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.     

Nascent polypeptide sequences that influence ribosome function

Ribosomes catalyze protein synthesis using transfer RNAs and auxiliary proteins. Historically, ribosomes have been considered nonspecific translational machines, having no regulatory functions. However, a new class of regulatory mechanisms has been discovered that is based on interactions occurring within the ribosomal peptide exit tunnel that result in ribosome stalling during translation of an appropriate mRNA segment. These discoveries reveal an unexpectedly dynamic role ribosomes play in regulating their own activity. By using nascent leader peptides in combination with bound specific amino acids or antibiotics, ribosome functions can be altered significantly resulting in regulated expression of downstream coding regions. This review summarizes relevant findings in recent articles and outlines our current understanding of nascent peptide-induced ribosome stalling in regulating gene expression.     

ermC leader peptide. Amino acid sequence critical for induction by translational attenuation

The ermC mRNA leader segment, which encodes a 19 amino acid leader peptide, MGIFSIFVISTVHYQPNKK, plays a key role in regulating expression of the ErmC methylase. The contribution of specific leader peptide amino acid residues to induction of ermC was studied using a model system in which the ErmC methylase was translationally fused to Escherichia coli beta-galactosidase as indicator gene. Codons of the ermC leader peptide were altered systematically by replacement of leader DNA segments with double-stranded DNA constructed from chemically synthesized oligonucleotides. Missense mutations that resulted in reduced efficiency of induction involved codons for amino acid residues 5 to 9 (-SIFVI-). Nonsense mutations causing termination of the leader peptide at codons 10 (-S-) or 12 (-V-) remained inducible. These findings suggest that the codons for residues 5 to 9 of the leader peptide comprise the critical region in which ribosomes stall in the presence of erythromycin.     

The Evolutionarily Conserved Eukaryotic Arginine Attenuator Peptide Regulates the Movement of Ribosomes That Have Translated It

Translation of the upstream open reading frame (uORF) in the 5′ leader segment of the Neurospora crassa arg-2 mRNA causes reduced initiation at a downstream start codon when arginine is plentiful. Previous examination of this translational attenuation mechanism using a primer-extension inhibition (toeprint) assay in a homologous N. crassa cell-free translation system showed that arginine causes ribosomes to stall at the uORF termination codon. This stalling apparently regulates translation by preventing trailing scanning ribosomes from reaching the downstream start codon. Here we provide evidence that neither the distance between the uORF stop codon and the downstream initiation codon nor the nature of the stop codon used to terminate translation of the uORF-encoded arginine attenuator peptide (AAP) is important for regulation. Furthermore, translation of the AAP coding region regulates synthesis of the firefly luciferase polypeptide when it is fused directly at the N terminus of that polypeptide. In this case, the elongating ribosome stalls in response to Arg soon after it translates the AAP coding region. Regulation by this eukaryotic leader peptide thus appears to be exerted through a novel mechanism of cis-acting translational control.     

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.     

Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation.

Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation. For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro. The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction. Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo. MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA. In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase. The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD. MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae. Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition. Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide. The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA.     

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.     

Characterization of a plasmid-specified ribosome methylase associated with macrolide resistance.

The ermC gene of plasmid pE194 specifies resistance to the macrolidelincosamide-streptogramin B antibiotics. This resistance, as well as synthesis of the 29,000 dalton protein product of ermC, has been shown to be induced by erythromycin. Weisblum and his colleagues have established that macrolide resistance is associated with a specific dimethylation of adenine in 23 S rRNA. We show that pE194 specifies an RNA methylase that can utilize either 50 S ribosomes or 23 S rRNA as substrates. Synthesis of this methylase is induced by low concentrations of erythromycin, and the enzyme is produced in elevated amounts by strains carrying a high copy number mutant of pE194. The methylase comigrates with the 29K ermC product on polyacrylamide gels. The purification and some properties of this methylase are described.