Biophysical and genetic experiments have defined how the Saccharomyces cerevisiae protein GAL4 and a subset of related proteins recognize specific DNA sequences. We assessed DNA sequence preferences of GAL4 and a related protein, PPR1, in an in vitro DNA binding assay. For GAL4, the palindromic CGG triplets at the ends of the 17-bp recognition site are essential for tight binding, whereas the identities of the internal 11 bp are much less important, results consistent with the GAL4-DNA crystal structure. Small reductions in affinity due to mutations at the center-most 5 bp are consistent with the idea that an observed constriction in the minor groove in the crystalline GAL4-DNA complex is sequence dependent. The crystal structure suggests that this sequence dependence is due to phosphate contacts mediated by arginine 51, as part of a network of hydrogen bonds. Here we show that the mutant protein GAL4(1-100)R51A fails to discriminate sites with alterations in the center of the site from the wild-type site. PPR1, a relative of GAL4, also recognizes palindromic CGG triplets at the ends of its 12-bp recognition sequence. The identities of the internal 6 bp do not influence the binding of PPR1. We also show that the PPR1 site consists of a 12-bp duplex rather than 16 bp as reported previously: the two T residues immediately 5 to the CGG sequence in each half site, although highly conserved, are not important for binding by PPR1. Thus, GAL4 and PPR1 share common CGG half sites, but they prefer DNA sequences with the palindromic CGG separated by the appropriate number of base pairs, 11 for GAL4 and 6 for PPR1.GAL4 and PPR1 (pyrimidine pathway regulator 1), two Saccharomyces cerevisiae transcription regulatory proteins, are members of a family of at least 12 fungal transcription factors containing a region termed the Zn 2 Cys 6 binuclear cluster (reviewed in reference 11). This region has six absolutely conserved cysteines and a number of other highly conserved residues. It has been shown for various members of the family that the Zn 2 Cys 6 region is essential for DNA binding (reviewed in references 15 and 24). The DNA sites of several members of the family, including those recognized by GAL4 and PPR1, contain palindromic CGG triplets within their DNA sites (Fig. 1a). Thus, GAL4 and PPR1 are members of a subset of Zn 2 Cys 6 -containing proteins whose members recognize twofold symmetric CGG half sites separated by distinct numbers of base pairs (24,29).GAL4 is an activator of transcription of various galactoseinducible genes. These genes possess GAL4 binding sites positioned in the upstream regions of their promoters (3, 4, 9). Examination of 16 known natural sites reveals a 17-bp pseudopalindromic consensus sequence (9). This consensus site is a high-affinity binding site for GAL4 derivatives in vitro (this study) and confers GAL4-dependent transcriptional activation in vivo when placed upstream of a test reporter gene, both in yeast cells (18, 21a) and in mammalian tissue culture cells (36). The site has highly co...
The biological activity of the transcription factor NF-B is mainly controlled by the IB proteins IB␣ and IB, which restrict NF-B in the cytoplasm and enter the nucleus where they terminate NF-B-dependent transcription. In this paper we describe the cloning and functional characterization of mouse IB. Mouse IB contains 6 ankyrin repeats required for its interaction with the Rel proteins and is expressed in different cell types where we found that it is up-regulated by NF-B inducers, as is the case for IB␣ and human IB. IB functions as a bona fide IB protein by restricting Rel proteins in the cytoplasm and inhibiting their in vitro DNA binding activity. Surprisingly, IB did not inhibit transcription of genes regulated by the p50͞p65 heterodimer efficiently, such as the human interferon- gene. However, IB was a strong inhibitor of interleukin-8 expression, a gene known to be regulated by p65 homodimers. In addition, IB appears to function predominantly in the cytoplasm to sequester p65 homodimers, in contrast with the other two members of the family, IB␣ and IB, which also function in the nucleus to terminate NF-B-dependent transcriptional activation.The transcription factor NF-B plays a major role in the activation of numerous genes involved in the function and development of the immune system, in the recruitment of leukocytes from the circulation into extravascular space, and in inflammatory and acute responses, etc. (reviewed in refs. 1-4). NF-B consists of homo-and heterodimeric proteins that belong to the Rel family of transcription factors. In mammals there are five Rel proteins, p50, p52, p65 (RelA), c-Rel, and RelB, all of which contain a so-called Rel homology region (RHR) that includes DNAbinding and dimerization domains and a nuclear localization signal (NLS). The Rel proteins are present in most cell types in an inactive cytosolic form. The cellular partitioning of NF-B is tightly regulated by the IB proteins, which are complexed with NF-B in the cytoplasm (5). The NF-B DNA binding activity can be induced by a large variety of extracellular signals, all of which culminate in the proteolytic degradation of IBs by the proteasome, thereby freeing NF-B to translocate to the nucleus, where it activates gene transcription (1-7).There are two known types of Rel complexes in the cytoplasm. The first type consists of heterodimers containing the p65 or c-Rel proteins associated with the precursors for p50 or p52 (p105 and p100, respectively). The second type consists of p65 and c-Rel homo-or heterodimers (with p50 or p52) associated with a member of the IB family. A common characteristic of the IB proteins is the presence of multiple copies of a motif, the ankyrin repeat, which interacts with the RHR. This interaction has two functional consequences. First, NF-B-IB complexes are sequestered in the cytoplasm, because the IBs mask the NLS through direct protein-protein interactions; and second, IBs can prevent NF-B from binding to the DNA in vitro and in vivo. The IB family consists of IB␣ (5 ankyrin rep...
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