Wolf Holtkamp

Structural basis for the inhibition of the eukaryotic ribosome

The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.

Cotranslational protein folding on the ribosome monitored in real time

Proteins shape up in the ribosome Proteins consist of linear chains of amino acids. These chains must fold into complex three-dimensional shapes to become functional. Holtkamp et al. “watched” how a small helical protein folds as it is being synthesized by the ribosome. The lengthening polypeptide passes out through the ribosome exit tunnel where folding starts. The initially compact structure quickly rearranges into a native three-dimensional structure as the polypeptide emerges from the tunnel. Science, this issue p. 1104 A small protein folds into a non-native form as it is synthesized on the ribosome before adopting its native shape. Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain—the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK—in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.

Dynamic switch of the signal recognition particle from scanning to targeting

Ribosomes synthesizing inner membrane proteins in Escherichia coli are targeted to the membrane by the signal recognition particle (SRP) pathway. By rapid kinetic analysis we show that after initial binding to the ribosome, SRP undergoes dynamic fluctuations in search of additional interactions. Non-translating ribosomes, or ribosomes synthesizing non-membrane proteins, do not provide these contacts, allowing SRPs to dissociate rapidly. A nascent peptide in the exit tunnel stabilizes SRPs in a standby state. Binding to the emerging signal-anchor sequence (SAS) of a nascent membrane protein halts the fluctuations of SRP, resulting in complex stabilization and recruitment of the SRP receptor. We propose a kinetic model where SRP rapidly scans all ribosomes until it encounters a ribosome exposing an SAS. Binding to the SAS switches SRP into the targeting mode, in which dissociation is slow and docking of the SRP receptor is accelerated.

GTP hydrolysis by EF‐G synchronizes tRNA movement on small and large ribosomal subunits

Elongation factor G (EF‐G) promotes the movement of two tRNAs and the mRNA through the ribosome in each cycle of peptide elongation. During translocation, the tRNAs transiently occupy intermediate positions on both small (30S) and large (50S) ribosomal subunits. How EF‐G and GTP hydrolysis control these movements is still unclear. We used fluorescence labels that specifically monitor movements on either 30S or 50S subunits in combination with EF‐G mutants and translocation‐specific antibiotics to investigate timing and energetics of translocation. We show that EF‐G–GTP facilitates synchronous movements of peptidyl‐tRNA on the two subunits into an early post‐translocation state, which resembles a chimeric state identified by structural studies. EF‐G binding without GTP hydrolysis promotes only partial tRNA movement on the 50S subunit. However, rapid 30S translocation and the concomitant completion of 50S translocation require GTP hydrolysis and a functional domain 4 of EF‐G. Our results reveal two distinct modes for utilizing the energy of EF‐G binding and GTP hydrolysis and suggest that coupling of GTP hydrolysis to translocation is mediated through rearrangements of the 30S subunit.

Dual use of GTP hydrolysis by elongation factor G on the ribosome

Elongation factor G (EF-G) is a GTPase that catalyzes tRNA and mRNA translocation during the elongation cycle of protein synthesis. The GTP-bound state of the factor on the ribosome has been studied mainly with non-hydrolyzable analogs of GTP, which led to controversial conclusions about the role of GTP hydrolysis in translocation. Here we describe a mutant of EF-G in which the catalytic His91 is replaced with Ala. The mutant EF-G does not hydrolyze GTP, but binds GTP with unchanged affinity, allowing us to study the function of the authentic GTP-bound form of EF-G in translocation. Utilizing fluorescent reporter groups attached to the tRNAs, mRNA, and the ribosome we compile the velocity map of translocation seen from different perspectives. The data suggest that GTP hydrolysis accelerates translocation up to 30-fold and facilitates conformational rearrangements of both 30S subunit (presumably the backward rotation of the 30S head) and EF-G that lead to the dissociation of the factor. Thus, EF-G combines the energy regime characteristic for motor proteins, accelerating movement by a conformational change induced by GTP hydrolysis, with that of a switch GTPase, which upon Pi release switches the conformations of EF-G and the ribosome to low affinity, allowing the dissociation of the factor.

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.

Co-translational protein folding: Progress and methods.

Proteins are synthesized as linear polymers and have to fold into their native structure to fulfil various functions in the cell. Folding can start co-translationally when the emerging peptide is still attached to the ribosome and is guided by the environment of the polypeptide exit tunnel and the kinetics of translation. Major questions are: When does co-translational folding begin? What is the role of the ribosome in guiding the nascent peptide towards its native structure? How does translation elongation kinetics modulate protein folding? Here we suggest how novel structural and biophysical approaches can help to probe the interplay between the ribosome and the emerging peptide and present future challenges in understanding co-translational folding.

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.

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

Abstract 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.

Dynamics of ribosomes and release factors during translation termination in E. coli

Release factors RF1 and RF2 promote hydrolysis of peptidyl-tRNA during translation termination. The GTPase RF3 promotes recycling of RF1 and RF2. Using single molecule FRET and biochemical assays, we show that ribosome termination complexes that carry two factors, RF1–RF3 or RF2–RF3, are dynamic and fluctuate between non-rotated and rotated states, whereas each factor alone has its distinct signature on ribosome dynamics and conformation. Dissociation of RF1 depends on peptide release and the presence of RF3, whereas RF2 can dissociate spontaneously. RF3 binds in the GTP-bound state and can rapidly dissociate without GTP hydrolysis from termination complex carrying RF1. In the absence of RF1, RF3 is stalled on ribosomes if GTP hydrolysis is blocked. Our data suggest how the assembly of the ribosome–RF1–RF3–GTP complex, peptide release, and ribosome fluctuations promote termination of protein synthesis and recycling of the release factors.