Effect of ermC leader region mutations on induced mRNA stability.

Induction of translation of the ermC gene product in Bacillus subtilis occurs upon exposure to erythromycin and is a result of ribosome stalling in the ermC leader peptide coding sequence. Another result of ribosome stalling is stabilization of ermC mRNA. The effect of leader RNA secondary structure, methylase translation, and leader peptide translation on induced ermC mRNA stability was examined by constructing various mutations in the ermC leader region. Analysis of deletion mutations showed that ribosome stalling causes induction of ermC mRNA stability in the absence of methylase translation and ermC leader RNA secondary structure. Furthermore, deletions that removed much of the leader peptide coding sequence had no effect on induced ermC mRNA stability. A leader region mutation was constructed such that ribosome stalling occurred in a position upstream of the natural stall site, resulting in induced mRNA stability without induction of translation. This mutation was used to measure the effect of mRNA stabilization on ermC gene expression.     

Induction of ermC requires translation of the leader peptide.

ermC confers resistance to macrolide-lincosamide streptogramin B antibiotics by specifying a ribosomal RNA methylase, which results in decreased ribosomal affinity for these antibiotics. ermC expression is induced by exposure to erythromycin. We have previously proposed a translational regulation model in which erythromycin causes stalling of a ribosome, which is translating a leader peptide. Stalling causes a conformation shift in the ermC mRNA which in turn unmasks the methylase ribosomal binding site. A prediction of this translational attenuation model for ermC induction was tested by replacing the second codon of the putative ermC leader peptide coding region by TAA. As expected, the introduction of this mutation resulted in an uninducible phenotype which was suppressible by two ochre suppressor mutations in Bacillus subtilis. It is concluded that translation through the leader peptide coding region, in frame with the predicted leader peptide, is required for ermC induction.     

Leader peptides of inducible chloramphenicol resistance genes from gram-positive and gram-negative bacteria bind to yeast and Archaea large subunit rRNA.

catA86 is the second gene in a constitutively transcribed, two-gene operon cloned from Bacillus pumilus . The region that intervenes between the upstream gene, termed the leader, and the catA86 coding sequence contains a pair of inverted repeat sequences which cause sequestration of the catA86 ribosome binding site in mRNA secondary structure. As a consequence, the catA86 coding sequence is untranslatable in the absence of inducer. Translation of the catA86 coding sequence is induced by chloramphenicol in Gram-positives and induction requires a function of the leader coding sequence. The leader-encoded peptide has been proposed to instruct its translating ribosome to pause at leader codon 6, enabling chloramphenicol to stall the ribosome at that site. Ribosome stalling causes destabilization of the RNA secondary structure, exposing the catA86 ribosome binding site, allowing activation of its translation. A comparable mechanism of induction by chloramphenicol has been proposed for the regulated cmlA gene from Gram-negative bacteria. The catA86 and cmlA leader-encoded peptides are in vitro inhibitors of peptidyl transferase, which is thought to be the basis for selection of the site of ribosome stalling. Both leader-encoded peptides have been shown to alter the secondary structure of Escherichia coli 23S rRNA in vitro. All peptide-induced changes in rRNA conformation are within domains IV and V, which contains the peptidyl transferase center. Here we demonstrate that the leader peptides alter the conformation of domains IV and V of large subunit rRNA from yeast and a representative of the Archaea. The rRNA target for binding the leader peptides is therefore conserved across kingdoms.     

Translation attenuation regulation of chloramphenicol resistance in bacteria — a review

The chloramphenicol (Cm)-inducible cat and cmlA genes are regulated by translation attenuation, a regulatory device that modulates mRNA translation. In this form of gene regulation, translation of the Cm^R coding sequence is prevented by mRNA secondary structure that sequesters its ribosome-binding site (RBS). A translated leader of nine codons precedes the secondary structure, and induction results when a ribosome becomes stalled at a specific site in the leader. Here we demonstrate that the site of ribosome stalling in the leader is selected by a cis effect of the nascent leader peptide on its translating ribosome.     

Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation

Neurospora crassa arg-2 mRNA contains an evolutionarily conserved upstream open reading frame (uORF) encoding the Arg attenuator peptide (AAP) that confers negative translational regulation in response to Arg. We examined the regulatory role of the AAP and the RNA encoding it using an N. crassa cell-free translation system. AAPs encoded by uORFs in four fungal mRNAs each conferred negative regulation in response to Arg by causing ribosome stalling at the uORF termination codon. Deleting the AAP non-conserved N terminus did not impair regulation, but deletions extending into the conserved region eliminated it. Introducing many silent mutations into a functional AAP coding region did not eliminate regulation, but a single additional nucleotide change altering the conserved AAP sequence abolished regulation. Therefore, the conserved peptide sequence, but not the mRNA sequence, appeared responsible for regulation. AAP extension at its C terminus resulted in Arg-mediated ribosomal stalling during translational elongation within the extended region and during termination. Comparison of Arg-mediated stalling at a rare or common codon revealed more stalling at the rare codon. These data indicate that the highly evolutionarily conserved peptide core functions within the ribosome to cause stalling; translational events at a potential stall site can influence the extent of stalling there.     

Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence.

Genes encoding chloramphenicol acetyltransferase in gram-positive bacteria are induced by chloramphenicol. Induction reflects an ability of the drug to stall a ribosome at a specific site in cat leader mRNA. Ribosome stalling at this site alters downstream RNA secondary structure, thereby unmasking the ribosome-binding site for the cat coding sequence. Here, we show that ribosome stalling in the cat-86 leader is a function of leader codons 2 through 5 and that stalling requires these codons to be presented in the correct reading frame. Codons 2 through 5 specify Val-Lys-Thr-Asp. Insertion of a second copy of the stall sequence 5' to the authentic stall sequence diminished cat-86 induction fivefold. Thus, the stall sequence can function in ribosome stalling when the stall sequence is displaced from the downstream RNA secondary structure. We suggest that the stall sequence may function in cat induction at two levels. First, the tetrapeptide specified by the stall sequence likely plays an active role in the induction strategy, on the basis of previously reported genetic suppression studies (W. W. Mulbry, N. P. Ambulos, Jr., and P.S. Lovett, J. Bacteriol. 171:5322-5324, 1989). Second, we show that embedded within the stall sequence of cat leaders is a region which is complementary to a sequence internal in 16S rRNA of Bacillus subtilis. This complementarity may guide a ribosome to the proper position on leader mRNA or potentiate the stalling event, or both. The region of complementarity is absent from Escherichia coli 16S rRNA, and cat genes induce poorly, or not at all, in E. coli.     

Regulation of the inducible chloramphenicol acetyltransferase gene of the Staphylococcus aureus plasmid pUB112.

Analyses of deletion mutants of the gene for chloramphenicol (Cm) acetyltransferase (CAT) carried by the staphylococcal plasmid pUB112 revealed a regulatory region, which is indispensable for Cm-inducible cat gene expression, located 70 bp in front of the CAT-coding sequence. This region consists of a possible ribosome binding site followed by an open reading frame coding for a peptide of nine amino acids and overlaps partially with an inverted repeat capable of forming a stem-loop structure. Deletion of the ribosome binding site and of parts of the open reading frame abolishes inducibility and results in a low-level cat gene expression, if the inverted repeat remains intact. Deletion of the 5' part of the possible stem leads to high-level constitutive CAT synthesis. The inverted repeat, therefore, exhibits negative control on cat gene expression whereas the preceding ribosome binding site is needed to enhance CAT synthesis in the presence of an inducer. These results suggest that translation of a leader peptide is a prerequisite for Cm-induced cat gene expression and that ribosome stalling on cat leader mRNA caused by Cm opens the stem-loop structure thereby releasing its negative effect on CAT synthesis.     

Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene.

