Translation initiation, a rate-limiting step in protein biosynthesis, involves the recognition, attachment and adaptation of the mRNA to the 30S subunit of the ribosome . Messenger RNA recognition is facilitated by the non-random distribution of purines about 5–10 nucleotides upstream the start codon [2–3]. This purine-rich sequence (typically 3–6 nucleotides long), known as the Shine-Dalgarno (SD) sequence, is also complementary to a conserved region at the 3’ end of the 16S rRNA located in the platform of the 30S subunit [4–5]. By complementary base pairing between the 16S rRNA and mRNA, the mRNA is attached to the 30S platform. Trans-acting initiation factors (IF1, IF2, and IF3) and ribosomal proteins mediate this attachment to the small subunit of the ribosome and help to unfold the mRNA for its accommodation in the channel of the ribosome. Although mutations in SD have been shown to alter protein expression levels up to 250-fold, SD itself is not obligatory for translation of some genes, e.g rpsA in Escherichia coli . In some of the cases where there is no complementarity between 16S rRNA and the sequence upstream of the mRNA start site, it has been shown that ribosomal protein S1 interacts with AU- rich regions to facilitate translation initiation .
A recent study in Lactococcus lactis revealed that in cases where mRNA-16S rRNA and/or mRNA-ribosomal protein interaction is absent, mRNA stability, or its lack thereof, contributes significantly in translation initiation efficiency [2, 6]. Hence, analyzing mRNA secondary structure is critical in understanding translation initiation, as the formation of highly stable hairpin structures around a start codon could not only occlude translation from that codon, but also drive translation from a weaker start codon with less secondary structure interference downstream [7–11]. Since SD serves as a recognition signal for the selection of the right reading frame for translation, it is expected that this sequence is somewhat sensitive to secondary structure formation.
A study of mRNA stability across alphaproteobacterial, gammaproteobacterial, cyanobacterial, plastid, metazoan mitochondrial, fungal mitochondrial and plant mitochondrial genomes was previously performed , and the results of randomly sampled 5000 genes from each group revealed that, on average, mRNAs without SD have less secondary structure than mRNAs with SD in organisms where SD-dependent and SD-independent translation coexist . Furthermore, in these organisms, mRNAs with and without SD generally have minimal secondary structure around the start codon, compared to the upstream and downstream regions of the start codon. The secondary structure analysis was based on predicting minimum free energy (MFE) of mRNAs with RNAfold function in the Vienna package , which is publicly available.
The objective of this study was to assess the influence of SD on the secondary structure of mRNAs in Rhodobacter sphaeroides. Two hypotheses were tested: 1) mRNAs with SD and mRNAs without SD retain similar stability, and 2) secondary structure around the start codon is minimized for mRNAs with SD and not for mRNAs without SD.
Generally, given the set of non-overlapping secondary structures, P, for a given sequence, S; Pr(P | S) = Boltzmann Distribution and N(Configurations) = [14–15]. Then probability of a base pair (i,j) for S is given by:
Minimum free energy (MFE) was used as a measure of secondary structure formation as previously described [12, 16]. MFE value is computed by adding up energy contributions of two consecutive base pairs according to nearest-neighbor-pairing rules [16–17]. The RNAfold function in Matlab Bioinformatics Toolbox [18–19], which implements the Turner energy model [16, 20–21], was used to compute the MFE values in this study. The RNAfold in Matlab incorporated some sequence-dependent adjustments in thermodynamic parameters to improve free energy minimization for RNA structure prediction .
This revised function performs better sequence knowledge-based computations of MFE, and a low MFE value for an input sequence indicates that the sequence is stable [22–24]. Furthermore, less secondary structure around the start codon and the SD of mRNAs would suggest that both the accessibility of the start codon and the exposure of the SD sequence for complementary pairing with 16S rRNA might be necessary for efficient translation initiation [8, 11, 25].