Structural conservation of putative functional motifs between Xenopus and human TFILLE-β
Structural conservation of putative functional motifs between Xenopus and human TFILLE-β
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非洲爪蟾和人类 TFILLE-β 之间假定功能基序的结构保守性
DOI：
10.1093/nar/20.16.4363
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发表时间：
1992
影响因子：
14.9
通讯作者：
M. Horikoshi
中科院分区：
文献类型：
--
作者：
Y. Ohkuma;S. Hashimoto;R. Roeder;M. Horikoshi
RNA聚合酶II的转录起始需要许多一般因素，包括TFIIE（综述见1）。先前的研究已经证明TFIIE由57 kD（TFIE-ci）和34 kD（TFIIE-3）亚基组成，其形成异源四聚体（Ci 2A）（2），需要α和亚基用于转录起始（3-5），结合RNA聚合酶II（6），并在RNA聚合酶H和TFIIF之后加入预起始复合物（5）。虽然对TFIIE在转录调控中的确切作用知之甚少，但TFIIE中结构-功能关系的表征可以提供相关信息。推导的人TFIIE-（（hTFIIE-，B））的氨基酸序列揭示了先前涉及DNA-蛋白质和/或蛋白质-蛋白质相互作用的几个基序（4）。为了鉴定高度进化保守的区域，从而推定功能的重要性，我们分离编码非洲爪蟾TFIIE-，B（xTFIIE-，B）的cDNA。以hTFIIE-β 3 cDNA为探针，从非洲爪蟾卵母细胞cDNA文库中分离到一个同源基因.在两条链上对含有完整开放阅读框（ORF）的克隆进行测序。该开放阅读框编码一个288个氨基酸的多肽，计算分子量为32.6 kD。xTFIIE-，B的氨基酸序列与hTFIIE-3的氨基酸序列的比较显示84.0%的同一性和93.1%的相似性，其中两个位点（五个残基）缺失和两个位点（两个残基）插入（参见图1）。一般来说，前面提到的所有假定的基序（4，5）都很保守，这表明它们在功能上是相关的。这些基序包括亮氨酸重复区（图1，方框d）、碱性区-螺旋-环基序（方框g）、另一个最近认识到的碱性区-螺旋-环基序（方框f）、潜在的核苷酸结合区（方框c）和与α-因子的序列相似性（方框B和e），包括与RNA聚合酶结合相关的亚区（2.1-2.2）（4）。虽然在氨基和羧基末端区域观察到相对频繁的氨基酸变化，特别是在富含丝氨酸的区域（图1，方框a）和第二个碱性区域-螺旋-环基序（方框g）内的碱性和环区域，但推定的功能特征（如电荷和潜在螺旋度）是高度保守的。除氨基和羧基端外，非洲爪蟾和人的氨基酸序列可以共线性比对，没有任何缺失和插入。对hTFIIE-，B和xTFIIE-，B中进化保守序列的了解将有助于结构-功能研究的设计。致谢我们感谢Robert Kovelman博士和亚历山大霍夫曼博士对手稿的批判性阅读，以及Jerry Dahsen博士和Doug A. Melton的cDNA文库。YO得到了人类前沿科学计划组织的奖学金支持，MH是亚历山大和亚历山大L。辛斯海默学者。这项研究得到了NIH对RGR和MH的赠款，以及皮尤信托基金对洛克菲勒大学的一般支持。
Transcription initiation by RNA polymerase II requires a number of general factors that include TFIIE (reviewed in 1). Previous studies have demonstrated that TFIIE is comprised of 57 kD (TFIE-ci) and 34 kD (TFIIE-(3) subunits which form a heterotetramer (Ci2A)(2), requires both a and subunits for transcription initiation (3-5), binds to RNA polymerase II (6), and joins the preinitiation complex after RNA polymerase H and TFIIF (5). Although little is known about the precise role of TFIIE in transcriptional regulation, the characterization of structure-function relationships in TFIIE could provide relevant information. The deduced amino acid sequence of human TFIIE-((hTFIIE-, B) revealed several motifs previously implicated in DNA-protein and/or protein-protein interactions (4). To identify regions of high evolutionary conservation, and thus of presumptive functional importance, we isolated cDNAs encoding Xenopus laevis TFIIE-, B (xTFIIE-, B). A hTFIIE-(3 cDNA was used as a probe to isolate a homologue from a Xenopus oocyte cDNA library. Clones containing a complete open reading frame (ORF) were sequenced on both strands. This putative ORF encodes a 288-amino acid polypeptide with a calculated molecular mass of 32.6 kD. Comparison of the amino acid sequence of xTFIIE-, B with that of hTFIIE-3 indicates 84.0% identity and 93.1% similarity with deletions at two sites (five residues) and insertions at two sites (two residues)(see Figure 1). Generally, all of the putative motifs noted previously (4, 5) are well conserved, suggesting that they are functionally relevant. These motifs include a leucine repeat region (Figure 1, box d), a basic region-helix-loop motif (box g), another recently-recognized basic region-helix-loop motif (box f), a potential nucleotide binding region (box c), and sequence similarities to a-factors (boxes b and e), including the subregion (2.1-2.2) implicated in binding to RNA polymerase (4). Although relatively frequent amino acid changes are observed in the amino-and carboxy-terminal regions, especially in the serine-rich region (Figure 1, box a) and the basic and loop regions within the second basic region-helix-loop motif (box g), putative functional features (such as charge and potential helicity) are highly conserved. Except for the amino-and carboxy-terminal regions, thewhole amino acid sequence ofXenopus and human can be alignedcolinearly without any deletions and insertions. Knowledge of the evolutionarily conserved sequences in hTFIIE-, B and xTFIIE-, B will help in the design of structure-function studies.ACKNOWLEDGMENTS We thank Dr Robert Kovelman and Alexander Hoffmann for critical reading of the manuscript, and Drs Jerry Thomsen and Doug A. Melton for a Xenopus laevis cDNA library. YO was supported by a fellowship from the Human Frontiers Scientific Program Organization and MH was an Alexandrine and Alexander L. Sinsheimer Scholar. This study was supported by NIH grants to RGR and MH and by general support from the Pew Trusts to the Rockefeller University.