Related Experiment Video
Updated: Aug 30, 2026
Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells
Published on: May 1, 2020
Phosphorylated positive transcription elongation factor b (P-TEFb) is tagged for inhibition through association with
Ruichuan Chen1, Zhiyuan Yang, Qiang Zhou
1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
Abstract:
The positive transcription elongation factor b (P-TEFb), comprising CDK9 and cyclin T, stimulates transcription of cellular and viral genes by phosphorylating RNA polymerase II. A major portion of nuclear P-TEFb is sequestered and inactivated by the coordinated actions of the 7SK snRNA and the HEXIM1 protein, whose induced dissociation from P-TEFb is crucial for stress-induced transcription and pathogenesis of cardiac hypertrophy. The 7SK.P-TEFb interaction, which can occur independently of HEXIM1 and does not by itself inhibit P-TEFb, recruits HEXIM1 for P-TEFb inactivation. To study the control of this interaction, we established an in vitro system that reconstituted the specific interaction of P-TEFb with 7SK but not other snRNAs. Using this system, together with an in vivo binding assay, we show that the phosphorylation of CDK9, on possibly the conserved Thr-186 in the T-loop, was crucial for the 7SK.P-TEFb interaction. This phosphorylation was not caused by CDK9 autophosphorylation or the general CDK-activating kinase CAK, but rather by a novel HeLa nuclear kinase. Furthermore, the stress-induced disruption of the 7SK.P-TEFb interaction was not caused by any prohibitive changes in 7SK but by the dephosphorylation of P-TEFb, leading to the loss of the key phosphorylation important for 7SK binding. Thus, the phosphorylated P-TEFb is tagged for inhibition through association with 7SK. We discuss the implications of this mechanism in controlling P-TEFb activity during normal and stress-induced transcription.
Insights
Phosphorylation of CDK9 by a novel kinase is essential for the positive transcription elongation factor b (P-TEFb) to bind 7SK snRNA, inhibiting transcription. Stress disrupts this interaction via P-TEFb dephosphorylation.
Area of Science:
- Molecular Biology
- Gene Regulation
- Biochemistry
Background:
- Positive transcription elongation factor b (P-TEFb), composed of CDK9 and cyclin T, is vital for cellular and viral gene transcription through RNA polymerase II phosphorylation.
- Nuclear P-TEFb is largely inactivated by 7SK snRNA and HEXIM1 protein, a complex dissociation critical for stress responses and cardiac hypertrophy pathogenesis.
- The 7SK.P-TEFb interaction, independent of HEXIM1, recruits HEXIM1 for P-TEFb inactivation, highlighting its regulatory role.
Purpose of the Study:
- To investigate the regulatory mechanisms controlling the interaction between P-TEFb and 7SK snRNA.
- To identify the specific phosphorylation events and kinases involved in P-TEFb interaction with 7SK snRNA.
- To elucidate the role of P-TEFb phosphorylation and dephosphorylation in stress-induced transcriptional regulation.
Main Methods:
- Development of an in vitro system to reconstitute the specific interaction between P-TEFb and 7SK snRNA.
- Utilization of in vivo binding assays to study P-TEFb and 7SK snRNA interactions.
- Analysis of CDK9 phosphorylation status and identification of the responsible kinase.
Main Results:
- CDK9 phosphorylation, potentially at Thr-186, is crucial for the P-TEFb.7SK snRNA interaction.
- A novel HeLa nuclear kinase, not CDK9 autophosphorylation or CAK, mediates the critical CDK9 phosphorylation.
- Stress-induced disruption of the 7SK.P-TEFb complex results from P-TEFb dephosphorylation, not changes in 7SK snRNA.
Conclusions:
- Phosphorylated P-TEFb is specifically targeted for inhibition through its association with 7SK snRNA.
- This phosphorylation-dependent interaction serves as a key regulatory mechanism for controlling P-TEFb activity.
- The findings provide insights into the control of P-TEFb activity during both normal and stress-induced transcription, with implications for pathogenesis.
Related Concept Videos
02:35Transcription Elongation Factors
The transcription elongation is regulated via pausing of RNA polymerase on several occasions during transcription. In bacteria, these halts are necessary because the transcription of DNA into mRNA is coupled to the translation of that mRNA into a...
02:42Transcription Attenuation in Prokaryotes
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
02:24Ribosome Profiling
Applications of ribosome profiling
Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
The technique helps...