Interaction of Fcp1 Phosphatase with Elongating RNA Polymerase II Holoenzyme, Enzymatic Mechanism of Action, and Genetic Interaction with Elongator

Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNAPII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated a ternary the Fcp1 the of Fcp1 in vitro, but a and Fcp1 in vivo. and a of transcription results that the transcription and a for Fcp1 in the of RNAPII Fcp1 de-phosphorylates the RNA polymerase II (RNAPII) C-terminal domain (CTD) in vitro, and mutation of the yeast FCP1 gene results in global transcription defects and increased CTD phosphorylation levels in vivo. Here we show that the Fcp1 protein associates with elongating RNAPII holoenzyme in vitro. Our data suggest that the association of Fcp1 with elongating polymerase results in CTD de-phosphorylation when the native ternary RNAPII0-DNA-RNA complex is disrupted. Surprisingly, highly purified yeast Fcp1 dephosphorylates serine 5 but not serine 2 of the RNAPII CTD repeat. Only free RNAPII0(Ser-5) and not RNAPII0-DNA-RNA ternary complexes act as a good substrate in the Fcp1 CTD de-phosphorylation reaction. In contrast, TFIIH CTD kinase has a pronounced preference for RNAPII incorporated a ternary the Fcp1 the of Fcp1 in vitro, but a and Fcp1 in vivo. and a of transcription results that the transcription and a for Fcp1 in the of RNAPII the RNA polymerase II (RNAPII) RNA polymerase C-terminal RNA polymerase C-terminal transcription the phosphorylation of the polymerase and the that RNAPII with and RNAPII in a with transcription as and In the of and RNAPII with and with that with elongating RNAPII RNA and a in as and C-terminal domain (CTD) of RNAPII is the for that the polymerase the CTD of the in yeast and in the CTD is serine 2 and serine 5 protein and and CTD the of RNAPII for a of transcription CTD the RNAPII CTD in gene a protein and the de-phosphorylates RNAPII in in a that is and Fcp1 is that is not Fcp1 transcription of the of and and in the of Fcp1 has the RNAPII in of that associates with and elongating In with that Fcp1 associates with the and of in that CTD phosphorylation and the association of as the complex as a of the of RNAPII in yeast of the complex highly yeast and the complex associates with complexes in and in transcription in the and of for the the of yeast and the yeast results that associates with the of yeast in and in that the gene of the complex and phosphorylation of the RNAPII CTD results the that is the polymerase and with the of the in elongating RNAPII the of CTD de-phosphorylation not the of we the that Fcp1 Here we that Fcp1 associates with the elongating RNAPII that holoenzyme and Fcp1 in vivo. show that TFIIH kinase RNAPII in a ternary complex as Fcp1 free serine RNAPII and that CTD de-phosphorylation a the CTD results the that the transcription and RNAPII in and and of elongating of and the in of elongating RNAPII holoenzyme of TFIIH Fcp1 and as purified the purified as TFIIH kinase with of RNAPII in 5 of in a for with TFIIH the kinase with and of in de-phosphorylation of the kinase and the for of the in a and a with of complexes with polymerase a in the of and a polymerase transcription in the of transcription of the with of in 5 5 of of for in a the transcription as substrate for Fcp1 TFIIH and the transcription the ternary complexes free polymerase the of of TFIIH as as free the as the polymerase phosphorylation as as the of with of as ternary as a substrate in 5 for in a of the of and for a and a with for with and and as of in FCP1 a and in that the as of the results that and in Fcp1 for of RNAPII of RNAPII yeast we de-phosphorylation of the of a of the of RNAPII de-phosphorylation phosphorylation of RNAPII in yeast for In contrast, that ternary as and of CTD de-phosphorylation and the that the RNAPII CTD of the ternary complex and a for in RNAPII the of RNAPII de-phosphorylation in we that protein as and the not of the of Fcp1 is in the of protein the CTD and results in a protein that has CTD in CTD de-phosphorylation and In the CTD de-phosphorylation and but in de-phosphorylation of the of de-phosphorylation not the as transcription in in and the in not results