Translation initiation region sequence preferences in Escherichia coli

Abstract

Background: The mRNA translation initiation region (TIR) comprises the initiator codon, Shine-Dalgarno (SD) sequence and translational enhancers. Probably the most abundant class of enhancers contains A/U-rich sequences. We have tested the influence of SD sequence length and the presence of enhancers on the efficiency of translation initiation.

Results: We found that during bacterial growth at 37 degrees C, a six-nucleotide SD (AGGAGG) is more efficient than shorter or longer sequences. The A/U-rich enhancer contributes strongly to the efficiency of initiation, having the greatest stimulatory effect in the exponential growth phase of the bacteria. The SD sequences and the A/U-rich enhancer stimulate translation co-operatively: strong SDs are stimulated by the enhancer much more than weak SDs. The bacterial growth rate does not have a major influence on the TIR selection pattern. On the other hand, temperature affects the TIR preference pattern: shorter SD sequences are preferred at lower growth temperatures. We also performed an in silico analysis of the TIRs in all E. coli mRNAs. The base pairing potential of the SD sequences does not correlate with the codon adaptation index, which is used as an estimate of gene expression level.

Conclusion: In E. coli the SD selection preferences are influenced by the growth temperature and not influenced by the growth rate. The A/U rich enhancers stimulate translation considerably by acting co-operatively with the SD sequences.

Figure 1

Sequences used in the current study. The SD sequences and enhancers were inserted in front of the ORF coding for green fluorescent protein (GFP). Different SD variants were constructed by mutating the sequence into complementary nucleotides. The enhancers used were the "A/U-rich enhancer" (the boxA sequence of rrnB [19, 33]) and its mutant with decreased activity ("weak enhancer") [19]. All SD variants in combination with the enhancers were inserted under control of the tac promoter regulated by IPTG.

Figure 2

The effect of the TIR on GFP synthesis. GFP synthesis directed by mRNAs lacking enhancer (A). GFP synthesis directed by mRNAs containing weak enhancers (B). GFP synthesis directed by mRNAs containing A/U-rich enhancers (C). Growth curve of the cultures shown on panel C (D). The bacterial cultures were inoculated and aliquots were taken at the indicated time points. GFP expression was induced in these aliquots for one hour and the fluorescence was measured. In addition, mRNA levels were monitored by real time PCR. The expression level was calculated according to the following formula: expression level = fluorescence/(molar amount of GFP mRNA/molar amount of EF-Tu mRNA).

Figure 3

Effect of the growth temperature on TIR selection. The cells were grown either at 20°C (A) or at 37°C (C). All the TIRs shown contain strong, A/U-rich enhancers. The bacterial cultures were inoculated and aliquots were taken at the indicated time points. GFP expression was induced in these aliquots for one hour and the fluorescence was measured. Relative fluorescence was calculated by dividing the fluorescence values measured for cells containing particular constructs by the fluorescence measured for the GAGG SD sequence. In addition, growth curves at 20°C (B) and 37°C (D) are shown.

Figure 4

TIR selection in different media. The cells were grown in either LB (A, B, C, D), MOPS medium containing glucose, "MOPS Glc" (E, F, G, H), or MOPS containing sodium acetate, "MOPS NaAcetate" (I, J, K, L) at 37°C. mRNAs lacking enhancer (A, E, I), containing the weak enhancer (B, F, J) or containing the strong A/U-rich enhancer (C, G, K) were tested. The bacterial cultures were inoculated and aliquots were taken at the indicated time points. GFP expression was induced in these aliquots for one hour (LB, MOPS Glc) or 3 hours (MOPS NaAcetate) and the fluorescence was measured. Relative fluorescence was calculated by dividing the fluorescence values measured for cells containing particular constructs by the fluorescence measured for the GAGG SD sequence. In addition, growth curves in different media are shown (D, H, L).

Figure 5

Distribution of the number of paired nucleotides in SD:aSD interactions and the CAI values for 4243 E. coli genes. The figure shows the number of genes (grey bars, left axis) and the average CAI with 95% confidence intervals (black dots, right axis).