SUMMARY Ribosomes that stall before completing peptide synthesis must be recycled and returned to the cytoplasmic pool. The protein Dom34 and cofactors Hbs1 and Rli1 can dissociate stalled ribosomes in vitro, but the identity of targets in the cell is unknown. Here we extend ribosome profiling methodology to reveal a high-resolution molecular characterization of Dom34 function in vivo. Dom34 removes stalled ribosomes from truncated mRNAs, but, in contrast, does not generally dissociate ribosomes on coding sequences known to trigger stalling, such as polyproline. We also show that Dom34 targets arrested ribosomes near the ends of 3´ UTRs. These ribosomes appear to gain access to the 3 UTR via a mechanism that does not require decoding of the mRNA. These results suggest that ribosomes frequently enter downstream noncoding regions and that Dom34 carries out the important task of rescuing them.

Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by phi values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native alpha-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion-collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution.

We compare the folding of representative members of a protein superfamily by experiment and simulation to investigate common features in folding mechanisms. The homeodomain superfamily of three-helical, single-domain proteins exhibits a spectrum of folding processes that spans the complete transition from concurrent secondary and tertiary structure formation (nucleation-condensation mechanism) to sequential secondary and tertiary formation (framework mechanism). The unifying factor in their mechanisms is that the transition state for (un)folding is expanded and very native-like, with the proportion and degree of formation of secondary and tertiary interactions varying. There is a transition, or slide, from the framework to nucleation-condensation mechanism with decreasing stability of the secondary structure. Thus, framework and nucleation-condensation are different manifestations of an underlying common mechanism.two-state ͉ three-state ͉ framework ͉ nucleation ͉ homeodomain A Holy Grail of protein folding is to find a single mechanism. Given the diversity of protein structure and the evolutionary pressure on function and not on folding rates, a unique mechanism for folding would seem unlikely. If there are simplifying features, then small, single-domain proteins may be the most likely to exhibit them. But such proteins seem to fold by two distinct mechanisms. The 6-85 repressor fragment (1) and the engrailed homeodomain (En-HD; ref.2) seem to fold by a classical diffusion-collision mechanism (3-5) whereby secondary structural elements form independently and then dock to form the tertiary structure. Chymotrypsin inhibitor 2, on the other hand, folds by nucleation-condensation, which is characterized by concerted consolidation of secondary and tertiary interactions as the whole domain collapses around an extended nucleus (6). It has been argued on general grounds that nucleation-condensation and diffusion-collision are different manifestations of a common mechanism in which secondary structure and tertiary structure form in parallel (7,8). Nucleationcondensation reflects the situation when secondary structure is inherently unstable in the absence of tertiary interactions whereas diffusion-collision becomes more probable with increasing stability of secondary structure.Studies of the folding of point mutants of a prototype protein are essential for discovering atomic level details of folding mechanisms and kinetics. Single-point mutants may even cause gross changes in the kinetics of folding, such as the transition from three-state to two-state folding (9). But, to extrapolate a general understanding of folding mechanisms, studies on members of the same fold family (different homologues sharing the same overall topology but with different primary structures) can be useful in finding correlations between amino acid sequences and three-dimensional structures (10-16). Although there can be different folding routes through different transition states for some proteins (17), it seems that mechanisms of folding are oft...

Summary Ribosome profiling is a powerful method for globally assessing the activity of ribosomes in a cell. Despite its application in many organisms, ribosome profiling studies in bacteria have struggled to obtain the resolution necessary to precisely define translational pauses. Here we report improvements that yield much higher resolution in E. coli profiling data, enabling us to more accurately assess ribosome pausing and refine earlier studies of the impact of polyproline motifs on elongation. We comprehensively characterize pausing at proline-rich motifs in the absence of elongation factor EFP. We find that only a small fraction of genes with strong pausing motifs have reduced ribosome density downstream and identify features that explain this phenomenon. These features allow us to predict which proteins likely have reduced output in the efp knockout strain.