Subunit communications crucial for the functional integrity of the yeast RNA polymerase II elongator (gamma-toxin target (TOT)) complex

In response to the Kluyveromyces lactis zymocin, the gamma-toxin target (TOT) function of the Saccharomyces cerevisiae RNA polymerase II (pol II) Elongator complex prevents sensitive strains from cell cycle progression. Studying Elongator subunit communications, Tot1p (Elp1p), the yeast homologue of human IKK-associated protein, was found to be essentially involved in maintaining the structural integrity of Elongator. Thus, the ability of Tot2p (Elp2p) to interact with the HAT subunit Tot3p (Elp3p) of Elongator and with subunit Tot5p (Elp5p) is dependent on Tot1p (Elp1p). Also, the association of core-Elongator (Tot1-3p/Elp1-3p) with HAP (Elp4-6p/Tot5-7p), the second three-subunit subcomplex of Elongator, was found to be sensitive to loss of TOT1 (ELP1) gene function. Structural integrity of the HAP complex itself requires the ELP4/TOT7, ELP5/TOT5, and ELP6/TOT6 genes, and elp6Delta/tot6Delta as well as elp4Delta/tot7Delta cells can no longer promote interaction between Tot5p (Elp5p) and Tot2p (Elp2p). The association between Elongator and Tot4p (Kti12p), a factor that may modulate the TOT activity of Elongator, requires Tot1-3p (Elp1-3p) and Tot5p (Elp5p), indicating that this contact requires a preassembled holo-Elongator complex. Tot4p also binds pol II hyperphosphorylated at its C-terminal domain Ser(5) raising the possibility that Tot4p bridges the contact between Elongator and pol II.     

延伸因子复合物对真核生物tRNA的修饰作用

延伸因子复合物(Elongator complex, Elp)由6个亚基蛋白Elp1～6组成,在真核细胞生物中呈现高度的进化保守,提示其具有重要的生物学功能。研究表明,Elp涉及多种细胞行为如转录延伸、细胞外分泌、端粒基因沉默和DNA损伤修复、神经系统的发育和功能等。然而越来越多的证据显示,Elp通过介导tRNA修饰影响翻译过程,从而间接调控上述细胞行为。在人类,ELP1/IKBKAP突变可导致家族性植物神经功能障碍症,ELP2、ELP3和ELP4基因的遗传变异也可能与其他神经退行性病变相关。本文对Elp的结构、Elp修饰tRNA和Elp相关疾病等的研究现状及其进展进行综述。     

Characterization of a Six-Subunit Holo-Elongator Complex Required for the Regulated Expression of a Group of Genes in Saccharomyces cerevisiae

The Elongator complex associated with elongating RNA polymerase II in Saccharomyces cerevisiae was originally reported to have three subunits, Elp1, Elp2, and Elp3. Using the tandem affinity purification (TAP) procedure, we have purified a six-subunit yeast Holo-Elongator complex containing three additional polypeptides, which we have named Elp4, Elp5, and Elp6. TAP tapping and subsequent purification of any one of the six subunits result in the isolation of all six components. Purification of Elongator in higher salt concentrations served to demonstrate that the complex could be separated into two subcomplexes: one consisted of Elp1, -2, and -3, and the other consisted of Elp4, -5, and -6. Deletions of the individual genes encoding the new Elongator subunits showed that only the ELP5 gene is essential for growth. Disruption of the two nonessential new Elongator-encoding genes, ELP4 and ELP6, caused the same phenotypes observed with knockouts of the original Elongator-encoding genes. Results of microarray analyses demonstrated that the gene expression profiles of strains containing deletions of genes encoding subunits of either Elongator subcomplex, in which we detected significantly altered mRNA expression levels for 96 genes, are very similar, implying that all the Elongator subunits likely function together to regulate a group of S. cerevisiae genes in vivo.     

RNA polymerase II elongator holoenzyme is composed of two discrete subcomplexes.

Elongator is a histone acetyltransferase complex that associates with the elongating form of RNA polymerase II. We purified Elongator to virtual homogeneity via a rapid three-step procedure based largely on affinity chromatography. The purified factor, holo-Elongator, is a labile six-subunit factor composed of two discrete subcomplexes: one comprised of the previously identified Elp1, Elp2, and Elp3 proteins and another comprised of three novel polypeptides, termed Elp4, Elp5, and Elp6. Disruption of the yeast genes encoding the new Elongator proteins confers phenotypes indistinguishable from those previously described for the other elp mutants, and concomitant disruption of genes encoding proteins in either subcomplex does not confer new phenotypes. Taken together, our results indicate that holo-Elongator is a functional entity in vitro as well as in vivo. Metazoan homologues of Elp1 and Elp3 have previously been reported. We cloned the human homologue of yeast ELP4 and show that this gene is ubiquitously expressed in human tissues.