OCTAMER AND SPH MOTIFS IN THE U1 ENHANCER COOPERATE TO ACTIVATE U1 RNA GENE-EXPRESSION
OCTAMER AND SPH MOTIFS IN THE U1 ENHANCER COOPERATE TO ACTIVATE U1 RNA GENE-EXPRESSION
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DOI：
10.1128/mcb.10.1.341
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发表时间：
1990-01-01
影响因子：
5.3
通讯作者：
STUMPH, WE
中科院分区：
文献类型：
--
作者：
ROEBUCK, KA;SZETO, DP;STUMPH, WE
鸡U1小核RNA基因的转录增强子延伸约50个碱基对，位于U1转录起始点上游180-230个碱基对。它由多个功能基序组成，包括一个GC盒、一个八聚体基序和一个新的SPH基序。通过卵母细胞表达实验研究了这三个不同的序列基序对增强子功能的贡献。在卵母细胞的非竞争性条件下，SPH基序能够在缺乏其他功能基序的情况下刺激U1 RNA转录，而八聚体基序本身则缺乏这一能力。然而，为了形成一个能稳定地被第二个竞争的小核RNA转录单位挑战的转录复合体，八聚体和SPH基序都是必需的。GC盒虽然是充分增强活性所必需的，但对于卵母细胞中稳定的复合体形成并不是必需的。用定点突变的方法研究了SPH基序的DNA序列要求。SPH基序的功能活性分布在八聚体的24个碱基对的3‘’区域，但特别依赖于位于SPH基序中心的SphI限制位点附近的序列。以鸡胚胎组织为材料，我们鉴定并部分纯化了一种称为SBF的因子，它与U1增强子的SPH基序特异结合。该因子在体外识别和结合突变的增强子DNA片段的能力与体内相应增强子序列的功能活性相关。
The transcriptional enhancer of a chicken U1 small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the U1 transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating U1 RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3'' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the U1 enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.