AU-rich sequences within 5' untranslated leaders enhance translation and stabilize mRNA in Escherichia coli

Abstract

We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.

FIG. 1.

Structures of the 5′ regions of chromosomally encoded lacZ mRNAs bearing extended SD sequences. (A) SD10H, construct carrying the start AUG codon (boldface) partly involved in a stable stem-loop structure. The 10-nt SD sequence is underlined. (B) SD10 and SD6 constructs, comprising a single-stranded AUG codon (boldface). The position of the BamHI site used to insert the AU-rich S1 targets is shown in boldface italic. (C) Inserts upstream of the SD sequence which serve as S1 targets (18): BoxA, rrnB transcriptional antiterminator BoxA (boldface) with natural flanking regions; CBoxA, an insert complementary to BoxA; MBoxA, a mutated variant of BoxA with a lower affinity to S1 (mutated nucleotides are circled).

FIG. 2.

Steady-state level and translation efficiency of lacZ mRNA depend on the structure of its translation initiation region but not simply on the length of the SD sequence. Translation initiation regions in front of lacZ are SD10H, SD10, BoxA-SD10, CBoxA-SD10, MBoxA-SD10, SD6, BoxA-SD6, and CBoxA-SD6 (Fig. 1). (A) Northern blot analysis of lacZ transcripts. Total RNA (10 μg) was analyzed on Northern blots. The full-length lacZ mRNA and two low-mass 5′-terminal RNA fragments (indicated by arrows) were revealed with an internal probe and a 5′-probe, respectively (see Materials and Methods). The bottom panel shows hybridization of the same membrane with the 23S rRNA-specific probe used as an internal control. (B) Relative abundances of the lacZ mRNAs (bars) directly correlates with their translation efficiency (graph). Relative abundance was evaluated as a ratio of a hybridization signal from the full-length lacZ mRNA to that from 23S rRNA. β-Galactosidase activities in strains (graph) are expressed in nanomoles of ONPG hydrolyzed per minute per milligram of total soluble cell protein. Averages from three independent assays are shown, with standard deviations not exceeding 15% of magnitude (18).

FIG. 3.

Northern blot analysis of the lacZ transcripts in strains bearing the rne-1(Ts) allele. Cultures were grown at 30°C to early log phase (A₆₀₀ of 0.3) and then shifted for 30 min to 42°C to inactivate RNase E before RNA isolation. Control cultures (only that for MBoxA-SD10 is shown) were grown at 30°C without a temperature shift. (A) A representative Northern blot for total RNA (about 10 μg) extracted from rne1 strains (indicated above the lanes). RNA was analyzed as described for Fig. 2. (B) The relative abundances of lacZ transcripts in rne1 strains are higher than that in the rne⁺ background (see Fig. 2B). Averages from two independent Northern blots are shown, with deviations not exceeding 20% of magnitudes.

FIG. 4.

Stabilization of lacZ mRNA by efficient translation. (A) Northern blot analysis of total RNA (10 μg) extracted from CBoxASD6, SD6, and SD6UUG strains at various times (above each lane) after addition of rifampin (400 μg per ml). RNA was analyzed on Northern blots as described for Fig. 2. (B) Decay of lacZ mRNAs bearing the same 6-nt SD sequence depends on their translation efficiency. A plot of the lacZ mRNA chemical stability versus time is shown. The β-galactosidase level in exponentially growing cells (in nanomoles of ONPG hydrolyzed per minute per milligram of total protein) is indicated below the graph. The relative chemical stability of lacZ mRNA at each time point after the rifampin block was evaluated as a ratio of the hybridization signal from the full-length lacZ mRNA to that from 23S rRNA. A logarithmic scale was used because of the large range covered by the data. Assuming exponential decay kinetics, the lacZ mRNA chemical half-lives were estimated to be 6.9, 3.18, and 2 min for CBoxASD6, SD6, and SD6UUG, respectively.