Effect of translational signals on mRNA decay in Bacillus subtilis

Abstract

A 254-nucleotide model mRNA, designated deltaermC mRNA, was used to study the effects of translational signals and ribosome transit on mRNA decay in Bacillus subtilis. DeltaermC mRNA features a strong ribosome-binding site (RBS) and a 62-amino-acid-encoding open reading frame, followed by a transcription terminator structure. Inactivation of the RBS or the start codon resulted in a fourfold decrease in the mRNA half-life, demonstrating the importance of ternary complex formation for mRNA stability. Data for the decay of deltaermC mRNAs with stop codons at positions increasingly proximal to the translational start site showed that actual translation--even the formation of the first peptide bond--was not important for stability. The half-life of an untranslated 3.2-kb deltaermC-lacZ fusion RNA was similar to that of a translated deltaermC-lacZ mRNA, indicating that the translation of even a longer RNA was not required for wild-type stability. The data are consistent with a model in which ribosome binding and the formation of the ternary complex interfere with a 5'-end-dependent activity, possibly a 5'-binding endonuclease, which is required for the initiation of mRNA decay. This model is supported by the finding that increasing the distance from the 5' end to the start codon resulted in a 2.5-fold decrease in the mRNA half-life. These results underscore the importance of the 5' end to mRNA stability in B. subtilis.

FIG. 1.

Schematic diagrams and half-lives of wild-type ΔermC mRNA and derivatives with a mutated RBS or a mutated start codon. The nucleotide sequence from the transcription start site to the translational start codon is shown at the top, with the RBS and start codon underlined. Half-lives (in minutes) are reported as means and standard deviations. N/A, not applicable.

FIG. 2.

Representative Northern blot analyses of ΔermC mRNAs. Total RNA was isolated from a wild-type strain (A), a start codon mutant (B), a mutant with a stop codon inserted immediately after the start codon (C), and a mutant with a 73-nt insertion at the 5′ end (D). RNA was isolated at 0, 2, 4, and 8 min after rifampin addition (times are indicated at top of panels). Also shown are blots reprobed for 5S rRNA, for normalization of the RNA quantity.

FIG. 3.

Schematic diagrams and half-lives of ΔermC mRNAs with stop codon insertions. Half-lives (in minutes) are reported as means and standard deviations.

FIG. 4.

Northern blot analysis of ΔermC-lacZ mRNA. The migration of full-length ΔermC-lacZ mRNA (FL) and two prominent intermediates (marked by asterisks) is indicated. Similar decay intermediates from other ermC-lacZ fusions have been observed elsewhere (11). The greater prominence of the lower decay intermediate in the mutant with the stop after position 1 may be due to increased processing by a 3′ exonuclease in the absence of competing ribosome flow. Values at top of panels indicate times in minutes.

FIG. 5.

Schematic diagrams and half-lives of ΔermC mRNAs with 5′ insertions and a deletion. The distance from the 5′ end to the start codon is indicated above the arrows. Half-lives (in minutes) are reported as means and standard deviations. N/A, not applicable.

FIG. 6.

Comparison of translation initiation regions of ΔermC, gsiB, and aprE. At the top left is the 10-nt sequence at the 3′ terminus of 16S rRNA, with the RBS regions of mRNAs lined up below this sequence. Shown at the right are the free energy of the RBS-16S rRNA interaction (ΔG₀), the mRNA half-life (t_1/2) (in minutes), the start codon sequence, the distance between the RBS and the start codon, the distance between the 5′ end and the start codon, and the presence or absence of a 5′ structure.

FIG. 7.

Model for interference with binding of the decay-initiating, 5′-end-dependent RNase. The RNase is depicted as a 5′-binding pocket attached to scissors representing the endonuclease activity.