The Mediator complex of Saccharomyces cerevisiae is required for both general and regulated transcription of RNA polymerase II (PolII) and is composed of two stable subcomplexes (Srb4 and Rgr1 subcomplexes). To decipher the function of each Mediator subcomplex and to delineate the functional relationship between the subcomplexes, we characterized the compositions and biochemical activities of PolII-Mediator complexes (holoenzymes) prepared from several Mediator mutant strains of S. cerevisiae. We found that holoenzymes devoid of a functional Gal11 module were defective for activated but not basal transcription in a reconstituted in vitro system. This activation-specific defect was correlated with a crippled physical interaction to transcriptional activator proteins, which could be bypassed by artificial recruitment of a mutant holoenzyme to a promoter. Consistent with this observation, a direct interaction between Gal11 and gene-specific transcriptional activator proteins was detected by far-Western analyses and column binding assays. In contrast, the srb5 deletion mutant holoenzyme was defective for both basal and activated transcription, despite its capacity for activator binding that is comparable to that of the wild-type holoenzyme. These results demonstrate that the Gal11 module of the Rgr1 subcomplex is required for the efficient recruitment of PolII holoenzyme to a promoter via activator-specific interactions, while the Srb4 subcomplex functions in the modulation of general polymerase activity.

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) converts the single-stranded (ϩ) viral RNA genome into a double-stranded proviral DNA prior to its integration into the host genomic DNA. The RT is a multifunctional enzyme with three recognized enzymatic activities of RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H activities. HIV-1 protease is an aspartic protease required for the proteolytic processing of the large Gag and Gag-Pol viral polyprotein precursors into the mature virion structural proteins, as well as the virion enzymes. These two enzymes, HIV-1 RT and protease, have key roles in HIV replication and inhibition of these enzymes along with HIV-1 integrase has been a major target of acquired immunodeficiency syndrome (AIDS) therapy. 1)In addition to the well-known nucleoside RT inhibitors (NRTIs), zidovudine (AZT), didanosine (ddC), zalcitabine (ddT), stavudine (d4T), lamivudine (3TC), and abacavir (ABC), non-nucleoside RT inhibitors (NNRTIs), such as nevirapine, delavirdine, and efavirenz have been formally approved to treat HIV infection. On the other hand, saquinavir, ritonavir, and indinavir are the three main HIV protease inhibitors available today. A number of inhibitors interacting with RT have been isolated from plants (baicalin, avarol, avarone, and psychotrine) and marine resources (illimaquinone, peyssonol, and KM043). [2][3][4] In the case of HIV-1 protease, natural products, such as mangostin, ursolic, and maslinic acid, have been reported to show inhibitory activity against this enzyme. 5) We previously reported the results of screening inhibitory activities of 47 types of Korean seaweed on HIV-1 RT and found that the EtOAc fraction of Ecklonia cava strongly inhibited the RDDP activity of this enzyme. 6) In this study, we report the isolation of four phlorotannin compounds containing dibenzo [1,4]dioxin elements in common from E. cava and the kinetic study of HIV-1 RT by 8,8Ј-bieckol (2) that showed the most potent inhibitory effect on this enzyme. MATERIALS AND METHODS MaterialsThe thalli of E. cava KJELLMAN were collected from the coasts of Korea including Sungsanpo, Wando, and Namhaedo from January 1999 to June 2000. After cleaning the surface of the thalli with water to remove visible epiphytes and dirt, samples were dried at 60°C for 12 h in an oven and then ground in a coffee grinder. This seaweed was identified by Prof. S. M. Boo of the Department of Biology, Chungnam National University, Korea, and Prof. Y. S. Oh of the Department of Aquaculture, Gyeongsang National University, Korea. A voucher specimen (SSI-06) was deposited in the Herbarium of the Department of Biological Sciences, Sungkyunkwan University.Extraction and Isolation The dried thalli (1 kg) of E. cava were extracted three times with 100% MeOH and evaporated in vacuo. The MeOH extract (180 g) was dissolved in water and partitioned with n-hexane. After the H 2 O layer was further partitioned with ethyl acetate, the organic solvent fraction was c...

A temperature-sensitive mutation was obtained in Med6p, a component of the mediator complex from the yeast Saccharomyces cerevisiae. The mediator complex has been shown to enable transcriptional activation in vitro. This mutation in Med6p abolished activation of transcription from four of five inducible promoters tested in vivo. There was no effect, however, on uninduced transcription, transcription of constitutively expressed genes, or transcription by RNA polymerases I and III. Mediator-RNA polymerase II complex isolated from the mutant yeast strain was temperature sensitive for transcriptional activation in a reconstituted in vitro system due to a defect in initiation complex formation. A database search revealed the existence of MED6-related genes in humans and Caenorhabditis elegans, suggesting that the role of mediator in transcriptional activation is conserved throughout the evolution.Regulation of mRNA synthesis requires intermediary proteins that transduce regulatory signals from upstream transcriptional activator proteins to basal transcription machinery at the core promoter. A reconstituted in vitro transcription system composed only of basal transcription machinery (core-RNA polymerase II and pure general transcription factors) does not respond to upstream transcriptional activator proteins even though it is fully functional for basal transcription (26,36). Three types of intermediary factors that enable the basal transcription machinery to respond to transcriptional activator proteins bound to regulatory DNA sequences have been identified: (i) TAF II s, which associate with TATA-binding protein (TBP) to form TFIID (12, 13; for a review, see reference 53); (ii) mediator, which associates with RNA polymerase II to form a holo-polymerase (11,25,26,28; for a review, see reference 4); and (iii) coactivators such as human upstream stimulatory activity (USA) (36; for a review, see reference 23), mammalian CBP/P300 (for a review, see reference 19), yeast ADA complex (17), and HMG proteins (42,46). The interaction of these multiprotein complexes with activators and general transcription factors is essential for transcriptional regulation (3, 12-14, 18, 19, 43, 48, 57). In this paper, we focus on the functional analysis of one intermediary factor from Saccharomyces cerevisiae called mediator.The existence of mediator was originally suggested from a squelching assay, in which the addition of one activator interferes with stimulation of transcription by another activator, suggesting that the two activators compete for a common target (11,25). Squelching is specific for the activation domains of transcriptional activator proteins and could be relieved by addition of a partially purified yeast protein fraction but not by addition of known components of the basal transcription machinery. These results identified a novel activity termed mediator that is distinct from proteins required for basal transcription and that is essential for transcriptional activation. Fractionation of the yeast mediator activity yielded a...

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