Modulation of TFIIH-associated kinase activity by complex formation and its relationship with CTD phosphorylation of RNA polymerase II.
BACKGROUND
The general transcription factor TFIIH plays important roles in initiation and the transition to elongation steps of transcription by RNA polymerase II (PolII). Both roles are dependent on the protein kinase, DNA-dependent ATPase and DNA helicase activities of TFIIH. However, how these enzyme activities of TFIIH contribute to transcription has remained elusive. TFIIH consists of nine subunits, and one of them, Cdk7, possesses kinase activity. Here the substrate specificities of TFIIH and two forms of the Cdk7-containing kinase complex are compared, and the relationship between transcription activity and the TFIIH-dependent phosphorylation of the carboxy terminal domain of the largest subunit of PolII (CTD) is studied.


RESULTS
We prepared TFIIH and two Cdk7-containing kinase complexes, Cdk7/Cyclin H and CAK (Cdk7/Cyclin H/MAT1). Consistent with previous reports, CAK strongly phosphorylated Cdk2, Cdk4, CTD and intact PolII. In contrast, Cdk7/Cyclin H, which lacks MAT1, did not phosphorylate these substrates, except for weak phosphorylation of Cdk2. The kinase activity of TFIIH displayed stronger substrate preference for Cdk4 than did CAK. In addition, TFIIH phosphorylation of PolII was stimulated by TFIIE both in solution and during preinitiation complex formation, whereas Cdk7/Cyclin H and CAK phosphorylation of PolII was not. In combination with other general transcription factors, TFIIH, but not Cdk7/CycH or CAK, promoted transcription on a linear DNA template. This transcription was well correlated with TFIIE stimulated TFIIH phosphorylation of serine at position 5 (Ser-5) within the heptapeptide repeat of the PolII CTD.


CONCLUSIONS
These results provide clues about the roles of CTD phosphorylation at Ser-5 in transcription.
Watanabe
Y
Y
Institute for Molecular and Cellular Biology and; The Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
Fujimoto
H
H
Watanabe
T
T
Maekawa
T
T
Masutani
C
C
Hanaoka
F
F
Ohkuma
Y
Y
D000818:Animals; D002478:Cells, Cultured; D018844:Cyclin-Dependent Kinases; D006801:Humans; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism*; D015398:Signal Transduction; D051758:Transcription Factor TFIIH; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D014157:Transcription Factors; D019473:Transcription Factors, TFII; D051758:Transcription Factor TFIIH; D011494:Protein Kinases; C060137:carboxy-terminal domain kinase; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D012319:RNA Polymerase II; D000095043:Cyclin-Dependent Kinase-Activating Kinase; C000722416:CDK7 protein, human