Abstract
The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5′ untranslated mRNA regions (5′-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5′-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5′-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5′-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.
Highlights
Shine-Dalgarno (SD) sequence of the prokaryotic mRNA is commonly regarded as a key element involved in the selection of the authentic start codons versus internal AUG codons
The ribosomal protein S1 binds to the A/U rich stretch of mRNA upstream of the start codon and is not universally conserved in bacteria: in Firmicutes which possess the largest fraction of genes preceded by SD sequence, the protein S1 is predicted to be nonfunctional in translation initiation [11]
Sequence logos detect SD sequence in the genomes of many prokaryote phyla The genome wide presence of SD sequence leads to the enrichment of adenine and guanine bases in sequence logo spanning the 25 to 210 bp region relative to the start codon of a gene [4,12]. Such enrichment can be clearly found in sequence logos of major bacterial phyla: Proteobacteria, Firmicutes, Actinobacteria, Thermotogae, Chloroflexi, and Aquificae (Fig. 1 A and S2, S6, S9, S10, S12, S14)
Summary
Shine-Dalgarno (SD) sequence of the prokaryotic mRNA is commonly regarded as a key element involved in the selection of the authentic start codons versus internal AUG codons It is usually 4–5 bp long, positioned 5–8 bp upstream from the start codon, and basepairs with the complementary sequence (anti SD) at the 39 end of 16S rRNA of the 30S ribosomal subunit. It is thought that 4–5 bp ShineDalgarno sequence-16S rRNA interaction is usually sufficient since SD sequence lengthening only rarely results in increased translation efficiency and longer, 8 or 10 bp interaction inhibits translation [1,5,6] It was shown, on the other hand, that E. coli can efficiently initiate translation in A/U rich translation initiation sites lacking SD sequence altogether [7] and that translation initiation of leaderless mRNA proceeds without mRNA-16S rRNA interaction [8]. The ribosomal protein S1 binds to the A/U rich stretch of mRNA upstream of the start codon and is not universally conserved in bacteria: in Firmicutes which possess the largest fraction of genes preceded by SD sequence, the protein S1 is predicted to be nonfunctional in translation initiation [11]
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