In
Bacillus subtilis, an RNase E sequence homologue is not identifiable in the genome. Nevertheless, indirect evidence for
B. subtilis RNase E activity has been obtained (
10), and we have shown, using the erythromycin (Em) resistance gene
ermC mRNA as a model, that access to the 5" end is critical for determining mRNA half life; Em-induced stalling of a ribosome near the 5" end of
ermC mRNA results in stabilization of the message (
3,
11). The specific set of codons in the
ermC leader region coding sequence is required for Em-induced ribosome stalling and resultant
ermC mRNA stabilization (
17,
22). Other
B. subtilis mRNAs have not been found to be stabilized by addition of Em.
The
B. subtilis chromosome contains a tetracycline (Tc) resistance gene (
32), designated
tet(L), encoding a multifunctional membrane protein that is a physiologically important monovalent cation/proton antiporter that catalyzes Na
+(K
+)/H
+ antiport (
18). Tet(L) protein is also capable of Tc/H
+ exchange, and the presence of chromosomal
tet(L) confers low-level resistance to Tc. Expression of
tet(L) is inducible by Tc (
7,
26), and inducibility is a function of the 124-nucleotide (nt)
tet(L) leader region, which encodes a 20-amino-acid open reading frame (Fig.
1).
Using
tet(L)-
lacZ fusions containing various mutations in the
tet(L) leader region, we developed a model for translational regulation that involves Tc-induced reinitiation of translation (
27). Specifically, the translational signals for the Tet(L) coding sequence function poorly, so that basal-level translation is low. On the other hand, the translational signals for the
tet(L) leader peptide function extremely efficiently, so that the leader peptide is translated constitutively at a high level. In the absence of Tc, ribosomes are continually translating the leader peptide, but this has either no effect or a negative effect on translation of the Tet(L) coding sequence. When a subinhibitory concentration (0.25 μg/ml) of Tc is added, Tc-bound ribosomes stall in the leader peptide coding sequence, allowing the leader region mRNA to assume a secondary structure (Fig.
1) that facilitates transfer of ribosomes from the leader peptide coding sequence to the Tet(L) coding sequence.
In the previous work, we measured a 12-fold translational induction, which represented only part of the total >20-fold induction by Tc. The results of tet(L)-lacZ transcriptional fusions suggested that induction of tet(L) gene expression included a component related to an increase in tet(L) mRNA.
In this report, we show that addition of a subinhibitory concentration of Tc to B. subtilis results in stabilization of tet(L) mRNA. The effect of Tc addition on the stability of various tet(L) RNAs containing leader region mutations was examined to probe the mechanism of Tc-induced stability. Addition of Tc also resulted in stabilization of several other cellular mRNAs.
DISCUSSION
The results reported here demonstrate an additional effect of Tc on gene expression in a gram-positive organism. It is well documented that binding of Tc to a ribosome inhibits translation by blocking the binding of aminoacyl-tRNA to the ribosomal A site (
14,
15). We show here that another effect of Tc is to increase mRNA stability. Although we have not proven directly that the stabilizing effect of Tc is related to its binding to ribosomes, we infer this from the fact that inactivation of the RBS1 sequence eliminated the stabilizing effect of Tc.
Previous work on Em-induced stability of
ermC mRNA has shown that initiation of mRNA decay in
B. subtilis occurs primarily from the 5" end (
3,
11). If the only effect of Tc were to inhibit elongation, one could suggest that the stabilizing effect of Tc is due to slowing of a ribosome by inhibiting the first elongation step after ternary complex formation, which would position the ribosome near the 5" end and block access of a decay-initiating RNase. However, data from leader region mutations of the mini-
tet(L) mRNA demonstrated that the effect of Tc on mRNA stability was a function of the ribosome-binding site (RBS) alone; insertion of a stop codon immediately after the leader peptide initiation codon, and even inactivation of the initiation codon itself, did not eliminate the stabilizing effect of Tc, while deletion of RBS1 completely abolished this effect (Table
1).
We speculate that binding of Tc to a ribosome causes the ribosome to engage in a more stable interaction with the RBS, thus providing greater protection against initiation of decay from the 5" end. In this regard, it is interesting that an analysis of UV cross-linking of 16S rRNA nucleotides revealed three cross-links that are affected by addition of Tc (
25). Two of these are at the top of helix 44, which lies close to the 3′-proximal helix 45 that is next to the anti-RBS sequence (
24).