Expression of the plasmid gene cat-86 is induced in Bacillus subtilis by two antibiotics, chloramphenicol and the nucleoside antibiotic amicetin. We proposed that induction by either drug causes the destabilization of a stem-loop structure in cat-86 mRNA that sequesters the ribosome-binding site for the cat coding sequence. The destabilization event frees the ribosome-binding site, permitting the initiation of translation of cat-86 mRNA. cat-86 induction is due to the stalling of a ribosome in a leader region of cat-86 mRNA, which is located 5' to the RNA stem-loop structure. A stalled ribosome that is active in cat-86 induction has its aminoacyl site occupied by leader codon 6. To test the hypothesis that a leader site 5' to codon 6 permits a ribosome to stall in the presence of an inducing antibiotic, we inserted an extra codon between leader codons 5 and 6. This insertion blocked induction, which was then restored by the deletion of leader codon 6. Thus, induction seems to require the maintenance of a precise spatial relationship between an upstream leader site(s) and leader codon 6. Mutations in the ribosome-binding site for the cat-86 leader, RBS-2, which decreased its strength of binding to 16S rRNA, prevented induction. In contrast, mutations that significantly altered the sequence of RBS-2 but increased its strength of binding to 16S rRNA did not block induction by either chloramphenicol or amicetin. We therefore suspected that the proposed leader site that permitted drug-mediated stalling was located between RBS-2 and leader codon 6. This region of the cat-86 leader contains an eight-nucleotide sequence (conserved region I) that is largely conserved among all known cat leaders. The codon immediately 5' to conserved region I differs, however, between amicetin-inducible and amicetin-noninducible cat genes. In amicetin-inducible cat genes such as cat-86, the codon 5' to conserved region I is a valine codon, GTG. The same codon in amicetin-noninducible cat genes is a lysine codon, either AAA or AAG. When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation. The potential role of the GTG codon in amicetin induction is discussed.     

Genetic Identification of Nascent Peptides That Induce Ribosome Stalling

Several nascent peptides stall ribosomes during their own translation in both prokaryotes and eukaryotes. Leader peptides that induce stalling can regulate downstream gene expression. Interestingly, stalling peptides show little sequence similarity and interact with the ribosome through distinct mechanisms. To explore the scope of regulation by stalling peptides and to better understand the mechanism of stalling, we identified and characterized new examples from random libraries. We created a genetic selection that ties the life of Escherichia coli cells to stalling at a specific site. This selection relies on the natural bacterial system that rescues arrested ribosomes. We altered transfer-messenger RNA, a key component of this rescue system, to direct the completion of a necessary protein if and only if stalling occurs. We identified three classes of stalling peptides: C-terminal Pro residues, SecM-like peptides, and the novel stalling sequence FXXYXIWPP. Like the leader peptides SecM and TnaC, the FXXYXIWPP peptide induces stalling efficiently by inhibiting peptidyl transfer. The nascent peptide exit tunnel and peptidyltransferase center are implicated in this stalling event, although mutations in the ribosome affect stalling on SecM and FXXYXIWPP differently. We conclude that ribosome stalling can be caused by numerous sequences and is more common than previously believed.     

Molecular mechanism of drug-dependent ribosome stalling

Inducible expression of the erm erythromycin resistance genes relies on drug-dependent ribosome stalling. The molecular mechanisms underlying stalling are unknown. We used a cell-free translation system to elucidate the contribution of the nascent peptide, the drug, and the ribosome toward formation of the stalled complex during translation of the ermC leader cistron. Toe-printing mapping, selective amino acid labeling, and mutational analyses revealed the peptidyl transferase center (PTC) as the focal point of the stalling mechanism. In the ribosome exit tunnel, the C-terminal sequence of the nascent peptide, critical for stalling, is in the immediate vicinity of the universally conserved A2062 of 23S rRNA. Mutations of this nucleotide eliminate stalling. Because A2062 is located in the tunnel, it may trigger a conformational change in the PTC, responding to the presence of a specific nascent peptide. The cladinose-containing macrolide antibiotic in the tunnel positions the nascent peptide for interaction with the tunnel sensory elements.     

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.