RESUMEN
The Cdk7 subunit of TFIIH phosphorylates RNA polymerase II (Pol II) during initiation, and, while recent studies show that inhibition of human Cdk7 negatively influences transcription, the mechanisms involved are unclear. Using in vitro transcription with nuclear extract, we demonstrate that THZ1, a covalent Cdk7 inhibitor, causes defects in Pol II phosphorylation, co-transcriptional capping, promoter proximal pausing, and productive elongation. THZ1 does not affect initiation but blocks essentially all Pol II large subunit C-terminal domain (CTD) phosphorylation. We found that guanylylation of nascent RNAs is length dependent and modulated by a THZ1-sensitive factor present in nuclear extract. THZ1 impacts pausing through a capping-independent block of DSIF and NELF loading. The P-TEFb-dependent transition into productive elongation was also inhibited by THZ1, likely due to loss of DSIF. Capping and pausing were also reduced in THZ1-treated cells. Our results provide mechanistic insights into THZ1 action and how Cdk7 broadly influences transcription and capping.
Asunto(s)
Antineoplásicos/química , Quinasas Ciclina-Dependientes/química , Fenilendiaminas/química , Pirimidinas/química , Iniciación de la Transcripción Genética , Antineoplásicos/farmacología , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Quinasas Ciclina-Dependientes/metabolismo , Células HeLa , Humanos , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fenilendiaminas/farmacología , Fosforilación , Regiones Promotoras Genéticas , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Pirimidinas/farmacología , ARN Polimerasa II/química , Procesamiento Postranscripcional del ARN , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/química , Factores de Elongación Transcripcional/metabolismo , Quinasa Activadora de Quinasas Ciclina-DependientesRESUMEN
Gdown1 is a substoichiometric subunit of RNA polymerase II (Pol II) that has been recently demonstrated to be involved in stabilizing promoter-proximal paused Pol II. It was shown to inhibit termination of Pol II by transcription termination factor 2 (TTF2) as well as block elongation stimulation by transcription factor IIF (TFIIF). Here, using in vitro transcription assays, we identified two functional domains in Gdown1. Although both are required to maintain a tight association with Pol II, the N- and C-terminal domains are responsible for blocking TTF2 and TFIIF, respectively. A highly conserved LPDKG motif found in the N-terminal domain of Gdown1 is also highly conserved in TTF2. Deletion of this motif eliminated the TTF2 inhibitory activity of Gdown1. We identified a phosphorylated form of Gdown1 with altered mobility in SDS-PAGE that appears during mitosis. A kinase in HeLa nuclear extract that caused the shift was partially purified. In vitro, Gdown1 phosphorylated by this kinase demonstrated reduced activity in blocking both TTF2 and TFIIF because of its reduced affinity for Pol II. Mass spectrometry identified Ser-270 as the site of this phosphorylation. An S270A mutation was not phosphorylated by the partially purified kinase, and an S270E mutation partially mimicked the properties of phospho-Gdown1. Gdown1 Ser-270 phosphorylation occurs predominately during mitosis, and we suggest that this would enable TTF2 to terminate all Pol II even if it is associated with Gdown1.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , ARN Polimerasa II/metabolismo , Serina/metabolismo , Factores de Transcripción/metabolismo , Terminación de la Transcripción Genética , Adenosina Trifosfatasas/química , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Unión Competitiva , Western Blotting , Proteínas de Unión al ADN/química , Células HeLa , Humanos , Espectrometría de Masas , Mitosis/genética , Datos de Secuencia Molecular , Mutación , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , ARN Polimerasa II/química , ARN Polimerasa II/genética , Homología de Secuencia de Aminoácido , Serina/química , Serina/genética , Factores de Transcripción/química , Factores de Transcripción TFII/química , Factores de Transcripción TFII/metabolismoRESUMEN
Phosphorylation of the C-terminal domain (CTD) of the large subunit of human RNA polymerase II (Pol II) is regulated during the transcription cycle by the combined action of specific kinases and phosphatases. Pol II enters into the preinitiation complex (PIC) unphosphorylated, but is quickly phosphorylated by Cdk7 during initiation. How phosphatases alter the pattern and extent of CTD phosphorylation at this early stage of transcription is not clear. We previously demonstrated the functional association of an early-acting, magnesium-independent phosphatase with early elongation complexes. Here we show that Ssu72 is responsible for that activity. We found that the phosphatase enters the transcription cycle during the formation of PICs and that Ssu72 is physically associated with very early elongation complexes. The association of Ssu72 with elongation complexes was stable to extensive washing with up to 200 mM KCl. Interestingly, Ssu72 ceased to function on complexes that contained RNA longer than 28 nt. However, when PICs were washed before initiation, the strict cutoff at 28 nt was lost. This suggests that factor(s) are important for the specific regulation of Ssu72 function during the transition between initiation and pausing. Overall, our results demonstrate when Ssu72 can act on early transcription complexes and suggest that Ssu72 may also function in the PIC prior to initiation.