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Binding to DNA of the RNA-polymerase II C-terminal domain allows discrimination between Cdk7 and Cdk9 phosphorylation.

Lolli G - Nucleic Acids Res. (2009)

Bottom Line: Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex.CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation.The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. graziano@biop.ox.ac.uk

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II regulates transcription through spatially and temporally coordinated events. Previous work had established that the CTD binds DNA but the significance of this interaction has not been determined. The present work shows that the CTD binds DNA in its unphosphorylated form, the form in which it is present in the pre-initiation complex. The CTD/DNA complex is recognized by and is phosphorylated by Cdk7 but not by Cdk9. Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex. The model has been tested by mutagenesis experiments. CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation. The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation.

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Cdk9 activity on CTD and pSer5-CTD. (A) CTD and pSer5-CTD were loaded on SDS–PAGE. Phosphorylation by Cdk7/CycH shifts the CTD band upwards. (B) Kinase assay. Cdk9/CycT1 phosphorylates equally well CTD and pSer5-CTD (generated by previous incubation with Cdk7/CycH). No further incorporation of 32P could be detected incubating pSer5-CTD with Cdk7/CycH indicating that the previous reaction went to completeness.
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Figure 5: Cdk9 activity on CTD and pSer5-CTD. (A) CTD and pSer5-CTD were loaded on SDS–PAGE. Phosphorylation by Cdk7/CycH shifts the CTD band upwards. (B) Kinase assay. Cdk9/CycT1 phosphorylates equally well CTD and pSer5-CTD (generated by previous incubation with Cdk7/CycH). No further incorporation of 32P could be detected incubating pSer5-CTD with Cdk7/CycH indicating that the previous reaction went to completeness.

Mentions: Cdk9 phosphorylates CTD Ser2 following CTD Ser5 phosphorylation by Cdk7. It is tempting to speculate that a docking site for CTD pSer5 on the Cdk9 surface would help Cdk9 in selecting such substrate. I therefore tested if prior phosphorylation of the CTD by Cdk7 made it a better substrate for Cdk9. Comparison of Cdk9 activity on unphosphorylated CTD and pSer5-CTD (obtained by previous incubation with Cdk7) did not show significant differences (Figure 5). The effect of phosphorylation by Cdk7 is not to make the CTD a better substrate for Cdk9. CTD binding and release from DNA and initiation transcription factors could be the event governing the right timing of Cdk7 and Cdk9 action on CTD.Figure 5.


Binding to DNA of the RNA-polymerase II C-terminal domain allows discrimination between Cdk7 and Cdk9 phosphorylation.

Lolli G - Nucleic Acids Res. (2009)

Cdk9 activity on CTD and pSer5-CTD. (A) CTD and pSer5-CTD were loaded on SDS–PAGE. Phosphorylation by Cdk7/CycH shifts the CTD band upwards. (B) Kinase assay. Cdk9/CycT1 phosphorylates equally well CTD and pSer5-CTD (generated by previous incubation with Cdk7/CycH). No further incorporation of 32P could be detected incubating pSer5-CTD with Cdk7/CycH indicating that the previous reaction went to completeness.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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Figure 5: Cdk9 activity on CTD and pSer5-CTD. (A) CTD and pSer5-CTD were loaded on SDS–PAGE. Phosphorylation by Cdk7/CycH shifts the CTD band upwards. (B) Kinase assay. Cdk9/CycT1 phosphorylates equally well CTD and pSer5-CTD (generated by previous incubation with Cdk7/CycH). No further incorporation of 32P could be detected incubating pSer5-CTD with Cdk7/CycH indicating that the previous reaction went to completeness.
Mentions: Cdk9 phosphorylates CTD Ser2 following CTD Ser5 phosphorylation by Cdk7. It is tempting to speculate that a docking site for CTD pSer5 on the Cdk9 surface would help Cdk9 in selecting such substrate. I therefore tested if prior phosphorylation of the CTD by Cdk7 made it a better substrate for Cdk9. Comparison of Cdk9 activity on unphosphorylated CTD and pSer5-CTD (obtained by previous incubation with Cdk7) did not show significant differences (Figure 5). The effect of phosphorylation by Cdk7 is not to make the CTD a better substrate for Cdk9. CTD binding and release from DNA and initiation transcription factors could be the event governing the right timing of Cdk7 and Cdk9 action on CTD.Figure 5.

Bottom Line: Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex.CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation.The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. graziano@biop.ox.ac.uk

ABSTRACT
The C-terminal domain (CTD) of RNA polymerase II regulates transcription through spatially and temporally coordinated events. Previous work had established that the CTD binds DNA but the significance of this interaction has not been determined. The present work shows that the CTD binds DNA in its unphosphorylated form, the form in which it is present in the pre-initiation complex. The CTD/DNA complex is recognized by and is phosphorylated by Cdk7 but not by Cdk9. Model-building studies indicate the structural mechanism underlying such specificity involves interaction of Cdk7 with DNA in the context of the CTD/DNA complex. The model has been tested by mutagenesis experiments. CTD dissociates from DNA following phosphorylation by Cdk7, allowing transcription initiation. The CTD then becomes accessible for further phosphorylation by Cdk9 that drives the transition to transcription elongation.

Show MeSH