Figure 1.
Genome-wide assessment of the binding affinities of the anti–Shine-Dalgarno sequence in the 16S rRNA to the 5′ UTRs of prokaryotic genes.
The 5′ UTR sequences from -22 to -2 were used for the computations. Shown are the normalized distributions of hybridization energies of α-proteobacterial, γ-proteobacterical, cyanobacterial and plastid genes. Four major peaks are clearly distinguishable in all taxonomic groups at the positions -5.9, -3.6, -1.4 and +1.5 kcal mol−1. The peaks (from left to right) correspond to: (i) mRNAs with the SD sequence AGGAG, (ii) mRNAs with the SD sequence GAGG, AGGA or GGAG, (iii) mRNAs with short SD-like sequences (AGG, GAG or GGA), which may engage in SD-type interactions with the 3′ end of the 16S rRNA and (iv) mRNAs without SD sequences
Figure 2.
The amount of RNA secondary structure predicted around the start codon in α-proteobacteria, γ-proteobacteria, cyanobacteria, plant, metazoan and fungal mitochondria, and plastids.
Position 0 is the first nucleotide of the start codon. Genes without an SD sequence are represented by green curves, those with an SD by blue curves. The line shows the running mean minimum free energy of 5000 genes, the shaded area around it indicates the standard error of the mean. The minimum free energy was determined using a sliding window covering 50 nucleotides. The difference in MFE between the green and blue curves upstream and downstream of the initiation region in the three bacterial groups is largely due to differences in AT-content between genomes within an individual organismal group and a correlation between AT-richness and prevalence of SD-independent translation. The MFE difference disappears when individual genomes are analyzed (Figure S1). Note that, in metazoan mitochondria, the peak of the minimum free energy is shifted into the coding region, because most transcripts in animal mitochondria are leaderless and lack a 5′ UTR.
Figure 3.
(A) Schematic map of the lacZ' cassette. Promoter (Prrn), 3′ UTR (rps16 3′ UTR) and coding region (lacZ') are marked. The arrow denotes the direction of transcription. The sequence between the BamHI and BglII restriction sites was exchanged in the various constructs (i. e., the 5′ UTR indicated by the cross-hatched box and the first 21 nt of the coding region indicated by the open box). (B) Sequences from -33 to +26 of the constructs based on the gene 10 5′ UTR and the 5′ end of the lacZ' coding region. The numbers indicate the pLS plasmid numbers. pLS67 contains the unmutated sequence. (C) Sequences from -33 to +26 of the constructs based on the galE 5′ UTR and 5′ end of the coding region. pLS68 harbors the wild-type sequence. (D) Sequences from -33 to +26 of the constructs based on the rpsA 5′ UTR and 5′ end of the coding region. pLS69 contains the wild-type sequence. Shine-Dalgarno sequences are shown in bold grey letters, the ATG start codon is indicated by bold black letters.
Figure 4.
Experimental testing of the unfoldedness hypothesis for SD–independent translation.
(A) Correlation between LacZ activity and accessibility of the start codon in constructs without a SD sequence. (B) Lack of a significant correlation between LacZ activity and accessibility of the start codon in constructs with a SD sequence. The 5′ UTRs on which the individual constructs were based are indicated by color-coding. The LacZ activity was normalized to the activity of the unmutated construct (g10L: pLS67; galE: pLS68; rpsA: pLS69; cp. Figure 3). The start codon accessibility was calculated as the probability that every position of the start codon is unbound and was determined by RNAplfold [34] using a window size of 50 nt and an unbound region size of 3 nt.
Figure 5.
Comparison of the minimum free energy (MFE) values of start codons, in-frame AUG codons, and out-of-frame AUG triplets in the Escherichia coli genome.
The middle line in the box is the median, the next two are the 1st and 3rd quartiles, the whiskers extend to 1.5 times the interquartile range of the box. Outliers are indicated by open circles. The outlier in the start codon group is the MFE value for the start codon of trmD (see text for details). Three in-frame AUGs (filled black circles) of murC, metG and trmD, respectively, were singled out for their close proximity and MFE to the start codon. Based on the low amount of structure around them, these AUGs are candidates for alternative translation initiation sites.