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A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

ABSTRACT: Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetracycline repressor (TetR) variants were identified in which binding of tetracycline triggers the repressor to associate with and not to dissociate from the operator DNA as in wild-type TetR. We investigated the origin of this activity reversal by limited proteolysis, CD spectroscopy and X-ray crystallography. We show that the TetR mutant Leu17Gly switches its function via a disorder-order mechanism that differs completely from the allosteric mechanism of wild-type TetR. Our study emphasizes how single point mutations can engender unexpected leaps in protein function thus enabling the appearance of new functionalities in proteins without the need for promiscuous intermediates.

SUBMITTER: Resch M

PROVIDER: S-EPMC2490752 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

Resch Marcus M Striegl Harald H Henssler Eva Maria EM Sevvana Madhumati M Egerer-Sieber Claudia C Schiltz Emile E Hillen Wolfgang W Muller Yves A YA

Nucleic acids research 20080628 13

Today's proteome is the result of innumerous gene duplication, mutagenesis, drift and selection processes. Whereas random mutagenesis introduces predominantly only gradual changes in protein function, a case can be made that an abrupt switch in function caused by single amino acid substitutions will not only considerably further evolution but might constitute a prerequisite for the appearance of novel functionalities for which no promiscuous protein intermediates can be envisaged. Recently, tetr ...[more]

PMID: 18587152

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Project description:Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by Streptomyces lincolnensis However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis The precise SLCG_2919 binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis The present study improves our understanding of molecular regulation for lincomycin biosynthesis.IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in Streptomyces species to improve antibiotic production.

Dataset Information

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

Publications

A protein functional leap: how a single mutation reverses the function of the transcription regulator TetR.

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