The results with Tc-induced stability of mini-
tet(L) mRNA in the S10 mutant strain (Fig.
3C) are consistent with the stabilizing effect of Tc being independent of the effect of Tc on translation. In vitro studies of translation with ribosomes containing the mutant S10 protein demonstrated resistance to the inhibitory effect of Tc (
31). Nevertheless, addition of Tc to the S10 mutant strain gave a similar mRNA stabilization as in the wild-type (Fig.
3C and
3D). This result is consistent with our hypothesis that Tc-induced stability is due to a Tc-induced alteration of the ribosome-RBS interaction, rather than through an effect on translation.
It was shown recently that binding of Tc to the
Thermus thermophilus 30S ribosomal subunit did not involve proteins but, rather, specific residues of 16S rRNA (
5). One of the proteins that lies nearest to the primary Tc binding site is the S10 protein, specifically the loop composed of residues 50 to 60 (
5). We used PCR to amplify the
rpsJ gene, which encodes the S10 protein, from
B. subtilis wild-type and S10 mutant strains in order to determine the actual mutation site. A comparison of the sequences showed that there was a mutation in codon 46 of the
rpsJ coding sequence gene, resulting in a Lys to Glu change. Thus, it is possible that this mutation confers resistance to the inhibitory effects on translation caused by Tc binding to nearby rRNA nucleotide residues, but does not alter the effect of Tc on mRNA stability. (It should be noted, however, that residues 45 to 56 of the
B. subtilis S10 protein constitute a region of the protein that has the least similarity to the
T. thermophilus S10 protein sequence, suggesting caution in making inferences about binding of Tc to
B. subtilis ribosomes from the data on
T. thermophilus ribosomes.)
Of particular interest was the fact that Tc-induced stability was observed even when translation of the body of the message was eliminated (RBS2 mutation on pYW107; Table
1). This reinforces the conclusion from our studies on induced
ermC mRNA stabilization, which indicated a primary role of the 5" end in determining mRNA half-life, independent of translation of the coding sequence (
3,
11). Furthermore, the insertion of a stop codon as 5"-proximal as the second codon of the leader peptide coding sequence had little effect on mini-
tet(L) mRNA stability, with or without Tc induction. Only when the leader peptide initiation codon was mutated to a nonfunctional ACG start codon was there a significant effect on uninduced mini-
tet(L) mRNA half-life (decrease from about 17 min to about 7 min).
It seems clear that the process of ribosome binding and initiation of translation, even without subsequent elongation, helps to confer wild-type stability on mRNA in B. subtilis. On the other hand, abolition of Tc induction was seen only when RBS1 was deleted, and this resulted in a further reduction of uninduced half-life to about 3 min. Thus, it appears that both RBS binding and initiation of translation contribute to basal mRNA stability, and binding of Tc enhances the effect of ribosome binding.
In earlier reports on the relationship of translation and mRNA decay in
E. coli, various antibiotics were used to either freeze ribosomes on mRNA or inhibit initiation and allow ribosomes to run off. Tc, which stabilizes polysomes, was found to stabilize mRNAs (
9,
28). However, in these studies the concentrations of antibiotics used could rapidly inhibit all protein synthesis (e.g., Tc at 100 μg/ml). As such, there is likely to be little relationship between those findings and the stabilization of
B. subtilis mRNA induced by the subinhibitory concentration of Tc used in our experiments.
Since Tc-regulated transcription is currently used for controlled gene expression in many eukaryotic systems (
1), we thought it important to demonstrate that the effect of Tc on mRNA stability that we observed in
B. subtilis did not also occur in higher organisms. In fact, Northern blot analysis of β-actin mRNA from
Saccharomyces cerevisiae cultures treated with Tc showed no difference in mRNA stability from untreated controls (unpublished results).
Presumably, binding of Tc to the bacterial ribosome is needed specifically for the stabilizing effect. We have not investigated whether this effect occurs in other gram-positive organisms. Nevertheless, it is interesting to speculate whether use of subinhibitory levels of Tc in clinical or agricultural settings might result in mRNA stabilization, thereby affecting cellular function.