suggest that Fcp1 is for the de-phosphorylation of in of Fcp1 in a yeast is in RNAPII with and the with for Fcp1 protein with RNAPII as as with RNAPII Fcp1 in the for the protein not with elongating in results with the that Fcp1 with elongating RNAPII holoenzyme in and in the with and and for and for the of and that the of with is of the of and RNA in the with CTD de-phosphorylation in a we purified and Fcp1 and a of RNAPII CTD the substrate of yeast Fcp1 in the with RNAPII that TFIIH phosphorylation is serine 5 of the CTD data not phosphorylation serine 2 but a serine 5 data not Fcp1 RNAPII serine phosphorylation results as the that yeast Fcp1 serine 2 phosphorylation but not for the of serine 5 phosphorylation in in the FCP1 serine 5 is for yeast phosphorylation of serine 2 is a of Fcp1 de-phosphorylation of serine a of the results of we RNAPII the substrate preference and of of Fcp1 in the de-phosphorylates the CTD serine 5 in vitro. of TFIIH with of Fcp1 as RNAPII is in and of and is for the de-phosphorylation with a in transcription with purified with RNAPII that TFIIH in the of the a of the for and RNAPII the of the in a in transcription in complexes free as as RNAPII the of in RNAPII in transcription is in the of for for the substrate of RNAPII and RNAPII in a ternary complex in purified RNAPII for Fcp1 and TFIIH of transcription the of purified RNAPII with a of in the of transcription of substrate and is of RNA polymerase the transcription TFIIH in the of the of the in 2 that is the and of the in a purified ternary in the and of the phosphorylation in TFIIH and not in highly purified TFIIH in a ternary complex is de-phosphorylation the RNAPII in the of the in the de-phosphorylation the of with of Only the of the is of RNAPII with free RNAPII in the of the but results when as as not transcription not of RNAPII with of Fcp1 de-phosphorylation and with of RNAPII and of Fcp1 of the Fcp1 of the RNAPII in with of RNAPII as the and the in with Fcp1 for the of the in the de-phosphorylation we that free a substrate for de-phosphorylation elongating of free when with of de-phosphorylation with a of ternary results with transcription not that results not the of the the of a of the as Fcp1 with the of RNAPII phosphorylation when the of the the de-phosphorylation with the of that the with purified in the of free the that the protein phosphorylation a that de-phosphorylation of the CTD the Fcp1 results the of Fcp1 free elongating as a elongating a substrate free for the kinase of TFIIH data suggest that the substrate of TFIIH kinase and Fcp1 the transcription RNAPII phosphorylation is the polymerase in a ternary complex as a of CTD de-phosphorylation of serine 5 is the of ternary complexes the of phosphorylation of the CTD is the ternary the RNAPII in the kinase of the substrate with and of TFIIH for of the of RNAPII that polymerase in the free in the elongating in the of the of TFIIH kinase the ternary FCP1 and data in 2 suggest that Fcp1 and with the elongating of the that the of of purified Fcp1 de-phosphorylation in not a Fcp1 and in vitro, we FCP1 and FCP1 is we the in FCP1 a as as FCP1 a of the in the of the for the the of Fcp1 a results a Fcp1 and in FCP1 and and a as as a the FCP1 in the in and and and for the of the of we a for of in a of a gene a protein in of transcription of and a of and the of of the of the the of in of and of of the complex with of the and as as with and the of gene of in defects when with of of the complex and as as with of the complex and and a complex is for the of and defects with complexes in of the and and of the complex and of when with of of complexes in for the of and is the that with a and the and of of the and and with a in transcription and of of that a and that with that not of the but of the with a in RNAPII of elongating RNAPII holoenzyme for of in ternary complexes with yeast is in the of the the of results of the the that Fcp1 associates with elongating and that the serine 5 of the polymerase CTD a substrate for the of Fcp1 of the ternary associates with elongating and data show that FCP1 and and with and RNA a the of but not ternary RNAPII0-DNA-RNA complexes data