Gil G, Osborne T F, Goldstein J L, Brown M S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1988 Dec 15;263(35):19009-19.
The gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis, is transcribed at a relatively high level when cellular sterols are depleted and is repressed when sterols accumulate. We have previously reported that the regulatory region of the hamster reductase gene contains eight different sequences that bind nuclear proteins as determined by DNase I footprinting assays. We here report the purification of a single activity that accounts for six of these footprints. This activity was found in a doublet of proteins (designated reductase promoter factor 1, RPF-1) that have apparent molecular weights of 33,000 and 35,000. They were isolated by DNA affinity chromatography using oligonucleotides corresponding to either of two footprinted sequences. The 33- and 35-kDa species were present as monomers, as indicated by gel filtration and gradient ultracentrifugation. Oligonucleotides corresponding to any one of the six footprinted sequences prevented the binding of RPF-1 to all of the other sequences, indicating that all six bind to a single site in RPF-1. The only sequence shared by all six footprinted sequences is the trinucleotide, TGG, both of whose guanosines made contact with RPF-1, as determined by methylation interference assays. The footprinted sequence that binds RPF-1 with highest affinity contains the palindrome, TGG(N7)CCA, which conforms to the consensus sequence for binding NF-1, a nuclear protein that stimulates replication of adeno-virus-2. Purified RPF-1 was shown to bind to the adenovirus NF-1 binding site with high affinity. Although the apparent molecular weight of the RPF-1 doublet was lower than the molecular weight range for NF-1 proteins (52,000-66,000), it is likely that the 33-35-kDa doublet is derived from a larger NF-1-like protein as a result of proteolysis. We conclude that RPF-1 belongs to a group of TGG-binding proteins that includes NF-1 and other proteins previously described as CCAAT binding proteins. This protein binds to six sites in the promoter region for hamster 3-hydroxy-3-methylglutaryl CoA reductase, where its function remains to be determined.
3-羟基-3-甲基戊二酰辅酶A还原酶是胆固醇生物合成的限速酶，当细胞内固醇缺乏时，该酶的基因转录水平相对较高，而当固醇积累时则受到抑制。我们之前报道过，通过DNA酶I足迹分析测定，仓鼠还原酶基因的调控区域包含八个与核蛋白结合的不同序列。我们在此报告一种单一活性的纯化，该活性占其中六个足迹的原因。这种活性存在于一对蛋白质（称为还原酶启动子因子1，RPF-1）中，其表观分子量分别为33,000和35,000。它们通过使用与两个足迹序列之一相对应的寡核苷酸进行DNA亲和层析分离得到。凝胶过滤和梯度超速离心表明，33 kDa和35 kDa的物种以单体形式存在。与六个足迹序列中任何一个相对应的寡核苷酸都能阻止RPF-1与所有其他序列的结合，这表明所有六个序列都结合到RPF-1中的一个单一位点。通过甲基化干扰分析确定，所有六个足迹序列共有的唯一序列是三核苷酸TGG，其两个鸟苷都与RPF-1接触。与RPF-1结合亲和力最高的足迹序列包含回文序列TGG(N7)CCA，它符合刺激腺病毒2复制的核蛋白NF-1的结合共有序列。纯化的RPF-1显示出与腺病毒NF-1结合位点具有高亲和力。尽管RPF-1双峰的表观分子量低于NF-1蛋白的分子量范围（52,000 - 66,000），但33 - 35 kDa的双峰很可能是由于蛋白水解作用从一个更大的NF-1样蛋白衍生而来。我们得出结论，RPF-1属于一组TGG结合蛋白，其中包括NF-1和其他先前被描述为CCAAT结合蛋白的蛋白质。这种蛋白质结合到仓鼠3-羟基-3-甲基戊二酰辅酶A还原酶启动子区域的六个位点，其功能仍有待确定。
