Interaction of human Spt4 Spt5 with transcription machinery

蛋白质编码基因的转录是由RNA聚合酶II(RNAPII)和通用转录因子(GTF)TFIIB、TFIID、TFIIE、TFIIF和TFIIH进行的(Orphanides等人，1996)。转录过程可以机械地分为不同的步骤。GTF与启动子的结合导致RNAPII的募集和转录的启动。接下来是启动子清除、延伸，最后是转录终止。Spt4与Spt5形成蛋白质复合体，调节体内的转录延伸(Hartzog等人，1996,1998)。最近，我们的生化分析发现，人类细胞中含有与Spt4/Spt5复合体对应的转录因子。我们报告了染色质特异的转录延伸因子FACT与RNA聚合酶II CTD激酶P-TEFb一起发挥作用，以缓解DRB敏感性诱导因子(DRB敏感性诱导因子)和负延伸因子(NELF)对转录的抑制。我们发现TFIIH的激酶活性对于这种活性是不必要的，这表明TFIIH介导的CTD磷酸化不参与FACT和DSIF/NELF活性的调节。因此，我们提出了一个新的转录调控网络，在该网络中，P-TEFb与FACT协同作用阻止了DSIF/NELF对转录的抑制。这项研究揭示了一个新的作用，即在裸露DNA上的转录调控中，它独立于其对染色质模板的活性。此外，本研究还揭示了P-TEFb和TFIIH在转录调控方面的功能差异。

Transcription of protein coding genes is carried out by RNA polymerase II (RNAPII) and the general transcription factors (GTFs) TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (Orphanides et al., 1996). The transcription process can be divided mechanistically into different steps. The binding of the GTFs to a promoter results in the recruitment of RNAPII and the initiation of transcription. This is followed by promoter clearance, elongation, and, finally, transcription termination. Spt4 forms a protein complex with Spt5 to regulate transcription elongation in vivo (Hartzog et al., 1996, 1998). Recently, our biochemical analysis revealed that human cells contain a counterpart to the Spt4/Spt5 complex.We report that the chromatin-specific transcription elongation factor FACT functions in conjunction with the RNA polymerase II CTD kinase P-TEFb to alleviate transcription inhibition by DSIF (DRB sensitivity-inducing factor) and NELF (negative elongation factor). We find that the kinase activity of TFIIH is dispensable for this activity, demonstrating that TFIIH-mediated CTD phosphorylation is not involved in the regulation of FACT and DSIF/NELF activities. Thus, we propose a novel transcriptional regulatory network in which DSIF/NELF inhibition of transcription is prevented by P-TEFb in cooperation with FACT.This study uncovers a novel role for FACT in the regulation of transcription on naked DNA that is independent of its activities on chromatin templates. In addition, this study reveals functional differences between P-TEFb and TFIIH in the regulation of transcription.