Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221

Shaw, William V.; Brenner, Daniel G.; LeGrice, Stuart F. J.; Skinner, Sarah E.; Hawkins, Alastair R.

doi:10.1016/0014-5793(85)80200-3

Cited by 64 publications

(27 citation statements)

References 30 publications

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“…RBS-2, GAAAGGA, exhibits a calculated AG of binding to 16S rRNA of -14 (26) and is therefore probably a moderately strong site of ribosome attachment (19,20). However, the sequence of RBS-2, as well as the sequences for ribosome-binding sites for other cat leaders (5,11,12,24), is atypical when compared with the ribosome-binding site for the cat coding sequence, AGGAGGA (Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1988

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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.

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Section: Resultsmentioning

confidence: 99%

“…These were designated conserved regions I and II. These sequences are taken from previously published data (2,5,11,12,24). Asterisks indicate translation termination condons.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 1988

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“…Sequences comparable to domains A and B exist 5' to each of four other chloramphenicol-inducible cat genes, but equivalent regulatory sequences do not exist upstream of the constitutively expressed Tn9 cat gene (2,3,9,16,21,34). Thus, cat gene inducibility correlates with a unique regulatory sequence.…”

Section: Cat-86 Induction Is Due To Activation Of Mrna Translationmentioning

confidence: 98%

Translational attenuation as the regulator of inducible cat genes

Lovett

1990

J Bacteriol

105

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“…Consequently, cat-86 mRNA is predicted to sequester RBS-3 within a stable stem-loop structure, where the RBS is apparently unavailable to base-pair with 16S rRNA. This predicted secondary structure of the mRNA in the vicinity of the RBS has been found to be characteristic of all the inducible cat genes that have been examined (5, 7, 8, 10,24). …”

mentioning

confidence: 65%

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

Ambulos¹,

Duvall²,

Lovett³

1986

J Bacteriol

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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 fromn 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 for 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.Chloramphenicol acetyltransferase (CAT) in grampositive bacteria is specified by genes designated cat, whose expression is induced by chloramphenicol (Cm) (23). One such gene, cat-86, was cloned from Bacillus pumilus DNA into Bacillus subtilis by using plasmid pUB110 as a vector (28). Subsequent replacement of the natural cat-86 promoter with a short multicloning site linker generated the promotercloning plasmid pPL703 (17) (Fig. 1). By use of pPL703 it has been shown that the sequences necessary for the Cminducible expression of cat-86 are independent of both the promoter that is selected to activate the gene and the cat-86 coding sequence (16). Accordingly, the inducible regulation of cat-86 results from sequences located within a 144-basepair (bp) regulatory region that intervenes between the coding sequence and the upstream site of promoter insertion.The nucleotide sequence of the 144-bp regulatory region demonstrates that the ribosofme bind...

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Chloramphenicol acetyltransferase gene of staphylococcal plasmid pC221

Cited by 64 publications

References 30 publications

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

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

Translational attenuation as the regulator of inducible cat genes

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

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