A widespread common protein fold packages ssRNA in virus particles with diverse morphology and genomic structure.

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo‐EM structure determination to show that folding of a β‐barrel protein begins with formation of a dynamic α‐helix inside the ribosome. As the growing peptide reaches the end of the tunnel, the N‐terminal part of the nascent chain refolds to a β‐hairpin structure that remains dynamic until its release from the ribosome. Contacts with the ribosome and structure of the peptidyl transferase center depend on nascent chain conformation. These results indicate that proteins may start out as α‐helices inside the tunnel and switch into their native folds only as they emerge from the ribosome. Moreover, the correlation of nascent chain conformations with reorientation of key residues of the ribosomal peptidyl‐transferase center suggest that protein folding could modulate ribosome activity.

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNA^Gly to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.

High-resolution structures at different stages, as well as biochemical, single molecule and computational approaches have highlighted the elasticity of tRNA molecules when bound to the ribosome. It is well acknowledged that the inherent structural flexibility of the tRNA lies at the heart of the protein synthesis process. Here, we review the recent advances and describe considerations that the conformational changes of the tRNA molecules offer about the mechanisms grounded in translation.

Introduction Protein synthesis is a central process for living entities. In all kingdoms of life, the key component of this process is the ribosome, a large macromolecular assembly composed of two distinctly sized subunits, whose cores are largely composed of ribosomal RNA, or rRNA. This molecular machine mediates the sequential incorporation of amino acids carried by the transfer RNAs (tRNAs) into the nascent protein chain. This process is also known as translation, because the genetic message encoded in the messenger RNA (mRNA) is deciphered into the language of proteins (Figure 7.1). The functional complexity of molecular machines such as the ribosome is coupled to their structural intricacy. Whereas initial imaging by electron microscopy showed nothing but dense granules ∼200Å in diameter (Palade, 1955), decades later the development of cryo-electron microscopy (cryo-EM), combined with image processing methodology, brought evidence for the extreme complexity of the ribosome with its multiple mobile and flexible parts. Recently, as the quality of electron density maps has greatly improved thanks to methodological advances in this field (see Chapter 2 in this volume by Joachim Frank), new intermediate states have been observed and the dynamic behavior of the ribosome and its ligands has been further characterized in conjunction with other biophysical techniques such as X-ray crystallography, kinetic analysis, and single-molecule fluorescent resonance energy transfer (smFRET) (see Figure 7.2). Considering that the 3D reconstructions obtained by cryo-EM are snapshots representing functional states along the translation pathway, it is now widely acknowledged that this experimental approach has been indispensable in unraveling essential processes that cooperate in translation. Furthermore, many structural insights that have come from cryo-EM work currently drive experimental design in a wide range of studies of the ribosome, from biochemical to crystallographic studies and single-molecule FRET.