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Site in the cat-86 regulatory leader that permits amicetin to induce expression of the gene
U J Kim1, N P Ambulos, E J Duvall
1Department of Biological Sciences, University of Maryland Baltimore County, Catonsville 21228.
Abstract:
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 When the GTG codon immediately 5' to conserved region I in cat-86 was changed to AAA, amicetin was no longer active in cat-86 induction, but chloramphenicol induction was unaffected by the mutation.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.
Insights
Antibiotic induction of plasmid gene cat-86 expression in Bacillus subtilis involves ribosome stalling. Specific leader sequences and codon positioning are crucial for this drug-mediated translational control.
Area of Science:
- Molecular Biology
- Microbiology
- Gene Regulation
Background:
- Plasmid gene cat-86 expression in Bacillus subtilis is induced by chloramphenicol and amicetin.
- Induction is proposed to involve destabilization of an mRNA stem-loop structure, freeing the ribosome-binding site.
- Ribosome stalling in the mRNA leader region is implicated in the induction mechanism.
Purpose of the Study:
- To investigate the role of specific leader sequences and ribosome stalling in cat-86 gene induction by antibiotics.
- To test the hypothesis that a leader site 5' to codon 6 is necessary for antibiotic-mediated ribosome stalling.
- To elucidate the role of the ribosome-binding site (RBS-2) and upstream sequences in drug-induced translation.
Main Methods:
- Site-directed mutagenesis was used to insert or delete codons within the cat-86 leader sequence.
- Mutations were introduced into the ribosome-binding site (RBS-2) to alter its interaction with 16S rRNA.
- Specific codons upstream of conserved region I were altered to investigate their role in amicetin induction.
Main Results:
- Insertion of an extra codon between leader codons 5 and 6 blocked induction, which was restored by deleting codon 6, indicating the importance of precise spatial relationships.
- Mutations decreasing RBS-2 binding to 16S rRNA prevented induction, while those increasing binding did not inhibit induction.
- Changing the valine codon (GTG) upstream of conserved region I to a lysine codon (AAA) abolished amicetin induction but not chloramphenicol induction.
Conclusions:
- Antibiotic induction of cat-86 expression requires a specific spatial arrangement between upstream leader sites and leader codon 6.
- The strength of RBS-2 binding to 16S rRNA is not the sole determinant of induction; specific upstream sequences are critical.
- The valine codon (GTG) immediately upstream of conserved region I plays a specific role in amicetin-mediated induction of cat-86.