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1.
J Bacteriol ; 180(16): 4166-70, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9696765

ABSTRACT

An unexpectedly high proportion of TGA nonsense mutations was obtained in a collection of chemically induced mutations in the spoIIR locus of Bacillus subtilis. Of 11 different mutations obtained, TGA mutations were found in four codons, whereas only three codons yielded missense mutations. Six suppressors of the TGA mutations were isolated, and five of the suppressing mutations were mapped to the prfB gene encoding protein release factor 2. These are the first mutations shown to map to the B. subtilis prfB locus. The sequence of the prfB gene was completed, and two revisions of the published sequence were made. The five prfB mutations also resulted in suppression of the catA86-TGA mutation to between 19 and 54% of the expression of catA86(+), compared to the readthrough level of 6% in the prfB+ strain. N-terminal sequencing of suppressed catA86-TGA-specified protein demonstrated that the amino acid inserted at UGA because of the prfB1 mutations was tryptophan.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Codon, Terminator/genetics , Sigma Factor , Suppression, Genetic , Transcription Factors , Amino Acid Sequence , Base Sequence , Codon, Nonsense/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial , Molecular Sequence Data , Sequence Homology, Amino Acid
2.
Nucleic Acids Res ; 25(9): 1720-6, 1997 May 01.
Article in English | MEDLINE | ID: mdl-9108153

ABSTRACT

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.


Subject(s)
Chloramphenicol Resistance/genetics , Gram-Negative Bacteria/genetics , Gram-Positive Bacteria/genetics , Protein Sorting Signals/metabolism , RNA, Ribosomal/metabolism , Base Sequence , DNA Footprinting , Halobacterium/genetics , Molecular Sequence Data , Nucleic Acid Conformation , Protein Binding , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , RNA, Fungal/chemistry , RNA, Fungal/metabolism , RNA, Ribosomal/chemistry , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/genetics
3.
Gene ; 179(1): 157-62, 1996 Nov 07.
Article in English | MEDLINE | ID: mdl-8955642

ABSTRACT

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 CmR 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.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol Resistance/genetics , Gene Expression Regulation, Bacterial , Protein Biosynthesis , Chloramphenicol O-Acetyltransferase/biosynthesis , Gram-Negative Bacteria/genetics , Gram-Positive Bacteria/genetics , Models, Genetic , Peptidyl Transferases/antagonists & inhibitors , RNA, Ribosomal, 23S/metabolism
4.
Microbiol Rev ; 60(2): 366-85, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8801438

ABSTRACT

Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene. The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome. The peptides are therefore cis-acting negative regulators of translation. The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome. A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria. Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA. Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions. Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation. Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection. Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA. uORFs associated with several eukaryotic genes inhibit downstream translation. When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon. This sets up a blockade to ribosome scanning which minimizes downstream translation. Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the known examples the active amino acid sequences function after their emergence from the ribosome, cis control of translation by the nascent peptide is gene specific; nearly all such regulatory peptides exert no obvious trans effects in cells. The in vitro biochemical activities of the cat/cmla leader peptides on ribosomes and rRNA suggest a mechanism through which the nascent peptide can modify ribosome behavior. Other cis-acting regulatory peptides may involve more complex ribosomal interactions.


Subject(s)
Gene Expression Regulation/physiology , Peptides/physiology , Ribosomes/metabolism , Amino Acid Sequence , Base Sequence , Molecular Sequence Data , Protein Biosynthesis/genetics
5.
Proc Natl Acad Sci U S A ; 92(19): 8650-4, 1995 Sep 12.
Article in English | MEDLINE | ID: mdl-7567991

