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Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

ABSTRACT: Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trimeric AT is active for binding to TRAP. Our chromatographic and spectroscopic data revealed that a large fraction of recombinantly overexpressed AT retains the N-formyl group (fAT), presumably due to incomplete N-formyl-methionine processing by peptide deformylase. Hydrodynamic parameters from NMR relaxation and diffusion measurements showed that fAT is exclusively trimeric (AT(3)), while (deformylated) AT exhibits slow exchange between both trimeric and dodecameric forms. We examined this equilibrium using NMR spectroscopy and found that oligomerization of active AT(3) to form inactive AT(12) is linked to protonation of the amino terminus. Global analysis of the pH dependence of the trimer-dodecamer equilibrium revealed a near physiological pK(a) for the N-terminal amine of AT and yielded a pH-dependent oligomerization equilibrium constant. Estimates of excluded volume effects due to molecular crowding suggest the oligomerization equilibrium may be physiologically important. Because deprotonation favors "active" trimeric AT and protonation favors "inactive" dodecameric AT, our findings illuminate a possible mechanism for sensing and responding to changes in cellular pH.

SUBMITTER: Sachleben JR

PROVIDER: S-EPMC2932582 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

Sachleben Joseph R JR McElroy Craig A CA Gollnick Paul P Foster Mark P MP

Proceedings of the National Academy of Sciences of the United States of America 20100816 35

Anti-TRAP (AT) is a small zinc-binding protein that regulates tryptophan biosynthesis in Bacillus subtilis by binding to tryptophan-bound trp RNA-binding attenuation protein (TRAP), thereby preventing it from binding RNA, and allowing transcription and translation of the trpEDCFBA operon. Crystallographic and sedimentation studies have shown that AT can homooligomerize to form a dodecamer, AT(12), composed of a tetramer of trimers, AT(3). Structural and biochemical studies suggest that only trim ...[more]

PMID: 20713740

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Project description:A complete transcript of the Bacillus subtilis pyr operon contains the following elements in 5' to 3' order: a 151-nucleotide (nt) untranslated leader; pyrR, encoding a 20-kDa protein; a 173-nt intercistronic region; pyrP, encoding a 46-kDa protein; a 145-nt intercistronic region; and eight overlapping cistrons encoding all of the six enzymes for de novo pyrimidine biosynthesis. Transcription is controlled by the availability of pyrimidines via an attenuation mechanism. There are three transcription terminators within the operon, each of which is preceded by another stem-loop structure, the antiterminator, whose formation would prevent formation of the terminator stem-loop. These are located in the leader, the pyrR-pyrP intercistronic region, and the pyrP-pyrB intercistronic region. Northern (RNA) blot analysis has identified transcripts of lengths which coincide with termination at these proposed attenuation sites and whose relative abundances vary in the expected pyrimidine-dependent manner. Each antiterminator contains a 50-base conserved sequence in its promoter-proximal half. Various transcriptional fusions of the pyr promoter and surrounding sequences to promoterless reporter genes support an attenuation mechanism whereby when pyrimidines are abundant, the PyrR protein binds to the conserved sequence in the pyr mRNA and disrupts the antiterminator, permitting terminator hairpin formation and promoting transcription termination. Deletion of pyrR from the chromosome resulted in the constitutive, elevated expression of aspartate transcarbamylase, which is encoded by pyrB, the third gene in the operon. Complementation of an E. coli upp mutant, as well as direct enzymatic assay, has demonstrated that pyrR also confers uracil phosphoribosyltransferase activity. Analysis of pyrR and upp deletion mutants demonstrated that upp, not pyrR, encodes the quantitatively important uracil phosphoribosyltransferase activity. The pyrP gene probably encodes an integral membrane uracil permease.

Dataset Information

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

Publications

Mechanism for pH-dependent gene regulation by amino-terminus-mediated homooligomerization of Bacillus subtilis anti-trp RNA-binding attenuation protein.

Similar Datasets

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