Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene cat-86 under control of the Bacillus subtilis 168 amylase promoter

The amyR1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' alpha B10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' alpha B10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' alpha B10 was isolated, p5' alpha B10 delta 1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR1 region and thus allows a more precise determination of the functional domain of amyR1.     

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.     

Induction of cat-86 by chloramphenicol and amino acid starvation in relaxed mutants of Bacillus subtilis

The chloramphenicol acetyltransferase gene cat-86 is induced through a mechanism that is a variation of classical attenuation. Induction results from the destabilization of an RNA stem-loop that normally sequesters the cat-86 ribosome-binding site. Destabilization of the stem-loop is due to the stalling of a ribosome in the leader region of cat-86 mRNA at a position that places the A site of the stalled ribosome at leader codon 6. Two events can stall ribosomes at the correct location to induce cat-86 translation: addition of chloramphenicol to cells and starvation of cells for the amino acid specified by leader codon 6. Induction by amino acid starvation is an anomaly because translation of the cat-86 coding sequence requires all 20 amino acids. To explain this apparent contradiction we postulated that amino acid starvation triggers intracellular proteolysis, thereby providing levels of the deprived amino acid sufficient for cat-86 translation. Here we show that a mutation in relA, the structural gene for stringent factor, blocks intracellular proteolysis that is normally triggered by amino acid starvation. The relA mutation also blocks induction of cat-86 by amino acid starvation, but the mutation does not interfere with chloramphenicol induction. Induction by amino acid starvation can be demonstrated in relA mutant cells if the depleted amino acid is restored at very low levels (e.g., 2 micrograms/ml). A mutation in relC, which may be the gene for ribosomal protein L11, blocks induction of cat-86 by either chloramphenicol or amino acid starvation. We believe this effect is due to a structural alteration of the ribosome resulting from the relC mutation and not to the relaxed phenotype of the cells.     

Analysis of the regulatory sequences needed for induction of the chloramphenicol acetyltransferase gene cat-86 by chloramphenicol and amicetin.

Induction of the chloramphenicol acetyltransferase gene cat-86 in Bacillus subtilis results from the activation of translation of cat-86 mRNA. The inducers, chloramphenicol and amicetin, are thought to enable ribosomes to destabilize a stem-loop structure in cat-86 mRNA that sequesters the ribosome binding site for the cat-86 coding sequence, designated RBS-3. The region of cat-86 mRNA which is 5' to the stem-loop contained two additional ribosome binding sites, RBS-1 and RBS-2, located 84 and 56 nucleotides, respectively, upstream from RBS-3. RBS-1 and RBS-2 were each followed by a potential translation initiation codon and a short open reading frame. Bal 31-generated deletions into the 5' end of the regulatory region that removed RBS-1 but did not enter RBS-2 caused a fourfold decrease in the uninduced and chloramphenicol-induced level of cat-86 expression and a more than 10-fold reduction in the amicetin-induced level of expression. Deletions that removed both RBS-1 and RBS-2 but did not enter the stem-loop abolished both chloramphenicol- and amicetin-inducible expression. These data indicate that RBS-2 and sequences 3' to RBS-2 are minimally essential to chloramphenicol induction. However, the presence of RBS-1 in the mRNA elevated the maximum level of expression obtained during chloramphenicol induction. These studies also demonstrate that induction of cat-86 by amicetin is highly dependent on RBS-1. To determine whether a correlation existed between RBS-1 and amicetin inducibility, we examined the sequence of the regulatory regions for two natural variants of cat-86, cat-66 and cat-57, which are chloramphenicol inducible but are very poorly induced by amicetin. Both contained nucleotide sequence differences from cat-86 in the vicinity of RBS-1 that would prevent translation of the leader peptide associated with RBS-1 in cat-86. In contrast, the regulatory regions got the three genes were virtually identical in the vicinity of RBS-2. These data indicate that efficient induction by amicetin requires sequences that are not essential for induction by chloramphenicol.     

The effects of deletions in the leader sequence of cat-86, a chloramphenicol-resistance gene isolated from Bacillus pumilus

The cat-86 gene of Bacillus pumilus, specifying a Cm-inducible CAT enzyme, was cloned previously into B. subtilis on plasmid pUB110. Various lines of evidence suggest that control of expression of this gene is at the level of translation and involves inverted complementary repeat sequences 5' to the initiation codon. A series of deletions have been generated in this region and their effects on the induction of cat-86 observed in B. subtilis, Escherichia coli and a number of ribosomal mutant strains of B. subtilis. The results indicate that the inverted complementary repeat sequences, which are capable of forming a stable stem-loop structure in the mRNA (delta G = -24.4 kcal/mol), form a barrier to translation in E. coli and B. subtilis.     

Cytoplasmic proteins interact with a translational control element in the protein-coding region of proopiomelanocortin mRNA

Previous studies have indicated that proopiomelanocortin (POMC) is translationally regulated. We proposed that the regulatory mechanism involves an interaction between trans-acting protein factors and a cis-acting stem-loop structure in the coding region of POMC mRNA. Functional interactions were tested by examining the translation of mouse POMC mRNA in a rabbit reticulocyte system. Specific binding was demonstrated with ultraviolet-crosslinking and RNA gel mobility shift assays. The evidence presented supports our hypothesis that the translational regulation of POMC gene expression involves recognition of the stem-loop by RNA-binding proteins. Furthermore, POMC stem-loop RNA-binding proteins specifically recognized a predicted stem-loop found in the coding region of corticotropin-releasing hormone, suggesting a novel mechanism of gene regulation that may extend to other neuropeptides as well.     

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.     

Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.     

Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of hepatitis C virus RNA

Sequences of the untranslated regions at the 5' and 3' ends (5'UTR and 3'UTR) of the hepatitis C virus (HCV) RNA genome are highly conserved and contain cis-acting RNA elements for HCV RNA replication. The HCV 5'UTR consists of two distinct RNA elements, a short 5'-proximal stem-loop RNA element (nucleotides 1 to 43) and a longer element of internal ribosome entry site. To determine the sequence and structural requirements of the 5'-proximal stem-loop RNA element in HCV RNA replication and translation, a mutagenesis analysis was preformed by nucleotide deletions and substitutions. Effects of mutations in the 5'-proximal stem-loop RNA element on HCV RNA replication were determined by using a cell-based HCV replicon replication system. Deletion of the first 20 nucleotides from the 5' end resulted in elimination of cell colony formation. Likewise, disruption of the 5'-proximal stem-loop by nucleotide substitutions abolished the ability of HCV RNA to induce cell colony formation. However, restoration of the 5'-proximal stem-loop by compensatory mutations with different nucleotides rescued the ability of the subgenomic HCV RNA to replicate in Huh7 cells. In addition, deletion and nucleotide substitutions of the 5'-proximal stem-loop structure, including the restored stem-loop by compensatory mutations, all resulted in reduction of translation by two- to fivefold, suggesting that the 5'-proximal stem-loop RNA element also modulates HCV RNA translation. These findings demonstrate that the 5'-proximal stem-loop of the HCV RNA is a cis-acting RNA element that regulates HCV RNA replication and translation.