suggest that Fcp1 with RNAPII for the that Fcp1 with elongating RNAPII in complex of the association of Fcp1 with elongating and that RNAPII when ternary we a with purified RNAPII de-phosphorylation in vitro. we that Fcp1 serine 5 but not serine 2 of the RNAPII that purified Fcp1 has with yeast FCP1 the serine 2 phosphorylation has serine 5 phosphorylation levels in in in with the serine 5 data the data show that Fcp1 de-phosphorylates free we that yeast Fcp1 has a preference for free elongating in with highly purified is pronounced for the CTD the of Fcp1 with ternary complexes is not the CTD for in as TFIIH kinase the substrate a pronounced preference for ternary complexes free substrate preference of TFIIH has not results that the transcription TFIIH RNAPII when is incorporated in a ternary complex and Fcp1 RNAPII serine 5 has the ternary complex in that the polymerase for a of that Fcp1 and the serine CTD that RNAPII is polymerase In with mutation Fcp1 the purified Fcp1 we that a protein the of the Fcp1 protein as RNAPII that TFIIH and free the CTD phosphorylation the and of the with the of CTD in the de-phosphorylation ternary complexes as substrate CTD de-phosphorylation the RNAPII and in the of the protein of the of and Fcp1 with the the as a when with the is the in the for the that Fcp1 a Fcp1 and the of in the of a and that the is that a of protein as the CTD in Fcp1 is that the of as the Fcp1 the not the of Fcp1 and but the of the Fcp1 of yeast in serine RNAPII in data show that and Fcp1 with elongating RNAPII In with the that and Fcp1 in a of Fcp1 the of for that the of Fcp1 and results that is for of the gene the of the CTD kinase results a for and in the of with as as with and has defects and of in we with and a of as and with the data a and RNAPII as as data that is in a and the RNA polymerase II (RNAPII) RNA polymerase C-terminal RNA polymerase C-terminal transcription the phosphorylation of the polymerase and the that RNAPII with and RNAPII in a with transcription as and In the of and RNAPII with and with that with elongating RNAPII RNA and a in as and C-terminal domain (CTD) of RNAPII is the for that the polymerase the CTD of the in yeast and in the CTD is serine 2 and serine 5 protein and and CTD the of RNAPII for a of transcription CTD the RNAPII CTD in gene a protein and the de-phosphorylates RNAPII in in a that is and Fcp1 is that is not Fcp1 transcription of the of and and in the of Fcp1 has the RNAPII in of that associates with and elongating In with that Fcp1 associates with the and of in that CTD phosphorylation and the association of as the complex as a of the of RNAPII in yeast of the complex highly yeast and the complex associates with complexes in and in transcription in the and of for the the of yeast and the yeast results that associates with the of yeast in and in that the gene of the complex and phosphorylation of the RNAPII CTD results the that is the polymerase and with the of the in elongating RNAPII the of CTD de-phosphorylation not the of we the that Fcp1 Here we that Fcp1 associates with the elongating RNAPII that holoenzyme and Fcp1 in vivo. show that TFIIH kinase RNAPII in a ternary complex as Fcp1 free serine RNAPII and that CTD de-phosphorylation a the CTD results the that the transcription and RNAPII in and and of elongating of and the in of elongating RNAPII holoenzyme of TFIIH Fcp1 and as purified the purified as TFIIH kinase with of RNAPII in 5 of in a for with TFIIH the kinase with and of in de-phosphorylation of the kinase and the for of the in a and a with of complexes with polymerase a in the of and a polymerase transcription in the of transcription of the with of in 5 5 of of for in a the transcription as substrate for Fcp1 TFIIH and the transcription the ternary complexes free polymerase the of of TFIIH as as free the as the polymerase phosphorylation as as the of with of as ternary as a substrate in 5 for in a of the of and for a and a with for with and and as of in FCP1 a and in that the as of the results that and in and RNAPII in and and of elongating of and the in of elongating RNAPII holoenzyme of TFIIH Fcp1 and as purified the TFIIH purified as TFIIH kinase