Björklund S, Hjortsberg K, Johansson E, Thelander L
Department of Medical Biochemistry and Biophysics, University of Umeå, Sweden.
Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11322-6. doi: 10.1073/pnas.90.23.11322.
Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins R1 and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase R1 gene was compiled from a number of overlapping lambda clones isolated from a Charon 4A mouse sperm genomic library. The R1-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the R1-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease S1 mapping of RNA. DNase I footprinting assays on the R1 promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Sp1, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent R1 gene expression.
哺乳动物核糖核苷酸还原酶（EC 1.17.4.1）由两个不同的亚基，即R1和R2蛋白组成，这两个亚基都是酶活性所必需的。小鼠核糖核苷酸还原酶R1基因的基因组结构是根据从Charon 4A小鼠精子基因组文库中分离出的多个重叠λ克隆编译而成的。编码R1的基因覆盖26 kb，由19个外显子组成。通过双脱氧核苷酸测序确定了所有外显子-内含子边界，结果表明内含子7起始于变异的GC而非GT。从编码R1基因的5'侧翼区域克隆并测序了约3.5 kb的DNA，并通过RNA的核酸酶S1图谱确定了转录起始位点。对R1启动子进行的DNase I足迹分析确定了两个几乎相同的23 bp长的蛋白质结合区域。通过凝胶阻滞迁移率变动分析和紫外线交联解析并部分鉴定了与其中一个23聚体区域结合的三种蛋白质复合物。一种复合物很可能包含Sp1，另一种复合物表现出S期特异性结合，这表明其在细胞周期依赖性R1基因表达中起直接作用。
