Unconventional Translation Strategies That Diversify The Bacterial Proteome

A common rule in genetics is that each gene encodes a single protein. Although this is usually the case, organisms have evolved unique strategies allowing them to make more than one protein from the same gene. With these strategies, cells diversify their proteome without changing the size of their genome. Two such interesting mechanisms are programmed ribosomal frameshifting and internal translation initiation. During frameshifting, the ribosome starts decoding the gene in the “normal” frame but then it changes and resumes translation in an alternative frame. The beginnings of the two proteins from a gene where frameshifting occurs are identical but their ends are different. In the case of internal initiation, translation can begin from the “normal” start site but also from a secondary one inside the gene. Thus, the dual initiation gives rise to two proteins with identical ends. Interestingly, only a few examples of frameshifting and internal initiation were known in bacteria. The goal of my PhD was to explore the prevalence, mechanisms and functional importance of frameshifting and internal initiation. We applied genome-wide approaches, biochemical experiments, proteomics, bioinformatics and genetics in the model microorganism Escherichia coli. We learned that: 1-Frameshifting regulates copper homeostasis in bacteria The frameshifting allows generation of a copper transporter and a copper chaperone from the copA gene. When frameshifting is disrupted, the cells suffer from copper toxicity. We also found that frameshifting could influence copper homeostasis in other organisms, including humans. 2-Internal initiation is a pervasive way to diversify the bacterial proteome We designed a genome-wide approach to “catch” ribosomes initiating translation, at normal or at internal start codons. With this method we found that not a few, as it was known before, but more than one hundred genes have functional internal start sites. Our results show that internal initiation is pervasive in bacteria and suggest that this mechanism may be exploited to generate proteins critical for cellular homeostasis. Importantly, our approach can be implemented to discover start sites in a wide range of bacterial genomes. Our work highlights the importance to explore the hidden alternative proteome of bacteria, shed light on translation regulation mechanisms and reveal novel proteins and cellular processes.