with of RNAPII in 5 of in a for with TFIIH the kinase with and of in de-phosphorylation of the kinase and the for of the in a and a with of complexes with polymerase a in the of and a polymerase transcription in the of transcription of the with of in 5 5 of of for in a the transcription as substrate for Fcp1 TFIIH and the transcription the ternary complexes free polymerase the of of TFIIH as as free the as the polymerase phosphorylation Fcp1 as as the of with of as ternary as a substrate in 5 for in a of the of and for a and a with for with and and as of in FCP1 a and in that the as of the results that and in Fcp1 for of RNAPII of RNAPII yeast we de-phosphorylation of the of a of the of RNAPII de-phosphorylation phosphorylation of RNAPII in yeast for In contrast, that ternary as and of CTD de-phosphorylation and the that the RNAPII CTD of the ternary complex and a for in RNAPII the of RNAPII de-phosphorylation in we that protein as and the not of the of Fcp1 is in the of protein the CTD and results in a protein that has CTD in CTD de-phosphorylation and In the CTD de-phosphorylation and but in de-phosphorylation of the of de-phosphorylation not the as transcription in in and the in not results suggest that Fcp1 is for the de-phosphorylation of in of Fcp1 in a yeast is in RNAPII with and the with for Fcp1 protein with RNAPII as as with RNAPII Fcp1 in the for the protein not with elongating in results with the that Fcp1 with elongating RNAPII holoenzyme in with CTD de-phosphorylation in a we purified and Fcp1 and a of RNAPII CTD the substrate of yeast Fcp1 in the with RNAPII that TFIIH phosphorylation is serine 5 of the CTD data not phosphorylation serine 2 but a serine 5 data not Fcp1 RNAPII serine phosphorylation results as the that yeast Fcp1 serine 2 phosphorylation but not for the of serine 5 phosphorylation in in the FCP1 serine 5 is for yeast phosphorylation of serine 2 is a of Fcp1 de-phosphorylation of serine a of the results of we RNAPII the substrate preference and of of Fcp1 in the de-phosphorylates the CTD serine 5 in vitro. of TFIIH with of Fcp1 as RNAPII is in and of and is for the de-phosphorylation with a in transcription with purified with RNAPII that TFIIH in the of the a of the for and RNAPII the of the in a in transcription in complexes free as as RNAPII the of in RNAPII in transcription is in the of for for the substrate of RNAPII and RNAPII in a ternary complex in purified RNAPII for Fcp1 and TFIIH of transcription the of purified RNAPII with a of in the of transcription of substrate and is of RNA polymerase the transcription TFIIH in the of the of the in 2 that is the and of the in a purified ternary in the and of the phosphorylation in TFIIH and not in highly purified TFIIH in a ternary complex is de-phosphorylation the RNAPII in the of the in the de-phosphorylation the of with of Only the of the is of RNAPII with free RNAPII in the of the but results when as as not transcription not of RNAPII with of Fcp1 de-phosphorylation and with of RNAPII and of Fcp1 of the Fcp1 of the RNAPII in with of RNAPII as the and the in with Fcp1 for the of the in the de-phosphorylation we that free a substrate for de-phosphorylation elongating of free when with of de-phosphorylation with a of ternary results with transcription not that results not the of the the of a of the as Fcp1 with the of RNAPII phosphorylation when the of the the de-phosphorylation with the of that the with purified in the of free the that the protein phosphorylation a that de-phosphorylation of the CTD the Fcp1 results the of Fcp1 free elongating as a elongating a substrate free for the kinase of TFIIH data suggest that the substrate of TFIIH kinase and Fcp1 the transcription RNAPII phosphorylation is the polymerase in a ternary complex as a of CTD de-phosphorylation of serine 5 is the of ternary complexes the of phosphorylation of the CTD is the ternary the RNAPII in the kinase of the substrate with and of TFIIH for of the of RNAPII that polymerase in the free in the elongating in the of the of TFIIH kinase the ternary FCP1 and data in 2 suggest that Fcp1 and with the elongating of the that the of of purified Fcp1 de-phosphorylation