A SYNTHETIC EARLY PROMOTER FROM A BACULOVIRUS - ROLES OF THE TATA BOX AND CONSERVED START SITE CAGT SEQUENCE IN BASAL LEVELS OF TRANSCRIPTION
A SYNTHETIC EARLY PROMOTER FROM A BACULOVIRUS - ROLES OF THE TATA BOX AND CONSERVED START SITE CAGT SEQUENCE IN BASAL LEVELS OF TRANSCRIPTION
复制标题
DOI：
10.1016/0042-6822(92)90916-d
复制
发表时间：
1992-10-01
期刊：
影响因子：
3.7
通讯作者：
ROHRMANN, GF
中科院分区：
文献类型：
--
作者：
BLISSARD, GW;KOGAN, PH;ROHRMANN, GF
许多杆状病毒早期基因和昆虫基因转录的RNA聚合酶II有一个保守的转录起始位点序列（CAGT）位于下游的共识TATA框。为了研究这两个基序在启动精确定位的基础转录中的功能和相互作用，从伪杉毒蛾核型多角体病毒（OpMNPV）的gp 64早期启动子的TATA盒和起始位点序列合成了43-nt的合成引物。合成的启动子准确地启动，并且也被杆状病毒转录激活因子IE 1反式激活。为了确定43-nt合成启动子内序列的作用，分析了一系列连接子扫描和间隔突变的转录活性、起始位点选择和反式激活。通过瞬时表达和报告基因分析，在体内检测了连接子扫描突变。为了研究转录起始位点的选择，启动子构建体被用于未感染的Spodoptera frugiperda（Sf 9）细胞核提取物的体外转录，体内和体外分析表明，TATA盒，而不是起始位点CAGT，是控制起始位点选择的主要元件。保守的起始位点CAGT序列的取代导致报告基因活性和体外转录物的减少，尽管转录物准确地启动。来自接头扫描和间隔突变的数据表明，保守的起始位点CAGT序列对于准确起始是不需要的，但是起始位点处的序列在起始效率中起重要作用。
Many baculovirus early genes and insect genes transcribed by RNA polymerase II have a conserved transcription start site sequence (CAGT) located downstream of a consensus TATA box. To examine the functions and interactions of these two motifs in initiating accurately positioned basal transcription, a 43-nt synthetic prompter was synthesized from the TATA box and start site sequences of the gp64 early promoter from theOrgyia pseudotsugatamulticapsid nuclear polyhedrosis virus (OpMNPV). The synthetic promoter initiated accurately and was also transactivated by the baculovirus transcriptional activator, IE1. To determine the roles of sequences within the 43-nt synthetic promoter, a series of linker-scanning and spacing mutations were analyzed for transcriptional activity, start site selection, and transactivation. Linker-scanning mutations were examinedin vivoby transient expression and reporter gene assays. To examine transcription start site selection, promoter constructs were used forin vitrotranscription in nuclear extracts from uninfectedSpodoptera frugiperda(Sf9) cells.In vivoandin vitroanalyses show that the TATA box, and not the start site CAGT, is the primary element controlling start site selection. Substitution of the conserved start site CAGT sequence resulted in a reduction of both reporter gene activity andin vitrotranscripts, although transcripts initiated accurately. Data from linker-scanning and spacing mutations indicate that the conserved start site CAGT sequences are not required for accurate initiation but sequences at the start site play an important role in initiation efficiency.