quality and localization of proteins. In this review, we summarize recent findings on mechanisms of translation elongation in bacteria, including decoding and recoding, peptide bond formation, tRNA-mRNA translocation, co-translational protein folding, interaction with protein biogenesis factors and targeting of ribosomes synthesizing membrane proteins to the plasma membrane. The data provide insights into how the ribosome shapes composition and quality of the cellular proteome.

Choreography of molecular movements during ribosome progression along mRNA.

During translation elongation, ribosome translocation along an mRNA entails rotations of the ribosomal subunits, swiveling motions of the small subunit (SSU) head and stepwise movements of the tRNAs together with the mRNA. Here, we reconstructed the choreography of the collective motions of the Escherichia coli ribosome during translocation promoted by elongation factor EF-G, by recording the fluorescence signatures of nine different reporters placed on both ribosomal subunits, tRNA and mRNA. We captured an early forward swiveling of the SSU head taking place while the SSU body rotates in the opposite, clockwise direction. Backward swiveling of the SSU head starts upon tRNA translocation and continues until the post-translocation state is reached. This work places structures of translocation intermediates along a time axis and unravels principles of the motions of macromolecular machines.

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon.

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY-SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP-RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY-FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex.

Fluctuations between multiple EF-G-induced chimeric tRNA states during translocation on the ribosome.

The coupled translocation of transfer RNA and messenger RNA through the ribosome entails large-scale structural rearrangements, including step-wise movements of the tRNAs. Recent structural work has visualized intermediates of translocation induced by elongation factor G (EF-G) with tRNAs trapped in chimeric states with respect to 30S and 50S ribosomal subunits. The functional role of the chimeric states is not known. Here we follow the formation of translocation intermediates by single-molecule fluorescence resonance energy transfer. Using EF-G mutants, a non-hydrolysable GTP analogue, and fusidic acid, we interfere with either translocation or EF-G release from the ribosome and identify several rapidly interconverting chimeric tRNA states on the reaction pathway. EF-G engagement prevents backward transitions early in translocation and increases the fraction of ribosomes that rapidly fluctuate between hybrid, chimeric and posttranslocation states. Thus, the engagement of EF-G alters the energetics of translocation towards a flat energy landscape, thereby promoting forward tRNA movement.

Lateral opening of the bacterial translocon on ribosome binding and signal peptide insertion.

Proteins are co-translationally inserted into the bacterial plasma membrane via the SecYEG translocon by lateral release of hydrophobic transmembrane segments into the phospholipid bilayer. The trigger for lateral opening of the translocon is not known. Here we monitor lateral opening by photo-induced electron transfer (PET) between two fluorophores attached to the two SecY helices at the rim of the gate. In the resting translocon, the fluorescence is quenched, consistent with a closed conformation. Ribosome binding to the translocon diminishes PET quenching, indicating opening of the gate. The effect is larger with ribosomes exposing hydrophobic transmembrane segments and vanishes at low temperature. We propose a temperature-dependent dynamic equilibrium between closed and open conformations of the translocon that is shifted towards partially and fully open by ribosome binding and insertion of a hydrophobic peptide, respectively. The combined effects of ribosome and peptide binding allow for co-translational membrane insertion of successive transmembrane segments.

Synchronous tRNA movements during translocation on the ribosome are orchestrated by elongation factor G and GTP hydrolysis.

The translocation of tRNAs through the ribosome proceeds through numerous small steps in which tRNAs gradually shift their positions on the small and large ribosomal subunits. The most urgent questions are: (i) whether these intermediates are important; (ii) how the ribosomal translocase, the GTPase elongation factor G (EF-G), promotes directed movement; and (iii) how the energy of GTP hydrolysis is coupled to movement. In the light of recent advances in biophysical and structural studies, we argue that intermediate states of translocation are snapshots of dynamic fluctuations that guide the movement. In contrast to current models of stepwise translocation, kinetic evidence shows that the tRNAs move synchronously on the two ribosomal subunits in a rapid reaction orchestrated by EF-G and GTP hydrolysis. EF-G combines the energy regimes of a GTPase and a motor protein and facilitates tRNA movement by a combination of directed Brownian ratchet and power stroke mechanisms.

Interplay between trigger factor and other protein biogenesis factors on the ribosome.

Nascent proteins emerging from translating ribosomes in bacteria are screened by a number of ribosome-associated protein biogenesis factors, among them the chaperone trigger factor (TF), the signal recognition particle (SRP) that targets ribosomes synthesizing membrane proteins to the membrane and the modifying enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). Here, we examine the interplay between these factors both kinetically and at equilibrium. TF rapidly scans the ribosomes until it is stabilized on ribosomes presenting TF-specific nascent chains. SRP binding to those complexes is strongly impaired. Thus, TF in effect prevents SRP binding to the majority of ribosomes, except those presenting SRP-specific signal sequences, explaining how the small amount of SRP in the cell can be effective in membrane targeting. PDF and MAP do not interfere with TF or SRP binding to translating ribosomes, indicating that nascent-chain processing can take place before or in parallel with TF or SRP binding.