Abstract
Bacterial translation initiation is influenced by base pairing between the Shine-Dalgarno (SD) sequence in the 5′ UTR of mRNA and the anti-SD (aSD) sequence at the free 3′ end of the 16S rRNA (3′ TAIL) due to: 1) the SD/aSD sequence binding location and 2) SD/aSD binding affinity. In order to understand what makes an SD/aSD interaction optimal, we must define: 1) terminus of the 3′ TAIL and 2) extent of the core aSD sequence within the 3′ TAIL. Our approach to characterize these components in Escherichia coli and Bacillus subtilis involves 1) mapping the 3′ boundary of the mature 16S rRNA using high-throughput RNA sequencing (RNA-Seq), and 2) identifying the segment within the 3′ TAIL that is strongly preferred in SD/aSD pairing. Using RNA-Seq data, we resolve previous discrepancies in the reported 3′ TAIL in B. subtilis and recovered the established 3′ TAIL in E. coli. Furthermore, we extend previous studies to suggest that both highly and lowly expressed genes favor SD sequences with intermediate binding affinity, but this trend is exclusive to SD sequences that complement the core aSD sequences defined herein.
Highlights
A recent model of SD/aSD interaction[10,11] (Fig. 1) suggests that optimal SD/aSD pairing may depend on three factors: 1) DtoStart (Fig. 1) which specifies the distance, in nucleotides, between the 16S rRNA 3′ terminus and the start codon, 2) SD/aSD binding affinity (Figs 1b), and 3) “leash” distance measured by D1 and D2 (Fig. 1)
We identify the 3′ TAIL in E. coli and
We BLASTed B. subtilis single reads from RNA-Seq run SRR1232437 against 85 nt at the 3′ terminus of the annotated B. subtilis 16S rDNA sequence (Fig. 2a, entry labelled 16S, NC_000964)
Summary
Elucidating the mature 16S rRNA 3′ tail using RNA-Seq data. B. subtilis using RNA-Seq data. To determine the extent of the core aSD sequence for both species, we examined the observed and expected usages for each site of the 3′ TAIL in base pairing with all putative SD sequences. E. coli has a lower preferred SD/aSD binding affinity relative to B. subtilis, SD sequences that complement CCUCC (−7.05 kcal/mol) are less selected for in the former than the latter due to the high binding affinity of the motif (Fig. 8b) Based on these observations, we suggest that the core aSD sequence is extended to 5′-CCUCCUUU-3′ in B. subtilis. 5′-AGGAG-3′ and 5′-GGAG-3′ in E. coli (Fig. 9), based on their 1) high usages, especially in HEGs, 2) intermediate binding affinity to core aSD sequences (5′-CCUCCUUU-3′ in B. subtilis, and 5′-CUCCUUA-3′ in E. coli), and 3) occurrences at optimal DtoStart locations. We speculate that this is due to the fact that intermediate levels of binding affinity are preferable
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