Coordination of -1 Programmed Ribosomal Frameshifting by Transcript and Nascent Chain Features Revealed by Deep Mutational Scanning

SummaryProgrammed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. In the alphavirus structural polyprotein, -1PRF is coordinated by a “slippery” sequence in the transcript, an RNA stem-loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4,530 mutations on -1PRF by deep mutational scanning. While most mutations within the slip-site and stem-loop disrupt -1PRF, mutagenic effects upstream of the slip-site are far more variable. Molecular dynamics simulations of polyprotein biogenesis suggest many of these mutations alter stimulatory forces on the nascent chain through their effects on translocon-mediated cotranslational folding. Finally, we provide evidence suggesting the coupling between cotranslational folding and -1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how -1PRF is coordinated by features within both the transcript and nascent chain.

Cotranslational folding of a periplasmic protein domain in Escherichia coli

In Gram-negative bacteria, periplasmic domains in inner membrane proteins are cotranslationally translocated across the inner membrane through the SecYEG translocon. To what degree such domains also start to fold cotranslationally is generally difficult to determine using currently available methods. Here, we apply Force Profile Analysis (FPA) – a method where a translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide – to follow the cotranslational translocation and folding of the large periplasmic domain of the E. coli inner membrane protease LepB in vivo. Membrane insertion of LepB’s two N-terminal transmembrane helices is initiated when their respective N-terminal ends reach 45-50 residues away from the peptidyl transferase center (PTC) in the ribosome. The main folding transition in the periplasmic domain involves all but the ~15 most C-terminal residues of the protein and happens when the C-terminal end of the folded part is ~70 residues away from the PTC; a smaller putative folding intermediate is also detected. This implies that wildtype LepB folds post-translationally in vivo, and shows that FPA can be used to study both co- and post-translational protein folding in the periplasm.

Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

Background The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. Results In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. Conclusions Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli

Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding-induced forces acting on the nascent polypeptide, have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond-containing E. coli protein alkaline phosphatase (PhoA) in a wild-type strain background and a strain background devoid of the periplasmic thiol:disulfide interchange protein DsbA. We find that folding-induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide-stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra-cytosolic compartment, like the periplasm.

Gradual Compaction of the Nascent Peptide During Cotranslational Folding on the Ribosome

ABSTRACTNascent polypeptides begin to fold in the constrained space of the ribosomal peptide exit tunnel. Here we use force profile analysis (FPA) and photo-induced energy-transfer fluorescence correlation spectroscopy (PET-FCS) to show how a small α-helical domain, the N-terminal domain of HemK, folds cotranslationally. Compaction starts vectorially as soon as the first α-helical segments are synthesized. As nascent chain grows, emerging helical segments dock onto each other and continue to rearrange at the vicinity of the ribosome. Inside or in the proximity of the ribosome, the nascent peptide undergoes structural fluctuations on the μs time scale. The fluctuations slow down as the domain moves away from the ribosome. Folding mutations have little effect on folding within the exit tunnel, but abolish the final domain stabilization. The results show the power of FPA and PET-FCS in solving the trajectory of cotranslational protein folding and in characterizing the dynamic properties of folding intermediates.