ABSTRACT

Peptides of 5 and 8 residues encoded by the leaders of attenuation regulated chloramphenicol-resistance genes inhibit the peptidyltransferase of microorganisms from the three kingdoms. Therefore, the ribosomal target for the peptides is likely to be a conserved structure and/or sequence. The inhibitor peptides "footprint" to nucleotides of domain V in large subunit rRNA when peptide-ribosome complexes are probed with dimethyl sulfate. Accordingly, rRNA was examined as a candidate for the site of peptide binding. Inhibitor peptides MVKTD and MSTSKNAD were mixed with rRNA phenol-extracted from Escherichia coli ribosomes. The conformation of the RNA was then probed by limited digestion with nucleases that cleave at single-stranded (T1 endonuclease) and double-stranded (V1 endonuclease) sites. Both peptides selectively altered the susceptibility of domains IV and V of 23S rRNA to digestion by T1 endonuclease. Peptide effects on cleavage by V1 nuclease were observed only in domain V. The T1 nuclease susceptibility of domain V of in vitro-transcribed 23S rRNA was also altered by the peptides, demonstrating that peptide binding to the rRNA is independent of ribosomal protein. We propose the peptides MVKTD and MSTSKNAD perturb peptidyltransferase center catalytic activities by altering the conformation of domains IV and V of 23S rRNA. These findings provide a general mechanism through which nascent peptides may cis-regulate the catalytic activities of translating ribosomes.


Subject(s)
Nucleic Acid Conformation , Oligopeptides/pharmacology , Peptidyl Transferases/antagonists & inhibitors , Protein Biosynthesis , RNA, Ribosomal, 23S/drug effects , Amino Acid Sequence , Base Sequence , Endonucleases/metabolism , Models, Genetic , Models, Molecular , Molecular Sequence Data , Oligopeptides/metabolism , Peptide Chain Initiation, Translational , Protein Binding , RNA, Ribosomal, 23S/metabolism
6.
J Bacteriol ; 177(12): 3616-8, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7768878

ABSTRACT

Expression of the chloramphenicol resistance gene cat-86 is regulated by translation attenuation. Among the three ribosomally targeted antibiotics that can induce the gene, only amicetin has an unknown mode of action. Here we demonstrate that the nucleoside antibiotic amicetin is an inhibitor of bacterial peptidyl transferase. Thus, the three inducers of cat-86, chloramphenicol, erythromycin, and amicetin, interact with the peptidyl transferase region of bacterial ribosomes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Chloramphenicol Resistance/genetics , Gene Expression Regulation, Bacterial/drug effects , Peptidyl Transferases/antagonists & inhibitors , Bacillus subtilis/drug effects , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Mutation , Pyrimidine Nucleosides/pharmacology , Ribosomes/drug effects
7.
J Biol Chem ; 270(22): 12957-60, 1995 Jun 02.
Article in English | MEDLINE | ID: mdl-7768882

ABSTRACT

The conformational mobility of glyoxalase I (Glx I) during catalysis has been probed using stable analogs of the enediol intermediate that forms along the reaction pathway: GSC(O)N(OH)R, where GS = glutathionyl and R = CH3 (1), C6H5 (2), C6H4Cl (3), or C6H4Br (4). For human erythrocyte Glx I, catalysis is unlikely to be coupled to major changes in protein secondary structure, as the circular dichroism spectrum of the enzyme (190-260 nm) is insensitive to saturating concentrations of either enediol analog or S-D-lactoylglutathione, the product of the Glx I reaction. However, a small conformational change is indicated by the fact that binding of enediol analog to the active site decreases intrinsic protein fluorescence by 11%, and protects the enzyme from proteolytic cleavage by Pronase E at the C-side of Ala-92 and Leu-93. In contrast, binding of S-D-lactoylglutathione does not affect protein fluorescence, and increases the rate of proteolytic cleavage by 1.5-fold. These observations are consistent with a model of catalysis in which a flexible peptide loop folds over and stabilizes the enediol intermediate bound to the active site. Indeed, a highly conserved sequence of amino acid residues is found near the proteolytic cleavage sites, for human Glx I (100-111) and Pseudomonas putida Glx I (93-105), that shows significant sequence homology to the "catalytic loop" of chicken muscle triosephosphate isomerase (TIM) (165-176). The active site base (Glu-165) of TIM, which catalyzes the proton transfer reaction during isomerization, corresponds in position to Glu-93 of P. putida Glx I. Consistent with a functional role for Glu-93, a mutant enzyme in which Glu-93 is replaced by Asp shows no detectable catalytic activity.


