Secondary structure of the ribosome binding site determines translational efficiency: a quantitative analysis.

ABSTRACT Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5′ end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.

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Unique features in the ribosome binding site sequence of the gram-positive Staphylococcus aureus beta-lactamase gene.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)68589-3 ◽

1981 ◽

Vol 256 (21) ◽

pp. 11283-11291 ◽

Cited By ~ 5

Author(s):

J.R. McLaughlin ◽

C.L. Murray ◽

J.C. Rabinowitz

Keyword(s):

Staphylococcus Aureus ◽

Binding Site ◽

Ribosome Binding Site ◽

Beta Lactamase ◽

Gram Positive ◽

Ribosome Binding ◽

Lactamase Gene

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pGR71 plasmid promotor sequence temporally regulated in Bacillus subtilis

Canadian Journal of Microbiology ◽

10.1139/w98-121 ◽

1998 ◽

Vol 44 (12) ◽

pp. 1186-1192

Author(s):

Guy Daxhelet ◽

Philippe Gilot ◽

Etienne Nyssen ◽

Philippe Hoet

Keyword(s):

Bacillus Subtilis ◽

Binding Site ◽

Transcription Initiation ◽

Promoter Sequence ◽

Initiation Site ◽

Chloramphenicol Acetyltransferase ◽

Ribosome Binding Site ◽

Chloramphenicol Resistance ◽

E Coli ◽

Ribosome Binding

pGR71, a composite of plasmids pUB110 and pBR322, replicates in Escherichia coli and in Bacillus subtilis. It carries the chloramphenicol resistance gene (cat) from Tn9, which is not transcribed in either host by lack of a promoter. The cat gene is preceded by a Shine-Dalgarno sequence functional in E. coli but not in B. subtilis. Deleted pGR71 plasmids were obtained in B. subtilis when cloning foreign viral DNA upstream of this cat sequence, as well as by BAL31 exonuclease deletions extending upstream from the cat into the pUB110 moiety. These mutant plasmids expressed chloramphenicol acetyltransferase (CAT), conferring on B. subtilis resistance to high chloramphenicol concentrations. CAT expression peaked at the early postexponential phase of B. subtilis growth. The transcription initiation site of cat, determined by primer extension, was located downstream of a putative promoter sequence within the pUB110 moiety. N-terminal amino acid sequencing showed that native CAT was produced by these mutant plasmids. The cat ribosome-binding site, functional in E. coli, was repositioned within the pUB110 moiety and had consequently an extended homology with B. subtilis 16S rRNA, explaining the production of native enzyme.Key words: chloramphenicol acetyltransferase, Bacillus subtilis, postexponential gene expression, plasmid pUB110, ribosome-binding site, transcriptional promoter.

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