in not a Fcp1 and in vitro, we FCP1 and FCP1 is we the in FCP1 a as as FCP1 a of the in the of the for the the of Fcp1 a results a Fcp1 and in FCP1 and and a as as a the FCP1 in the in and and and for the of the of we a for of in a of a gene a protein in of transcription of and a of and the of of the of the the of in of and of of the complex with of the and as as with and the of gene of in defects when with of of the complex and as as with of the complex and and a complex is for the of and defects with complexes in of the and and of the complex and of when with of of complexes in for the of and is the that with a and the and of of the and and with a in transcription and of of that a and that with that not of the but of the with a in RNAPII Fcp1 for of RNAPII of RNAPII yeast we de-phosphorylation of the of a of the of RNAPII de-phosphorylation phosphorylation of RNAPII in yeast for In contrast, that ternary as and of CTD de-phosphorylation and the that the RNAPII CTD of the ternary complex and a for in RNAPII In the of RNAPII de-phosphorylation in we that protein as and the not of the of Fcp1 is in the of protein the CTD and results in a protein that has CTD in CTD de-phosphorylation and In the CTD de-phosphorylation and but in de-phosphorylation of the of de-phosphorylation not the as transcription in in and the in not results suggest that Fcp1 is for the de-phosphorylation of in vitro. of Fcp1 in a yeast is in RNAPII with and the with for Fcp1 protein with RNAPII as as with RNAPII Fcp1 in the for the protein not with elongating in results with the that Fcp1 with elongating RNAPII holoenzyme in CTD with CTD de-phosphorylation in a we purified and Fcp1 and a of RNAPII CTD the substrate of yeast Fcp1 in the with RNAPII that TFIIH phosphorylation is serine 5 of the CTD data not phosphorylation serine 2 but a serine 5 data not Fcp1 RNAPII serine phosphorylation results as the that yeast Fcp1 serine 2 phosphorylation but not for the of serine 5 phosphorylation in in the FCP1 serine 5 is for yeast phosphorylation of serine 2 is a of Fcp1 de-phosphorylation of serine a of the results of we RNAPII the substrate preference and of of Fcp1 in the for the de-phosphorylation with a in transcription with purified with RNAPII that TFIIH in the of the a of the for and RNAPII the of the in a in transcription in complexes free as as RNAPII the of in RNAPII in transcription is in the of for for the substrate of RNAPII and RNAPII in a ternary complex in with Fcp1 for the of the in the de-phosphorylation we that free a substrate for de-phosphorylation elongating of free when with of de-phosphorylation with a of ternary results with transcription not that results not the of the the of a of the as Fcp1 with the of RNAPII phosphorylation when the of the the de-phosphorylation with the of that the with purified in the of free the that the protein phosphorylation a that de-phosphorylation of the CTD the Fcp1 results the of Fcp1 In free elongating as a elongating a substrate free for the kinase of TFIIH data suggest that the substrate of TFIIH kinase and Fcp1 the transcription RNAPII phosphorylation is the polymerase in a ternary complex as a of CTD de-phosphorylation of serine 5 is the of ternary complexes the of FCP1 and data in 2 suggest that Fcp1 and with the elongating of the that the of of purified Fcp1 de-phosphorylation in not a Fcp1 and in vitro, we FCP1 and FCP1 is we the in FCP1 a as as FCP1 a of the in the of the for the the of Fcp1 a results a Fcp1 and in vivo. the of the of we a for of in a of a gene a protein in of transcription of and a of and the of of the of the the of in of and of of the complex with of the and as as with and the of gene of in defects when with of of the complex and as as with of the complex and and a complex is for the of and defects with complexes in of the and and of the complex and of when with of of complexes in for the of and is the that with a and the and of elongating RNAPII holoenzyme for of in ternary complexes with yeast is in the of the the of results of the the that Fcp1 associates with elongating and that the serine 5 of the polymerase CTD a substrate for the of Fcp1 of the ternary