Inability of Prevotella bryantii to form a functional Shine-Dalgarno interaction reflects unique evolution of ribosome binding sites in Bacteroidetes

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.

Figure 1. Sequence logo analysis of start codon upstream regions.

A: Sequence logos showing the presence of SD sequence in some well known bacteria. Sequence logos of Bacteroidetes (B) and Chlorobi (C) demonstrate the lack of SD sequence. The position 0 represents the nucleotide position of start codon.

Figure 2. Start codon upstream sequences of plasmid borne reporter genes.

The sequence starts with the PstI site of the pRH3 or pUC19 vector (shown in italics). The stop codon at the start of the upstream sequence is underlined and the start codon of nucB or hMGFP is in boldface. A and B: nucB constructs with added (A) or removed (B) SD sequences. C: start codon upstream sequences containing SD sequences that are involved in formation of mRNA secondary structures with different stability. The sequences starting with mg were used in E. coli to translate the MGFP. D: Start codon upstream sequences of plasmid pRH3 borne nucB constructs containing SD sequence length of 10, 8, and 6 nucleotides. E: nucB upstream sequence used to asses the effect of secondary structure 20 bp upstream of the start codon. The secondary structure element was inserted into construct 0 from C after the designated adenosine.

Figure 3. Folding energy distribution of start codon upstream regions in P. bryantii B₁4 genome compared to distributions of successive methionine upstream regions.