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 (or ) gene from () 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 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 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 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 , 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.