Subject(s)
Lactoylglutathione Lyase/metabolism , Triose-Phosphate Isomerase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Chickens , Circular Dichroism , Humans , Hydrolysis , Kinetics , Lactoylglutathione Lyase/chemistry , Lactoylglutathione Lyase/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Triose-Phosphate Isomerase/chemistry , Triose-Phosphate Isomerase/genetics
8.
Gene ; 150(1): 93-6, 1994 Dec 02.
Article in English | MEDLINE | ID: mdl-7959071

ABSTRACT

The gene encoding glyoxalase I (GlxI) from Pseudomonas putida has been cloned into the high-expression plasmid pBTacI. In the presence of IPTG, JM109 cells transformed with this vector give expression levels of GlxI 4000-fold higher than wild-type Escherichia coli. Contrary to a previous report, the nucleotide sequence of the gene encodes a 173-amino-acid polypeptide. Edman analysis indicates that the predicted N-terminal methionine is lost post-translationally to yield a 19407-Da protein. Mass spectrometry of the intact protein, and of the peptides generated from treatment with CNBr, does not indicate any additional post-translational modifications of the enzyme. Contrary to previous conclusions, there are no major regions of dissimilarity between the human and bacterial enzymes.


Subject(s)
Genes, Bacterial , Lactoylglutathione Lyase/genetics , Pseudomonas putida/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Escherichia coli , Humans , Mass Spectrometry , Molecular Sequence Data , Plasmids , Pseudomonas putida/enzymology , Sequence Homology, Amino Acid
9.
J Bacteriol ; 176(22): 7115-7, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7961481

ABSTRACT

Placing a translation stop codon at the ribosomal pause site in the leader of the attenuation-regulated cat-86 gene activates cat expression in the absence of the inducer, chloramphenicol. Genetic experiments have shown that this phenomenon depends on the amino acid sequence of the leader-encoded peptide and could readily be explained if the peptide was an inhibitor of translation termination. Here we demonstrate that the cat-86 leader pentapeptide is an in vitro inhibitor of translation termination in addition to its previously described antipeptidyltransferase activity.


Subject(s)
Chloramphenicol Resistance/genetics , Peptide Chain Termination, Translational/drug effects , Protein Sorting Signals/pharmacology , Amino Acid Sequence , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Genes, Bacterial/genetics , Models, Genetic , Molecular Sequence Data , RNA, Transfer, Met/metabolism
11.
J Bacteriol ; 176(20): 6238-44, 1994 Oct.
Article in English | MEDLINE | ID: mdl-7928994

ABSTRACT

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.


Subject(s)
Bacterial Proteins , Chloramphenicol O-Acetyltransferase/biosynthesis , Gene Expression Regulation, Bacterial , Oligopeptides/pharmacology , Peptidyl Transferases/antagonists & inhibitors , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Amino Acid Sequence , Bacteria/genetics , Base Sequence , Chloramphenicol/pharmacology , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol Resistance/genetics , Conserved Sequence , Enzyme Induction , Erythromycin/metabolism , Eukaryotic Cells/physiology , Methyltransferases/metabolism , Molecular Sequence Data , Prokaryotic Cells/physiology , RNA, Ribosomal, 23S/genetics , Ribosomes/metabolism , Saccharomyces cerevisiae/genetics , Species Specificity
12.
Proc Natl Acad Sci U S A ; 91(12): 5612-6, 1994 Jun 07.
Article in English | MEDLINE | ID: mdl-7515506

ABSTRACT

The chloramphenicol (Cm)-inducible cmlA gene of Tn1696 specifies nonenzymatic resistance to Cm and is regulated by attenuation. The first eight codons of the leader specify a peptide that inhibits peptidyl transferase in vitro. Functionally similar, but less inhibitory, peptides are encoded by the leaders of Cm-inducible cat genes. However, the cat and cmlA coding sequences are unrelated and specify proteins of unrelated function. The inhibition of peptidyl transferase by the leader peptides is additive with that of Cm. Erythromycin competes with the inhibitory action of the peptides, and erythromycin and the peptides footprint to overlapping sites at the peptidyl transferase center of 23S rRNA. It is proposed that translation of the cmlA and cat leaders transiently pauses upon synthesis of the inhibitor peptides. The predicted site of pausing is identical to the leader site where long-term occupancy by a ribosome (ribosome stalling) will activate downstream gene expression. We therefore propose the inducer, Cm, converts a peptide-paused ribosome to the stalled state. We discuss the idea that cooperativity between leader peptide and inducer is necessary for ribosome stalling and may link the activation of a specific drug-resistance gene with a particular antibiotic.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol Resistance , Gene Expression Regulation, Bacterial , Peptide Chain Elongation, Translational/drug effects , Peptidyl Transferases/antagonists & inhibitors , Ribosomes/metabolism , Amino Acid Sequence , Bacillus subtilis , Base Sequence , Emetine/pharmacology , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Bacterial/genetics , RNA, Messenger/genetics , RNA, Ribosomal, 23S/metabolism , Thermus
13.
Mol Microbiol ; 12(2): 181-6, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8057843