associates with elongating and data show that FCP1 and and with and RNA a the of but not ternary RNAPII0-DNA-RNA complexes data suggest that Fcp1 with RNAPII for the that Fcp1 with elongating RNAPII in complex of the association of Fcp1 with elongating and that RNAPII when ternary we a with purified RNAPII de-phosphorylation in vitro. we that Fcp1 serine 5 but not serine 2 of the RNAPII that purified Fcp1 has with yeast FCP1 the serine 2 phosphorylation has serine 5 phosphorylation levels in in in with the serine 5 data the data show that Fcp1 de-phosphorylates free we that yeast Fcp1 has a preference for free elongating in with highly purified is pronounced for the CTD the of Fcp1 with ternary complexes is not the CTD for in as TFIIH kinase the substrate a pronounced preference for ternary complexes free substrate preference of TFIIH has not results that the transcription TFIIH RNAPII when is incorporated in a ternary complex and Fcp1 RNAPII serine 5 has the ternary complex in that the polymerase for a of that Fcp1 and the serine CTD that RNAPII is polymerase In with mutation Fcp1 the purified Fcp1 we that a protein the of the Fcp1 protein as RNAPII that TFIIH and free the CTD phosphorylation the and of the with the of CTD in the de-phosphorylation ternary complexes as substrate CTD de-phosphorylation the RNAPII and in the of the protein of the of and Fcp1 with the the as a when with the is the in the for the that Fcp1 a Fcp1 and the of in the of a and that the is that a of protein as the CTD in Fcp1 is that the of as the Fcp1 the not the of Fcp1 and but the of the Fcp1 of yeast in serine RNAPII in data show that and Fcp1 with elongating RNAPII In with the that and Fcp1 in a of Fcp1 the of for that the of Fcp1 and results that is for of the gene the of the CTD kinase results a for and in the of with as as with and has defects and of in we with and a of as and with the data a and RNAPII as as data that is in a and of elongating RNAPII holoenzyme for of in ternary complexes with yeast is in the of the the of results of the the that Fcp1 associates with elongating and that the serine 5 of the polymerase CTD a substrate for the of Fcp1 of the ternary associates with elongating and data show that FCP1 and and with and RNA a the of but not ternary RNAPII0-DNA-RNA complexes data suggest that Fcp1 with RNAPII for the that Fcp1 with elongating RNAPII in complex of the association of Fcp1 with elongating and that RNAPII when ternary we a with purified RNAPII de-phosphorylation in vitro. we that Fcp1 serine 5 but not serine 2 of the RNAPII that purified Fcp1 has with yeast FCP1 the serine 2 phosphorylation has serine 5 phosphorylation levels in in in with the serine 5 data the data show that Fcp1 de-phosphorylates free we that yeast Fcp1 has a preference for free elongating in with highly purified is pronounced for the CTD the of Fcp1 with ternary complexes is not the CTD for in as TFIIH kinase the substrate a pronounced preference for ternary complexes free substrate preference of TFIIH has not results that the transcription TFIIH RNAPII when is incorporated in a ternary complex and Fcp1 RNAPII serine 5 has the ternary complex in that the polymerase for a of that Fcp1 and the serine CTD that RNAPII is polymerase In with mutation Fcp1 the purified Fcp1 we that a protein the of the Fcp1 protein as RNAPII that TFIIH and free the CTD phosphorylation the and of the with the of CTD in the de-phosphorylation ternary complexes as substrate CTD de-phosphorylation the RNAPII and in the of the protein of the of and Fcp1 with the the as a when with the is the in the for the that Fcp1 a Fcp1 and the of in the of a and that the is that a of protein as the CTD in Fcp1 is that the of as the Fcp1 the not the of Fcp1 and but the of the Fcp1 of yeast in serine RNAPII in Our data show that and Fcp1 with elongating RNAPII In with the that and Fcp1 in a of Fcp1 the of for that the of Fcp1 and results that is for of the gene the of the CTD kinase results a for and in the of with as as with and has defects and of in we with and a of as and with the data a and RNAPII as as data that is in a and

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