Xenopus transcription factor IIIA (TFIIIA) binds to over 50 base pairs in the internal control region of the 5 S rRNA gene, yet the binding energy for this interaction (⌬G 0 ؍ ؊12.8 kcal/mol) is no greater than that exhibited by many proteins that occupy much smaller DNA targets. Despite considerable study, the distribution of the DNA binding energy among the various zinc fingers of TFIIIA remains poorly understood. By analyzing TFIIIA mutants with disruptions of individual zinc fingers, we have previously shown that each finger contributes favorably to binding (Del Rio, S., Menezes, S. R., and Setzer, D. R. (1993) J. Mol. Biol. 233, 567-579). Those results also suggested, however, that simultaneous binding by all nine zinc fingers of TFIIIA may involve a substantial energetic cost. Using complementary N-and C-terminal fragments and full-length proteins containing pairs of disrupted fingers, we now show that energetic interference indeed occurs between zinc fingers when TFIIIA binds to the 5 S rRNA gene and that the greatest interference occurs between fingers at opposite ends of the protein in the TFIIIA⅐5 S rRNA gene complex. Some, but not all, of the thermodynamically unfavorable strain in the TFIIIA⅐5 S rRNA gene complex may be derived from bending of the DNA that is necessary to accommodate simultaneous binding by all nine zinc fingers of TFIIIA. The energetics of DNA binding by TFIIIA thus emerges as a compromise between individual favorable contacts of importance along the length of the internal control region and long range strain or distortion in the protein, the 5 S rRNA gene, or both that is necessary to accommodate the various local interactions. Xenopus transcription factor IIIA (TFIIIA)1 is a multifunctional protein that recognizes the major cis-acting transcriptional control element in 5 S rRNA genes (the internal control region, or ICR) and thereby nucleates the formation of transcription complexes that result in the synthesis of 5 S rRNA (1-3). It also binds to 5 S rRNA itself to form a ribonucleoprotein storage particle that accumulates in Xenopus oocytes (4,5) and that may mediate feedback regulation of 5 S rRNA gene expression in somatic cells (5, 6). As the first sequence-specific DNA-binding protein to be purified from eukaryotic cells (1) and the archetypal zinc finger protein (7), TFIIIA has been an influential model protein for understanding the mechanisms of sequence-specific DNA and RNA recognition.The nine zinc fingers of TFIIIA define the sequence features that can be used to recognize zinc finger motifs in other proteins (7). These include two cysteine and two histidine residues with conserved spacing; these four amino acids coordinate a Zn 2ϩ ion (8) that contributes substantially to the stability of the folded domain (9 -11). Three conserved hydrophobic residues are also likely to stabilize TFIIIA-like zinc finger domains through interactions in a hydrophobic pocket. The large number of zinc fingers in TFIIIA has made its structural analysis difficult, and structural studies of ...
The authors report the first DNA-based diagnosis of Bartonella henselae cultured from a brain lesion in a patient with acquired immune deficiency syndrome. This human immunodeficiency virus-infected patient presented with altered mental status, fever, and diabetes insipidus. Magnetic resonance imaging revealed multifocal parenchymal and leptomeningeal involvement, which was confirmed on studies of tissue biopsy samples. Using the polymerase chain reaction and gene sequencing techniques, the authors definitively demonstrated the presence of B. henselae in the brain tissue biopsy specimen.
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