The tertiary structure of G3PAT comprises two domains, the larger of which, domain II, features an extensive cleft lined by hydrophobic residues and contains at one end a cluster of positively charged residues flanked by a H(X)(4)D motif, which is conserved amongst many glycerolipid acyltransferases. We predict that these hydrophobic and positively charged residues represent the binding sites for the fatty acyl substrate and the phosphate moiety of the glycerol 3-phosphate, respectively, and that the H(X)(4)D motif is a critical component of the enzyme's catalytic machinery.
Telomeric DNA of Tetrahymena thermophila consists of a long stretch of (TTGGGG) n double-stranded repeats with a single-stranded (TTGGGG) 2 3 overhang at the end of the chromosome. We have identified and characterized a protein that specifically binds to a synthetic telomeric substrate consisting of duplex DNA and the 3 telomeric repeat overhang. This protein is called TEP (telomere end-binding protein). A change from G to A in the third position of the TTGGGG overhang repeat converts the substrate to a human telomere analog and reduces the binding affinity approximately threefold. Changing two G's to C's in the TTGGGG repeats totally abolishes binding. However, permutation of the Tetrahymena repeat sequence has only a minor effect on binding. A duplex structure adjacent to the 3 overhang is required for binding, although the duplex need not contain telomeric repeats. TEP does not bind to G-quartet DNA, which is formed by many G-rich sequences. TEP has a greatly reduced affinity for RNA substrates. The copy number of TEP is at least 2 ؋ 10 4 per cell, and it is present under different conditions of cell growth and development, although its level varies. UV cross-linking experiments show that TEP has an apparent molecular mass of ϳ65 kDa. Unlike other telomere end-binding proteins, TEP is sensitive to high salt concentrations.Telomeres are the natural ends of eukaryotic chromosomes. They protect chromosomes from nuclease degradation and from end-to-end ligation, ensure complete replication of chromosomes, and are involved in chromosome organization and nuclear architecture (2,22,34,47). Telomeres typically contain an array of short (5-to 8-bp) sequence repeats which are G rich in the strand that extends to the 3Ј end of the chromosome (2,22,34,47). In those cases studied in molecular detail, it has been shown that the G-rich strand forms a 3Ј single-stranded overhang of 12 to 16 nucleotides at the chromosomal terminus (20,25,32). Most telomeric sequences fit the consensus C 1-8 (T/A) 1-4 (2, 22, 34, 47).It is important to characterize proteins that bind to telomeres because they are intimately involved in telomere-mediated chromosome stabilization. Moreover, telomere-binding proteins must interact with telomerase, an enzyme involved in telomere replication and maintenance, whose activity is implicated in both cancer and aging (10). Telomeric DNA is associated with two types of proteins in vivo. Internal telomerebinding proteins interact with the duplex region of telomeric repeats. These include PPT, identified in Physarum polycephalum, and RAP1, identified in the yeast Saccharomyces cerevisiae (3,4,8,9,27). PPT is a 10-kDa heat-stable protein that binds specifically to the duplex region of the telomeric sequence (T 2 AG 3 ) n and is thought to cover the length of the telomere (8). RAP1 is a multifunctional protein that, in addition to binding telomeric repeats in yeast cells, binds to the upstream activating sequences of many genes and to silencer elements. Underexpression of RAP1 reduces telomere length, whereas...
G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.
Glycerol-3-phosphate 1-acyltransferase (E.C. 2.3.1.15; G3PAT) catalyses the incorporation of an acyl group from either acyl-acyl carrier proteins (acylACPs) or acylCoAs into the sn-1 position of glycerol 3-phosphate to yield 1-acylglycerol 3-phosphate. Crystals of squash G3PAT have been obtained by the hanging-drop method of vapour diffusion using PEG 4000 as the precipitant. These crystals are most likely to belong to space group P2(1)2(1)2(1), with approximate unit-cell parameters a = 61.1, b = 65.1, c = 103.3 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected in-house using a MAR 345 imaging-plate system.
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