Bienkiewicz E A, Moon Woody A, Woody R W
Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523, USA.
J Mol Biol. 2000 Mar 17;297(1):119-33. doi: 10.1006/jmbi.2000.3545.
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandemly repeated copies of a heptapeptide with the Y(1)S(2)P(3)T(4)S(5)P(6)S(7) consensus sequence. This repeat contains two overlapping SPXX motifs that can adopt a beta-turn conformation. In addition, each CTD repeat contains the PXXP sequence characteristic of the left-handed helix of polyproline II (P(II)) found in SH3 domain ligands and the PXY sequence that is the target for WW domains. We have studied CTD fragments using circular dichroism (CD) to characterize the conformation of the CTD in water and in the hydrogen bond-promoting solvent trifluoroethanol (TFE). In water, an eight-repeat fragment is predominantly unordered, but at 32 degrees C has P(II) and beta-turn contents estimated to be about 15 % and less than 10 %, respectively. In 90 % TFE, the beta-turn fraction is estimated to be about 75 %, the remainder being unordered and P(II) conformations. The Tyr side-chains are ordered to a significant extent in 90 % TFE. Replacement of the fully conserved Pro residues by alpha-aminoisobutyric acid leads to a large increase in beta-turn. Replacement of Ser2 by Ala does not substantially alter the CTD conformation in water or TFE. Ser5 replacement by Ala increases the P(II) content in water and affects the conformation in TFE-rich solutions. Phosphorylation of Ser2 and Ser5 has little effect in water, but Ser2 affects the conformation in TFE-rich solution in much the same way as Ser5-->Ala substitution. The CD of the full-length murine CTD in water is similar to that of the eight-repeat fragment, indicating little difference in conformation with increasing chain length beyond eight repeats. The roles of P(II) and beta-turn in the interaction of CTD with its target proteins (mediator and RNA-processing components) are discussed. The most likely interactions are between P(II) and WW or SH3 domains, or with some unknown P(II)-binding motif.
RNA聚合酶II最大亚基的C末端结构域(CTD)由具有Y(1)S(2)P(3)T(4)S(5)P(6)S(7)共有序列的七肽串联重复拷贝组成。该重复序列包含两个重叠的SPXX基序,它们可形成β-转角构象。此外,每个CTD重复序列包含在SH3结构域配体中发现的聚脯氨酸II(P(II))左手螺旋特征的PXXP序列以及WW结构域的靶序列PXY。我们使用圆二色性(CD)研究了CTD片段,以表征CTD在水中和促进氢键形成的溶剂三氟乙醇(TFE)中的构象。在水中,一个八重复片段主要是无序的,但在32℃时,其P(II)和β-转角含量估计分别约为15%和不到10%。在90%的TFE中,β-转角部分估计约为75%,其余为无序和P(II)构象。在90%的TFE中,酪氨酸侧链在很大程度上是有序的。用α-氨基异丁酸取代完全保守的脯氨酸残基会导致β-转角大幅增加。用丙氨酸取代Ser2在水或TFE中不会显著改变CTD构象。用丙氨酸取代Ser5会增加水中的P(II)含量,并影响富含TFE溶液中的构象。Ser2和Ser5的磷酸化在水中影响很小,但Ser2对富含TFE溶液中构象的影响与Ser5被丙氨酸取代的情况非常相似。全长小鼠CTD在水中的CD与八重复片段的相似,表明超过八次重复后,随着链长增加,构象差异不大。讨论了P(II)和β-转角在CTD与其靶蛋白(中介体和RNA加工成分)相互作用中的作用。最可能的相互作用是P(II)与WW或SH3结构域之间,或与一些未知的P(II)结合基序之间。