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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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Primary and tertiary structure comparison for Cdk7, Cdk2 and Cdk9. (A) Sequence alignment. Cdk7 amino acids contacting the CTD/DNA complex (distance <3.5 Å) are shown in red (polar contacts) and orange (van der Waals contacts). The model of CTD/DNA recognition by Cdk7 was tested using Cdk2 mutants. Mutated residues are shown in green in the Cdk2 sequence. (B) Electrostatic surface representation. Black circles delimitate β3αC loops of the three kinases. Differently from Cdk2 and Cdk9, the Cdk7 β3αC loop is positively charged.
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Figure 3: Primary and tertiary structure comparison for Cdk7, Cdk2 and Cdk9. (A) Sequence alignment. Cdk7 amino acids contacting the CTD/DNA complex (distance <3.5 Å) are shown in red (polar contacts) and orange (van der Waals contacts). The model of CTD/DNA recognition by Cdk7 was tested using Cdk2 mutants. Mutated residues are shown in green in the Cdk2 sequence. (B) Electrostatic surface representation. Black circles delimitate β3αC loops of the three kinases. Differently from Cdk2 and Cdk9, the Cdk7 β3αC loop is positively charged.

Mentions: The CTD/DNA complex inserts between the two kinase lobes, both of which contribute to the binding via van der Waals and polar interactions (Figure 2B). The catalytic dyad Asp137-Lys139, responsible for the phospho-transfer from ATP to the substrate, is conserved in all Cdks as well as Gly21 that interacts with CTD via its peptide backbone. Other interactions involve Gln172 and four residues of the β3-αC loop that is longer and more basic in Cdk7 than in the other Cdks (residues 45–57 in Cdk7; Figures 2B and 3). The interaction between the β3-αC loop and DNA spans four bases of the same DNA strand (Table 2). It is largely responsible for the calculated energy of interaction between Cdk7 and the CTD/DNA complex and gives the correct orientation to the CTD/DNA complex to accommodate Ser5 inside Cdk7 active site. These interactions could increase Cdk7 affinity for CTD/DNA selecting it as a better substrate rather than the CTD alone.Figure 3.


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

Lolli G - Nucleic Acids Res. (2009)

Primary and tertiary structure comparison for Cdk7, Cdk2 and Cdk9. (A) Sequence alignment. Cdk7 amino acids contacting the CTD/DNA complex (distance <3.5 Å) are shown in red (polar contacts) and orange (van der Waals contacts). The model of CTD/DNA recognition by Cdk7 was tested using Cdk2 mutants. Mutated residues are shown in green in the Cdk2 sequence. (B) Electrostatic surface representation. Black circles delimitate β3αC loops of the three kinases. Differently from Cdk2 and Cdk9, the Cdk7 β3αC loop is positively charged.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
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Figure 3: Primary and tertiary structure comparison for Cdk7, Cdk2 and Cdk9. (A) Sequence alignment. Cdk7 amino acids contacting the CTD/DNA complex (distance <3.5 Å) are shown in red (polar contacts) and orange (van der Waals contacts). The model of CTD/DNA recognition by Cdk7 was tested using Cdk2 mutants. Mutated residues are shown in green in the Cdk2 sequence. (B) Electrostatic surface representation. Black circles delimitate β3αC loops of the three kinases. Differently from Cdk2 and Cdk9, the Cdk7 β3αC loop is positively charged.
Mentions: The CTD/DNA complex inserts between the two kinase lobes, both of which contribute to the binding via van der Waals and polar interactions (Figure 2B). The catalytic dyad Asp137-Lys139, responsible for the phospho-transfer from ATP to the substrate, is conserved in all Cdks as well as Gly21 that interacts with CTD via its peptide backbone. Other interactions involve Gln172 and four residues of the β3-αC loop that is longer and more basic in Cdk7 than in the other Cdks (residues 45–57 in Cdk7; Figures 2B and 3). The interaction between the β3-αC loop and DNA spans four bases of the same DNA strand (Table 2). It is largely responsible for the calculated energy of interaction between Cdk7 and the CTD/DNA complex and gives the correct orientation to the CTD/DNA complex to accommodate Ser5 inside Cdk7 active site. These interactions could increase Cdk7 affinity for CTD/DNA selecting it as a better substrate rather than the CTD alone.Figure 3.

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