Reading frame maintenance by the ribosome during stalling

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PROJECT SUMMARY/ABSTRACT In all domains of life, decoding of the genetic information into peptides is accomplished by the ribosome, which reads the messenger RNA (mRNA) three nucleotides at a time. Following careful selection of the aminoacyl-tRNA that matches this triplet codon, the ribosome must precisely move to reading the next codon. Precise translocation is not an easy task given the multiple coordinated movements of the mRNA, tRNA and the ribosomal subunits that must occur. Failure to do so results in so-called frameshifting errors, which are detrimental to proteostasis as they result in errant protein products that bear no resemblance to the encoded ones. Notably, much of what we know about reading-frame maintenance comes from studies on programmed or “intentional” frameshifting. These studies revealed that sequence and structural features of the mRNA and its interaction with elements of the ribosome, translation factors and the tRNA contribute to these events. Although many of these elements are unique to each mRNA, almost all frameshifting events rely on ribosome stalling. Cellular response to stalls has been almost exclusively in the context of quality control and ribosome rescue. In particular, stalls are recognized by ubiquitin ligases when they cause ribosome collisions. In principle, colliding ribosomes can also provide structural impediments required for frameshifting; indeed, we recently showed that collisions can lead to efficient +1 frameshifting, suggesting that cells must have evolved factors to maintain reading frame during translation stalls. As ribosomes appear to stall frequently under stress, these mechanisms are more than likely to become critical for cell survival and recovery. This proposal is focused on one recently identified mechanism that involves the highly conserved multi-protein bridging factor (Mbf1). Our preliminary studies suggest that the factor prevents collision-mediated +1 frameshifting. This, together with a preliminary cyoEM structure of a Mbf1-bound ribosome, forms the basis of our major hypothesis that Mbf1 recognizes collided ribosomes to prevent them from altering the reading frame of the leading one. We will test this hypothesis through three aims. In the first one, we will assess how altering ribosome density and mRNA-sequence and -structural features modulate the function of Mbf1 in an effort to establish a relationship between ribosome collisions and frameshifting. In the second aim, using modified ribosome-profiling approaches to assess frameshifting transcriptome-wide, we will dissect the role of Mbf1 in preventing frameshifting occurring at stochastic collisions as well as those experienced under stress. In the third aim, the mechanism of Mbf1 recruitment to stalled ribosomes will be studied using a battery of biochemical and biophysical approaches. We are most interested in investigating how the factor alters the function and the structure of the translation machinery. Collectively, our interdisciplinary approach builds and expands on established expertise and resources, which we plan to use to uncover important details about how conserved factors are recruited to ribosomes to modulate their function under stress.