P-TEFb-Mediated Phosphorylation of hSpt5 C-Terminal Repeats Is Critical for Processive Transcription Elongation

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan.
Molecular Cell (Impact Factor: 14.02). 02/2006; 21(2):227-37. DOI: 10.1016/j.molcel.2005.11.024
Source: PubMed


Human DSIF, a heterodimer composed of hSpt4 and hSpt5, plays opposing roles in transcription elongation by RNA polymerase II (RNA Pol II). Here, we describe an evolutionarily conserved repetitive heptapeptide motif (consensus = G-S-R/Q-T-P) in the C-terminal region (CTR) of hSpt5, which, like the C-terminal domain (CTD) of RNA Pol II, is highly phosphorylated by P-TEFb. Thr-4 residues of the CTR repeats are functionally important phosphorylation sites. In vitro, Thr-4 phosphorylation is critical for the elongation activation activity of DSIF, but not to its elongation repression activity. In vivo, Thr-4 phosphorylation is critical for epidermal growth factor (EGF)-inducible transcription of c-fos and for efficient progression of RNA Pol II along the gene. We consider this phosphorylation to be a switch that converts DSIF from a repressor to an activator. We propose the "mini-CTD" hypothesis, in which phosphorylated CTR is thought to function in a manner analogous to phosphorylated CTD, serving as an additional code for active elongation complexes.

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    • "The exact molecular mechanism underlying this switch remains to be determined, but, in vitro, Cdk9 phosphorylates both DSIF and NELF (Kim and Sharp 2001; Fujinaga et al. 2004) in addition to the Ser2, Thr4, Ser5, and Ser7 residues of the Pol II CTD (Ramanathan et al. 2001; Glover-Cutter et al. 2009; Hsin et al. 2011). Phosphorylation by Cdk9 of the Spt5 C-terminal repeat (CTR) regions potentiates the elongation stimulatory activity of DSIF in vitro (Yamada et al. 2006). P-TEFb has also been reported to modify transcriptional activators and coactivators (Alarcon et al. 2009; Dobrovolna et al. 2012) and a component of a histone modification pathway (Shchebet et al. 2012). "
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    ABSTRACT: The transcription cycle of RNA polymerase II (Pol II) is regulated at discrete transition points by cyclin-dependent kinases (CDKs). Positive transcription elongation factor b (P-TEFb), a complex of Cdk9 and cyclin T1, promotes release of paused Pol II into elongation, but the precise mechanisms and targets of Cdk9 action remain largely unknown. Here, by a chemical genetic strategy, we identified ∼ 100 putative substrates of human P-TEFb, which were enriched for proteins implicated in transcription and RNA catabolism. Among the RNA processing factors phosphorylated by Cdk9 was the 5 ′ -to-3 ′ “ torpedo ” exoribonuclease Xrn2, required in transcription termination by Pol II, which we validated as a bona fide P-TEFb substrate in vivo and in vitro. Phosphorylation by Cdk9 or phos- phomimetic substitution of its target residue, Thr439, enhanced enzymatic activity of Xrn2 on synthetic substrates in vitro. Conversely, inhibition or depletion of Cdk9 or mutation of Xrn2-Thr439 to a nonphosphorylatable Ala residue caused phenotypes consistent with inefficient termination in human cells: impaired Xrn2 chromatin localization and increased readthrough transcription of endogenous genes. Therefore, in addition to its role in elongation, P-TEFb regulates termination by promoting chromatin recruitment and activation of a cotranscriptional RNA processing enzyme, Xrn2.
    Preview · Article · Jan 2016 · Genes & development
    • "However, NusG functions as a monomer in bacteria as the Spt4 homologue does not exist in this domain of life (Ponting, 2002). NusG and Spt4:Spt5 are widely accepted as elongation factors that stimulate transcription elongation and RNAP processivity (Kwak et al., 2003; Mooney et al., 2009; Yamada et al., 2006; Zhu et al., 2007); however, these proteins have also been reported to suppress transcription elongation under certain conditions (Core and Lis, 2008; Margaritis and Holstege, 2008; Sevostyanova et al., 1047-8477/Ó 2015 Elsevier Inc. All rights reserved. "
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    ABSTRACT: Spt5 (NusG in bacteria) is the only RNA polymerase-associated factor known to be conserved in all three domains of life. In archaea and eukaryotes, Spt5 associates with Spt4, an elongation factor that is absent in bacteria, to form a functional heterodimeric complex. Previous studies suggest that the Spt4:Spt5 complex interacts directly with DNA at the double-stranded DNA exit tunnel of RNA polymerase to regulate gene transcription. In this study, the DNA-binding ability of Spt4:Spt5 from the archaeon Methanocaldococcus jannaschii was confirmed via nuclear magnetic resonance chemical shift perturbation and fluorescence polarization assays. Crystallographic analysis of the full-length MjSpt4:Spt5 revealed two distinct conformations of the C-terminal KOW domain of Spt5. A similar alkaline region was found on the Spt4:Spt5 surface in both crystal forms, and identified as double-stranded DNA binding patch through mutagenesis-fluorescence polarization assays. Based on these structural and biochemical data, the Spt4:Spt5-DNA binding model was built for the first time.
    No preview · Article · Oct 2015 · Journal of Structural Biology
    • "DSIF, which consists of Spt4 and Spt5, promotes pausing in conjunction with NELF and also serves as a factor required for increasing the catalytic rate of transcription elongation by Pol II. P-TEFb is a kinase that phosphorylates the C-terminal domain (CTD) of RPB1, the largest subunit of Pol II, the C-terminal domain of SPT5, and the NELF-E subunit to promote the transition to transcription elongation (Fujinaga et al., 2004; Marshall et al., 1996; Yamada et al., 2006). The RPB1 CTD contains a heptad repeat (consensus Tyr1- Ser2-Pro3-Thr4-Ser5-Pro6-Ser7) that is subject to extensive post-translational modifications, most notably phosphorylation at Ser2 and Ser5 residues (Smith and Shilatifard, 2013). "
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    ABSTRACT: The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II's C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Aug 2015 · Cell
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