Analysis of tetra- and hepta-nucleotides motifs promoting -1 ribosomal frameshifting in Escherichia coli.
Bottom Line: Depending on the signal, the frameshifting frequency can vary over a wide range, from less than 1% to more than 50%.While motif efficiency varies widely, a major distinctive rule of bacterial -1 frameshifting is that the most efficient motifs are those allowing cognate re-pairing of the A site tRNA from ZZN to ZZZ.The outcome of the genomic search is a set of 69 gene clusters, 59 of which constitute new candidates for functional utilization of -1 frameshifting.
Affiliation: School of Biochemistry and Cell biology, University College Cork, Cork, Ireland.Show MeSH
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Mentions: In addition, the region 30 nt upstream of the motif was checked for the presence of a conserved SD-like sequence and the region extending 200 nt downstream of the frameshift site was analysed for the presence of a conserved RNA secondary structure; our criteria for a conserved stimulator was the presence of such a structure in at least 50% of the genes of a cluster. The SD-like sequences to be searched 6–17 nt upstream of the motif were those for which a stimulatory effect was experimentally demonstrated (Materials and Methods) (32). A conserved SD was found in 8 out of the 56 non-mobile clusters and in 3 out of the 13 IS and prophage clusters (Figure 8C, Supplementary Tables S4 and S8). In contrast, a potential stimulatory structure was predicted in a larger proportion of clusters: a conserved hairpin is present in the 8 IS clusters, in 3 out of 5 of the phage clusters and in 42 out of the 56 non-mobile genes clusters (see Materials and Methods for the parameters used to define the hairpin structure). Nine clusters possess both types of stimulators. To characterize further the predicted structures, we compared them with IS3 family members possessing a frameshift site and an associated stimulatory structure (9,10). To assess structures of different sizes, we used a single parameter, ΔGhp.nt−1; which is the ΔGunfold@37°C value of the hairpin divided by the number of nucleotides in the structure. An overall comparison showed that taken together the hairpins of our 53 clusters had a lower ΔGhp.nt−1 than those from a set of 271 IS3 family members (0.317 ± 0.114 versus 0.452 ± 0.124 kcal.mol−1.nt−1; Supplementary Table S8 and Figure S2). For a more refined comparison, 20 IS3 members were selected because they have a hairpin ranging from 17 to 131 nt. The average ΔG.nt−1 (ΔGav.nt−1) downstream of the frameshift motif was determined as detailed in Materials and Methods for these ISs as well as for our 53 clusters. The difference between ΔGhp.nt−1 and ΔGav.nt−1, ΔΔG.nt−1, was calculated and plotted against the size of the structure (Figure 9). It appeared that all the IS hairpins have a positive ΔΔG.nt−1 value (≥0.09 kcal.mol−1.nt−1) indicating that the hairpin segment is more structured than average, as expected if there is selective pressure for its maintenance (Figure 9A). The distribution of ΔΔG.nt−1 values is clearly not the same for our 53 clusters, especially the non-mobile genes clusters (Figure 9B): only 14 of them are at or above the 0.09 kcal.mol−1.nt−1 threshold value defined by the IS set. The remaining 28 clusters, as well as 2 phage clusters and 1 IS cluster, appeared to have a local folding level close or even below average. This suggests that their respective potential hairpins may not have been selected for but are fortuitous, non-biologically relevant, structures.
Affiliation: School of Biochemistry and Cell biology, University College Cork, Cork, Ireland.