ABSTRACT

Inducible cat genes from Gram-positive bacteria are regulated by translation attenuation. The inducer chloramphenicol stalls a ribosome at a specific site in the leader of cat transcripts; this destabilizes a downstream stem-loop structure that normally sequesters the ribosome-binding site for the cat structural gene. The five-amino-acid peptide MVKTD that is synthesized when a ribosome has translated to the leader induction site is an inhibitor of peptidyl transferase in vitro. Thus, the peptide may be the in vivo determinant of the site of ribosome stalling. Here we provide evidence that the leader pentapeptide can exert a cis-effect on its translating ribosome in vivo. Converting leader codon 6 to the ochre codon results in expression of cat-86 in the absence of inducer. We term this autoinduction. Autoinduction is abolished by mutations that change the amino-acid sequence of the leader peptide but have no, or little, effect on the sequence of nucleotides at the leader stall site. In contrast, four nucleotide changes within the leader site occupied by the stalled ribosome that result in synonymous codon replacements do not diminish autoinduction. Our evidence indicates that the cat-86 leader pentapeptide can alter the function of its translating ribosome.


Subject(s)
Codon , Peptide Chain Elongation, Translational , Peptide Chain Termination, Translational , Protein Sorting Signals/genetics , Ribosomes/metabolism , Amino Acid Sequence , Bacillus subtilis/genetics , Base Sequence , Chloramphenicol/pharmacology , Frameshift Mutation , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Peptide Chain Elongation, Translational/drug effects , Point Mutation
14.
Gene ; 140(1): 79-83, 1994 Mar 11.
Article in English | MEDLINE | ID: mdl-8125344

ABSTRACT

Induction of chloramphenicol (Cm) acetyltransferase-encoding genes (cat) by Cm has been proposed to result from the destabilization of a stem-loop that sequesters the ribosome-binding site for the cat coding sequence. Destabilization is caused by the stalling of a ribosome at a specific site in the leader of cat transcripts that immediately precedes the stem-loop. By use of in vivo dimethylsulfate probing of cat-86 leader mRNA, we demonstrate the existence of the stem-loop structure in cat transcripts isolated from uninduced cells and its release during induction. Leader mutations chosen to provide the mRNA with an alternative folding pattern that destabilizes the stem-loop cause constitutive cat expression. Our results establish the occurrence in vivo of the stem-loop in cat-86 transcripts and its role as a negative regulator of cat expression.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation , Mutagenesis , Nucleic Acid Conformation , RNA, Messenger/chemistry , Base Sequence , Chloramphenicol/pharmacology , Gene Expression Regulation/drug effects , Molecular Sequence Data , Protein Biosynthesis , RNA, Messenger/genetics , Terminator Regions, Genetic
15.
J Bacteriol ; 175(17): 5309-13, 1993 Sep.
Article in English | MEDLINE | ID: mdl-7690023

ABSTRACT

The site of ribosome stalling in the leader of cat transcripts is critical to induction of downstream translation. Site-specific stalling requires translation of the first five leader codons and the presence of chloramphenicol, a sequence-independent inhibitor of ribosome elongation. We demonstrate in this report that a synthetic peptide (the 5-mer) corresponding to the N-terminal five codons of the cat-86 leader inhibits peptidyl transferase in vitro. The N-terminal 2-, 3-, and 4-mers and the reverse 5-mer (reverse amino acid sequence of the 5-mer) are virtually without effect on peptidyl transferase. A missense mutation in the cat-86 leader that abolishes induction in vivo corresponds to an amino acid replacement in the 5-mer that completely relieves peptidyl transferase inhibition. In contrast, a missense mutation that does not interfere with in vivo induction corresponds to an amino acid replacement in the 5-mer that does not significantly alter peptidyl transferase inhibition. Our results suggest that peptidyl transferase inhibition by the nascent cat-86 5-mer peptide may be the primary determinant of the site of ribosome stalling in the leader. A model based on this concept can explain the site specificity of ribosome stalling as well as the response of induction to very low levels of the antibiotic inducer.


