Predicting Shine–Dalgarno Sequence Locations Exposes Genome Annotation Errors

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Abstract
In prokaryotes, Shine–Dalgarno (SD) sequences, nucleotides upstream from start codons on messenger RNAs (mRNAs) that are complementary to ribosomal RNA (rRNA), facilitate the initiation of protein synthesis. The location of SD sequences relative to start codons and the stability of the hybridization between the mRNA and the rRNA correlate with the rate of synthesis. Thus, accurate characterization of SD sequences enhances our understanding of how an organism's transcriptome relates to its cellular proteome. We implemented the Individual Nearest Neighbor Hydrogen Bond model for oligo–oligo hybridization and created a new metric, relative spacing (RS), to identify both the location and the hybridization potential of SD sequences by simulating the binding between mRNAs and single-stranded 16S rRNA 3′ tails. In 18 prokaryote genomes, we identified 2,420 genes out of 58,550 where the strongest binding in the translation initiation region included the start codon, deviating from the expected location for the SD sequence of five to ten bases upstream. We designated these as RS+1 genes. Additional analysis uncovered an unusual bias of the start codon in that the majority of the RS+1 genes used GUG, not AUG. Furthermore, of the 624 RS+1 genes whose SD sequence was associated with a free energy release of less than −8.4 kcal/mol (strong RS+1 genes), 384 were within 12 nucleotides upstream of in-frame initiation codons. The most likely explanation for the unexpected location of the SD sequence for these 384 genes is mis-annotation of the start codon. In this way, the new RS metric provides an improved method for gene sequence annotation. The remaining strong RS+1 genes appear to have their SD sequences in an unexpected location that includes the start codon. Thus, our RS metric provides a new way to explore the role of rRNA–mRNA nucleotide hybridization in translation initiation. More than 30 years ago researchers first discovered a sequence of messenger RNA (mRNA) nucleotides in bacteria that ribosomes recognize as a signal for where to begin protein synthesis. Today, genome annotation software takes advantage of this finding and uses it to help identify the location of start codons. Because these sequences, now referred to as Shine–Dalgarno (SD) sequences, are always upstream from start codons, annotation programs look for them in the region 5′ to these candidate sites. In a comprehensive analysis of 18 bacterial genomes, the authors show that when looking for SD sequences, it sometimes pays off to analyze unlikely locations. By examining the region that immediately surrounds the start codon for SD sequences, the authors identify many mis-annotated genes and in so doing offer a method to help check for these in future annotation projects.