Cotranslational folding of proteins on the ribosome.

Many proteins in the cell fold cotranslationally within the restricted space of the polypeptide exit tunnel or at the surface of the ribosome. A growing body of evidence suggests that the ribosome can alter the folding trajectory in many different ways. In this review, we summarize the recent examples of how translation affects folding of single-domain, multiple-domain and oligomeric proteins. The vectorial nature of translation, the spatial constraints of the exit tunnel, and the electrostatic properties of the ribosome-nascent peptide complex define the onset of early folding events. The ribosome can facilitate protein compaction, induce the formation of intermediates that are not observed in solution, or delay the onset of folding. Examples of single-domain proteins suggest that early compaction events can define the folding pathway for some types of domain structures. Folding of multi-domain proteins proceeds in a domain-wise fashion, with each domain having its role in stabilizing or destabilizing neighboring domains. Finally, the assembly of protein complexes can also begin cotranslationally. In all these cases, the ribosome helps the nascent protein to attain a native fold and avoid the kinetic traps of misfolding

Long-range signalling in activation of the translational GTPase EF-Tu.

Elongation factor Tu (EF-Tu) is a GTPase that delivers aminoacyl-tRNA (aa-tRNA) to the ribosome during protein synthesis. The factor is activated in response to the correct recognition of the mRNA codon by the anticodon of the aa-tRNA. The mechanism of signalling between the decoding centre of the ribosome and the site of GTP hydrolysis in EF-Tu where GTP hydrolysis takes place is not known. New high-resolution cryo-electron microscopic (cryo-EM) structures of the ribosome complex with aa-tRNA and EF-Tu blocked by the antibiotic kirromycin provide insights into the mechanistic details of the long-range signalling responsible for GTPase activation

Translation in prokaryotes.

This review summarizes our current understanding of translation in prokaryotes, focusing on the mechanistic and structural aspects of each phase of translation: initiation, elongation, termination, and ribosome recycling. The assembly of the initiation complex provides multiple checkpoints for messenger RNA (mRNA) and start-site selection. Correct codon–anticodon interaction during the decoding phase of elongation results in major conformational changes of the small ribosomal subunit and shapes the reaction pathway of guanosine triphosphate (GTP) hydrolysis. The ribosome orchestrates proton transfer during peptide bond formation, but requires the help of elongation factor P (EF-P) when two or more consecutive Pro residues are to be incorporated. Understanding the choreography of transfer RNA (tRNA) and mRNA movements during translocation helps to place the available structures of translocation intermediates onto the time axis of the reaction pathway. The nascent protein begins to fold cotranslationally, in the constrained space of the polypeptide exit tunnel of the ribosome. When a stop codon is reached at the end of the coding sequence, the ribosome, assisted by termination factors, hydrolyzes the ester bond of the peptidyl-tRNA, thereby releasing the nascent protein. Following termination, the ribosome is dissociated into subunits and recycled into another round of initiation. At each step of translation, the ribosome undergoes dynamic fluctuations between different conformation states. The aim of this article is to show the link between ribosome structure, dynamics, and function

The ribosome as a versatile catalyst: Reactions at the peptidyl transferase center.

In all contemporary organisms, the active site of the ribosome—the peptidyl transferase center—catalyzes two distinct reactions, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the hydrolysis of peptidyl-tRNA with the help of a release factor. However, when provided with appropriate substrates, ribosomes can also catalyze a broad range of other chemical reaction, which provides the basis for orthogonal translation and synthesis of alloproteins from unnatural building blocks. Advances in understanding the mechanisms of the two ubiquitous reactions, the peptide bond formation and peptide release, provide insights into the versatility of the active site of the ribosome. Release factors 1 and 2 and elongation factor P are auxiliary factors that augment the intrinsic catalytic activity of the ribosome in special cases

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

Elongation factor P: Function and effects on bacterial fitness.

The elongation phase of translation is promoted by three universal elongation factors, EF-Tu, EF-Ts, and EF-G in bacteria and their homologs in archaea and eukaryotes. Recent findings demonstrate that the translation of a subset of mRNAs requires a fourth elongation factor, EF-P in bacteria or the homologous factors a/eIF5A in other kingdoms of life. EF-P prevents the ribosome from stalling during the synthesis of proteins containing consecutive Pro residues, such as PPG, PPP, or longer Pro clusters. The efficient and coordinated synthesis of such proteins is required for bacterial growth, motility, virulence, and stress response. EF-P carries a unique post-translational modification which contributes to its catalytic proficiency. The modification enzymes, which are lacking in higher eukaryotes, provide attractive new targets for the development of new, highly specific antimicrobials