Subject(s)
Chloramphenicol O-Acetyltransferase/metabolism , Peptidyl Transferases/antagonists & inhibitors , Protein Sorting Signals/metabolism , Amino Acid Sequence , Bacillus subtilis/enzymology , Bacillus subtilis/genetics , Base Sequence , Chloramphenicol/pharmacology , Chloramphenicol O-Acetyltransferase/genetics , Drug Resistance, Microbial/genetics , Enzyme Induction , Erythromycin/pharmacology , Kinetics , Lincomycin/pharmacology , Molecular Sequence Data , Peptidyl Transferases/drug effects , Protein Sorting Signals/genetics , RNA, Bacterial , Ribosomes/metabolism
16.
Mol Microbiol ; 8(6): 1063-9, 1993 Jun.
Article in English | MEDLINE | ID: mdl-7689687

ABSTRACT

Induction of cat-86 translation results from the stalling of a ribosome at a discrete location in the leader region of the transcript. Stalling destabilizes an adjacent region of secondary structure that sequesters the cat-86 ribosome binding site, thereby activating cat-86 translation. Two well characterized antibiotics, chloramphenicol and erythromycin, induce cat-86 by stalling a ribosome at the appropriate leader site. Here we demonstrate differences between the two antibiotics with respect to induction. First, induction by chloramphenicol is dependent on nucleotides in the leader sequence that are different from those necessary for erythromycin induction. Second, variants of Bacillus subtilis that are chloramphenicol resistant because of chromosome mutations permit cat-86 induction by chloramphenicol, whereas erythromycin-resistance host mutations block or greatly reduce cat-86 induction by erythromycin. Third, selected strains of B. subtilis bearing alterations in proteins of the 50S ribosomal subunit interfere with cat-86 induction by chloramphenicol, yet these strains are chloramphenicol sensitive. Lastly, induction by chloramphenicol is not reversed by removal of the antibiotic whereas erythromycin induction is reversible. The data indicate that chloramphenicol induction results from an effect of the drug that is not identical to its role as a general inhibitor of ribosome elongation. Induction by erythromycin, on the other hand, could not be distinguished from its antibiotic activity.


Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/biosynthesis , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol/pharmacology , Gene Expression Regulation, Bacterial/drug effects , Protein Biosynthesis/drug effects , Bacillus subtilis/drug effects , Bacterial Proteins/genetics , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol Resistance/genetics , Enzyme Induction/drug effects , Erythromycin/pharmacology , Nucleic Acid Conformation , Pyrimidine Nucleosides/pharmacology , RNA, Bacterial/genetics , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Ribosomal Proteins/genetics , Ribosomes/drug effects
17.
Mol Microbiol ; 6(19): 2769-76, 1992 Oct.
Article in English | MEDLINE | ID: mdl-1279359

