Lessons from RatA: Why the Basics in Molecular Biology Are Still Crucial!

Since the first bacterial genomes were sequenced and annotated over 25 years ago, sequencing technologies have rapidly advanced in both speed and cost efficiency. To date, over two million annotated bacterial genomes have been deposited in the National Center for Biotechnology Information (NCBI) database. Yet, there are many genes with unknown functions and, furthermore, conflicting results have been published for many investigated genes. One example is the ratA (or pasT) gene from Escherichia coli (E. coli) K-12 strains. Initially identified as a ribosome-targeting toxin, later studies described RatA as the bacterial homolog of the mitochondrial Coq10 protein and, therefore, beneficial for E. coli cells during aerobic growth. This study shows that these conflicting results originated from a mis-annotation of the start codon in the genomic sequence. Overexpression of the ratA gene as currently annotated leads to the synthesis of two RatA protein variants, a toxic and a non-toxic one. This study further identifies the endogenous ratA promoter and shows that only the shorter, non-toxic variant of RatA is synthesized during different growth phases specifically under aerobic conditions. Our findings thereby not only solidify the role of RatA in E. coli, but also demonstrate the importance of first validating the basics of molecular biology when investigating a previously poorly described gene, even in times of advanced high-throughput techniques.