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Functional interactions within yeast mediator and evidence of differential subunit modifications
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik.
2003 (Engelska)Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, nr 6, s. 3831-3839Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

It is possible to recruit RNA polymerase II to a target promoter and, thus, activate transcription by fusing Mediator subunits to a DNA binding domain. To investigate functional interactions within Mediator, we have tested such fusions of the lexA DNA binding domain to Med1, Med2, Gal11, Srb7, and Srb10 in wild type, med1, med2, gal11, sin4, srb8, srb10, and srb11 strains. We found that lexA-Med2 and lexA-Gal11 are strong activators that are independent of all Mediator subunits tested. lexA-Srb10 is a weak activator that depends on Srb8 and Srb11. lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. An unexpected finding was that lexA-VP16 differs from Gal4-VP16 in that it is independent of the activator binding Mediator module. Both lexA-Med1 and lexA-Srb7 are stably associated with Med4 and Med8, which suggests that they are incorporated into Mediator. Med4 and Med8 exist in two mobility forms that differ in their association with lexA-Med1 and lexA-Srb7. Within purified Mediator, Med4 is present as a phosphorylated lower mobility form. Taken together, these results suggest that assembly of Mediator is a multistep process that involves conversion of both Med4 and Med8 to their low mobility forms.

Ort, förlag, år, upplaga, sidor
2003. Vol. 278, nr 6, s. 3831-3839
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
URN: urn:nbn:se:umu:diva-4195DOI: 10.1074/jbc.M206946200PubMedID: 12468546OAI: oai:DiVA.org:umu-4195DiVA, id: diva2:143191
Tillgänglig från: 2004-11-03 Skapad: 2004-11-03 Senast uppdaterad: 2018-06-09Bibliografiskt granskad
Ingår i avhandling
1. Studies of Functional Interactions within Yeast Mediator and a Proposed Novel Mechanism for Regulation of Gene Expression
Öppna denna publikation i ny flik eller fönster >>Studies of Functional Interactions within Yeast Mediator and a Proposed Novel Mechanism for Regulation of Gene Expression
2004 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The yeast Mediator complex is required for transcriptional regulation both in vivo and in vitro and the identification of similar complexes from metazoans indicates that its function is conserved through evolution. Mediator subunit composition and structure is well characterized both by biochemical, genetic and biophysical methods. In contrast, little is known about the mechanisms by which Mediator operates and how the complex is regulated. The aim of my thesis was to elucidate how Mediator functions at the molecular level and to investigate functional interactions within Mediator.

It is possible to recruit RNA polymerase II to a target promoter and thus to activate transcription by fusing Mediator subunits to a DNA binding domain. In order to investigate functional interactions within Mediator, we made such fusion proteins where different Mediator subunits were fused to the DNA binding domain of lexA. The expression of a reporter gene containing binding sites for lexA was subsequently measured in both a wild type strain and in strains where genes encoding specific Mediator subunits had been disrupted. We found that lexA-Med2 and lexA-Gal11 are strong activators that function independently of all Mediator subunits tested. On the other hand, lexA-Srb10 is a weak activator that depends on Srb8 and Srb11 and lexA-Med1 and lexA-Srb7 are both cryptic activators that become active in the absence of Srb8, Srb10, Srb11, or Sin4. Both lexA-Med1 and lexA-Srb7 proteins showed a stable association with the Mediator subunits Med4 and Med8 in wild type cells and in all deletion strains tested, indicating that they were functionally incorporated into the Mediator complex. We also showed that both Med4 and Med8 exist in two forms that differed in electrophoretic mobility and that these forms differed in their ability to associate with Mediator immuno-purified from the LEXA-SRB7 and LEXA-MED1 strains. Dephosphorylation assays of purified Mediator indicated that the two mobility forms of Med4 corresponded to the phosphorylated and unphosphorylated forms of the Med4 protein respectively.

Some of the data presented in this study as well as previous genetic and biochemical data obtained in our lab suggested a functional link between the Med1, Med2, Srb10 and Srb11 proteins. We extended these findings by showing that the Srb10 kinase phosphorylates the Med2 protein at residue serine 208, both in vitro and in vivo. We also showed that a point mutation of the single phosphorylation site to an alanine or to an aspartic acid residue altered the gene expression of a specific set of genes. Taken together, these data indicate that posttranslational modification of Mediator subunits is a so far uncharacterized mechanism for regulation of gene expression.

In order to study the function of the Srb7 subunit of Mediator, we isolated a temperature sensitive strain where the amino acids 2 to 8 of srb7 were deleted. The Mediator subunits Nut2 and Med7 were isolated as high copy suppressor of srb7-∆(2-8) and we were also able to show that Srb7 interacted with Nut2 and Med7 both in a 2-hybrid system and in co-immuno precipitation experiments using recombinantly expressed proteins. Interestingly, a deletion of amino acids 2 to 8 of Srb7 abolishes its interaction with both Med7 and Nut2 in vitro. Med4 also interacted with Srb7 in the 2-hybrid system and surprisingly, the first eight amino acids of Srb7 were shown to be sufficient for this interaction.

Ort, förlag, år, upplaga, sidor
Umeå: Medicinsk biokemi och biofysik, 2004. s. 71
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 923
Nyckelord
Biochemistry, Mediator, Transcriptional regulation, Srb10, Cdk8, Med2, Srb7, Med21, RNA pol II, Biokemi
Nationell ämneskategori
Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)
Forskningsämne
medicinsk biokemi
Identifikatorer
urn:nbn:se:umu:diva-348 (URN)91-7305-743-6 (ISBN)
Disputation
2004-11-26, KB3A9, KBC-Huset, Umeå Universitet, Umeå, 09:00
Opponent
Handledare
Tillgänglig från: 2004-11-03 Skapad: 2004-11-03Bibliografiskt granskad

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