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 §1§ Crystal structure of Bacillus subtilis YabJ, a purine regulatory protein
and member of the highly conserved YjgF family  §1§

    1. [16]Sangita Sinha [17]*,
    2. [18]Pekka Rappu [19]†,
    3. [20]S. C. Lange [21]*,
    4. [22]Pekka Mäntsälä [23]†,
    5. [24]Howard Zalkin [25]‡, and
    6. [26]Janet L. Smith [27]* , [28]§

    1.


    Departments of ^*Biological Sciences and ^‡Biochemistry, Purdue
    University, West Lafayette, IN 47907; and ^†Department of
    Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2,
    FIN-20014 Turku, Finland

    1. Communicated by Michael G. Rossmann, Purdue University, West
       Lafayette, IN (received for review July 15, 1999)


   [29]Next Section

 §2§ Abstract §2§

   The yabJ gene in Bacillus subtilis is required for adenine-mediated
   repression of purine biosynthetic genes in vivo and codes for an
   acid-soluble, 14-kDa protein. The molecular mechanism of YabJ is
   unknown. YabJ is a member of a large, widely distributed family of
   proteins of unknown biochemical function. The 1.7-Ã… crystal structure
   of YabJ reveals a trimeric organization with extensive buried
   hydrophobic surface and an internal water-filled cavity. The most
   important finding in the structure is a deep, narrow cleft between
   subunits lined with nine side chains that are invariant among the 25
   most similar homologs. This conserved site is proposed to be a binding
   or catalytic site for a ligand or substrate that is common to YabJ and
   other members of the YER057c/YjgF/UK114 family of proteins.

   Purine biosynthesis in Bacillus subtilis is regulated by the purine
   operon repressor gene, purR. The purR operon consists of purR and yabJ,
   an ORF of unknown function. The repressor PurR binds to control regions
   upstream of the transcription start sites and regulates transcription
   of purEKBC(orf)QLFMNHD in the pur operon, of purR and yabJ in the purR
   operon, and of purA ([30]1). PurR binding to control-site DNA is
   blocked by 5-phosphoribosyl-1-pyrophosphate (PRPP), a central
   nucleotide metabolite that acts as an inducer of pur operon and purA
   transcription. 5-phosphoribosyl-1-pyrophosphate is the starting
   material for purine biosynthesis. No other metabolite, nucleotide,
   nucleoside, or nucleotide base is known to affect PurR DNA binding in
   vitro ([31]2).

   Recently, yabJ, the distal coding sequence in the purR operon, was
   shown to affect PurR function in vivo ([32]3). Regulation of
   transcription of purine biosynthetic genes is sensitive to levels of
   some nutrients and depends on the interrelated pools of
   5-phosphoribosyl-1-pyrophosphate, nucleotides, nucleosides, and
   nucleotide bases. Adenine uptake by B. subtilis indirectly increases
   PurR repression of pur operon and purA transcription through changes to
   the 5-phosphoribosyl-1-pyrophosphate pool. YabJ is also required for
   adenine-mediated repression of purA in vivo ([33]3). The mechanism by
   which YabJ stimulates adenine-mediated repression of purA by PurR is
   unknown. However, YabJ and PurR may be cotranslated and produced in
   stoichiometric quantities in vivo because the last codon of purR
   overlaps the initiation codon of yabJ.

   YabJ belongs to a widely distributed family of proteins of unknown
   function. Members of the YER057c/YjgF/UK114 family of proteins are
   identified by sequence similarity, having 20–98% pairwise identity. The
   family is named for genes or proteins in yeast (YER057c), Escherichia
   coli (yjgF), and goat (UK114). All are ≈14-kDa proteins and do not
   occur as domains of larger proteins. Many YjgF proteins are
   acid-soluble. A variety of biological processes have been reported to
   be influenced by YjgF proteins, in addition to the YabJ function in
   purine regulation. Homologs occur in bacteria, animals, and fungi but
   are absent from the genomes of most archaea and of several parasitic
   prokaryotes that also lack many biosynthetic pathways. No YjgF homolog
   has yet been detected in a plant, although its presence in a
   cyanobacterium ([34]4) suggests that it may also occur in plants. No
   three-dimensional structures of YjgF proteins have been published,
   although crystallization of a rat homolog has been reported ([35]5).
   Here we present the 1.7-Ã… crystal structure of B. subtilis YabJ. The
   structure reveals a conserved cleft on the protein surface that will
   aid in elucidation of a function for this family of proteins.
   [36]Previous Section[37]Next Section

 §2§ Materials and Methods §2§

 §3§ Cloning and Overexpression of yabJ.  §3§

   The yabJ gene was amplified by PCR from chromosomal DNA of a wild-type
   B. subtilis strain. The 446-bp PCR product was confirmed by sequencing
   and was cloned into a T7 expression vector to produce plasmid pDR1. E.
   coli strain BL21(DE3)/pLysS was transformed with pDR1. Strain
   BL21(DE3)/pLysS/pDR1 was grown at 37°C in LB medium supplemented with
   100 μg/ml ampicillin to an OD[600] of 0.6, was induced with 0.4 mM
   isopropyl β-d-thiogalactoside, and was grown for 10 hr at 30°C. Cells
   were harvested by centrifugation and were resuspended in 50 mM Trisâ‹…HCl
   (pH 8.0), 2 mM EDTA, 10 mM MgCl[2], and 10 μg/ml DNase I (5 ml of
   buffer per gram of wet cells). The cell pellet was frozen at −20°C.

 §3§ Purification of YabJ. §3§

   Frozen BL21(DE3)/pLysS/pDR1 cells were thawed and incubated at room
   temperature for 15 min. The lysate was clarified by centrifugation at
   20,000 × g for 30 min. All purification steps were performed at room