Structural analysis of proteins regulating transcription of purine biosynthesis genes in Bacillus subtilis

Sangita Sinha, Purdue University

Abstract

The Bacillus subtilis pur and purA operons encode genes required for de novo biosynthesis of purines from PRPP. Two proteins, the 285-residue PurR and the 125-residue YabJ, encoded by the purR operon, regulate transcription of the pur, purA and purR operons. A homodimer of the transcription initiation repressor, PurR, binds to unknown specificity elements in a long segment of control-site DNA of these operons, introducing positive supercoils in the DNA. PRPP induces these operons by inhibiting DNA binding by PurR. YabJ modulates PurR function in vivo by an unknown mechanism. This research presents the 2.3 Å PurR and the 1.7 Å YabJ X-ray crystal structures, solved by SeMet MAD and Hg MAD techniques, respectively. Structures of both proteins provide information important to elucidating their functions. PurR is a two-domain protein. The highly basic, N-terminal, helix-turn-helix (HTH) domain probably binds DNA. This domain has no sequence similarity to any DNA-binding domain of known structure, hence, its fold classification and the protein elements responsible for binding DNA were unknown prior to PurR structure determination. An extensive hydrophobic interface between the PurR HTH domains appears to fix their “recognition” helices in an unusual orientation, suggesting a novel mode of DNA binding relative to other dimeric HTH DNA binding proteins. The C-terminal, type I phosphoribosyltransferase (PRTase) domain binds PRPP as an effector molecule. Unlike the catalytic PRTases, no enzymatic activity has been detected for PurR, possibly because of the absence of structural elements for binding a nucleophilic co-substrate. YabJ is the first structural representative of a large, widely distributed family of unknown function. The presence of YabJ homologs in all organisms except certain parasitic eubacteria, suggests that they play an important, yet hitherto unidentified, role in an essential cellular process. The extensive subunit interface of YabJ indicates an unexpected trimeric biological state. The electronegative YabJ surface potential suggests a direct interaction with DNA is unlikely. Residues conserved amongst various YabJ homologs map to a single, solvent-accessible pocket, suggestive of a ligand- or substrate-binding site. Despite this detailed information provided by the YabJ structure, the biological role of this protein remains an intriguing mystery.

Degree

Ph.D.

Advisors

Smith, Purdue University.

Subject Area

Biophysics|Molecular biology|Biochemistry

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