ABSTRACT

Chloramphenicol activates translation of cat-86 mRNA by stalling a ribosome in the leader of individual transcripts. Stalling triggers two sequential events: the destabilization of a region of secondary structure that sequesters the cat ribosome-binding site (RBS-C), and the initiation of cat translation. The site of drug-dependent ribosome stalling is dictated by the leader sequence, crb; crb causes a ribosome to stall with its aminoacyl site at leader codon 6. We demonstrate that induction requires the maintenance of a precise spatial relationship between crb and sequences within the left inverted repeat of the secondary structure. Therefore, destabilization of the secondary structure during chloramphenicol induction may result from the interaction of a stalled ribosome with a specific sequence in the secondary structure rather than from non-specific masking of RNA sequences. cat-86 regulation also depends on the distance that separates crb from RBS-C. This interval of 33 nucleotides was incrementally increased and decreased by mutations within a loop in the secondary structure. Shortening the distance between crb and RBS-C by three nucleotides reduced induction by half and a deletion of nine nucleotides abolished induction. Insertion mutations were without effect on induced expression but elevated basal expression. The results indicate that when the A site of a ribosome occupies leader codon 6 the secondary structure is destabilized and there is no interference with entry of a second ribosome at RBS-C. The data further demonstrate that when the A site of a ribosome in the leader is within 30 nucleotides of RBS-C, cat expression decreases. This decrease probably results from competition of the leader ribosome with the ribosome initiating cat translation. Our observations demonstrate that in wild-type cat-86 the distances between crb and the secondary structure, and between crb and RBS-C provide the precise spacing necessary to achieve three interdependent effects: the destabilization of the RNA secondary structure by a ribosome stalled at crb; a lack of competition between a ribosome stalled at crb and the initiating ribosome; and maintenance of a low, but measurable, basal level of cat expression. The spatial relationships identified as necessary for the regulation of cat-86 are conserved in the regulatory regions for five other inducible cat genes.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Protein Biosynthesis , RNA, Messenger/genetics , Regulatory Sequences, Nucleic Acid , Ribosomes/metabolism , Base Sequence , Chloramphenicol O-Acetyltransferase/biosynthesis , Codon , Enzyme Induction , Gene Deletion , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Messenger/chemistry , Repetitive Sequences, Nucleic Acid
18.
J Bacteriol ; 173(24): 7881-6, 1991 Dec.
Article in English | MEDLINE | ID: mdl-1720771

ABSTRACT

The induction of cat-86 by chloramphenicol has been proposed to follow the translational attenuation model. In the absence of inducer, the cat-86 gene is transcribed but remains phenotypically unexpressed because the transcripts sequester the ribosome binding site for the cat coding sequence in a stable stem-loop structure, preventing translation initiation. The translational attenuation model proposes that the natural inducer, chloramphenicol, stalls a ribosome in the leader region of cat transcripts, which causes localized melting of the downstream stem-loop structure, allowing initiation of translation of the cat-86 coding sequence. Although it is established that ribosome stalling in the cat-86 leader can induce translation of the coding sequence, several subsequent steps predicted by the model remain to be experimentally confirmed. As a consequence, the present evidence for cat-86 regulation can also be explained by two other potential control devices, ribosome hopping and translational frameshifting. Here we describe experiments designed to determine whether the alternatives to translational attenuation regulate cat-86. The results obtained are inconsistent with both competing models and are consistent with predictions made by the translational attenuation model.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Enzymologic , Protein Biosynthesis , Ribosomes/metabolism , Bacillus subtilis/genetics , Base Sequence , Blotting, Western , Molecular Sequence Data , Nucleic Acid Conformation , Protein Sorting Signals/genetics , Protein Sorting Signals/metabolism , RNA, Bacterial/metabolism
20.
Gene ; 105(1): 113-7, 1991 Aug 30.
Article in English | MEDLINE | ID: mdl-1937000

ABSTRACT

The translational attenuation regulatory model suggests a mechanism that can explain the induction of cat-86 by chloramphenicol (Cm). In this model, Cm serves to stall a ribosome at a specific site in a leader region of cat-86 transcripts. The stalled ribosome is thought to destabilize a downstream region of RNA secondary structure that normally sequesters the cat-86 ribosome-binding site (RBS-3). Three mutations in codon 4 of the cat-86 leader have been identified which result in constitutive cat expression. Each of the three mutations generates a likely -10 promoter sequence in the leader. Twenty nucleotides (nt) upstream is the wild-type sequence, 5'-TTGAAA, which differs from the consensus sigA -35 domain by only a single nt. The transcription start point from the resulting mutant promoter is within the DNA region that normally specifies the RNA secondary structure that sequesters cat-86 RBS-3. Thus, the basis for the constitutive phenotype is the absence of the RNA secondary structure in the transcripts driven by the promoter generated through mutagenesis of leader codon 4.


Subject(s)
Chloramphenicol O-Acetyltransferase/genetics , Codon/genetics , Gene Expression Regulation, Bacterial/genetics , Plasmids/genetics , Transcription, Genetic/genetics , Amino Acid Sequence , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Base Sequence , Chloramphenicol/pharmacology , Chloramphenicol Resistance , Gene Expression Regulation, Bacterial/drug effects , Molecular Sequence Data , Mutation/genetics , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , Protein Biosynthesis/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomes/drug effects , Ribosomes/metabolism
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