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1.  The Mediator Complex and Transcription Elongation 
Biochimica et biophysica acta  2012;1829(1):69-75.
Mediator is an evolutionarily conserved multisubunit RNA polymerase II (Pol II) coregulatory complex. Although Mediator was initially found to play a critical role in regulation of the initiation of Pol II transcription, recent studies have brought to light an expanded role for Mediator at post-initiation stages of transcription.
Scope of review
We provide a brief description of the structure of Mediator and its function in the regulation of Pol II transcription initiation, and we summarize recent findings implicating Mediator in the regulation of various stages of Pol II transcription elongation.
Major conclusions
Emerging evidence is revealing new roles for Mediator in nearly all stages of Pol II transcription, including initiation, promoter escape, elongation, pre-mRNA processing, and termination.
General significance
Mediator plays a central role in the regulation of gene expression by impacting nearly all stages of mRNA synthesis.
PMCID: PMC3693936  PMID: 22983086
2.  Purification of RNA Polymerase II General Transcription Factors from Rat Liver 
Methods in enzymology  1996;273:194-207.
Eukaryotic messenger RNA synthesis is a complex biochemical process requiring the concerted action of multiple “general” transcription factors (TFs) that control the activity of RNA polymerase II at both the initiation1 and elongation2,3 stages of transcription. Because the general transcription factors are present at low levels in mammalian cells, their purification is a formidable undertaking. For this reason we explored the feasibility of using rat liver as a source for purification of the general factors. Rat liver has proven to be an ideal model system for biochemical studies of transcription initiation and elongation by RNA polymerase II (Figs. 1 and 2). In our hands the yield of general transcription factors per gram of rat liver is roughly equivalent to their yield per gram of cultured HeLa cells. Moreover, we have been able to develop convenient and reproducible methods for preparation of rat liver extracts from as much as 1 kg of liver per day. Because it is both technically difficult and expensive to obtain such quantities of cultured cells on a daily basis, rat liver provides a significant logistic advantage for purification of the general transcription factors.
PMCID: PMC3618461  PMID: 8791613
3.  Origins and Activity of the Mediator Complex 
The Mediator is a large, multisubunit RNA polymerase II transcriptional regulator that was first identified in Saccharomyces cerevisiae as a factor required for responsiveness of Pol II and the general initiation factors to DNA binding transactivators. Since its discovery in yeast, Mediator has been shown to be an integral and highly evolutionarily conserved component of the Pol II transcriptional machinery with critical roles in multiple stages of transcription, from regulation of assembly of the Pol II initiation complex to regulation of Pol II elongation. Here we provide a brief overview of the evolutionary origins of Mediator, its subunit composition, and its remarkably diverse collection of activities in Pol II transcription.
PMCID: PMC3207015  PMID: 21821140
4.  Transcription Initiated by RNA Polymerase II and Purified Transcription Factors from Liver 
The Journal of Biological Chemistry  1990;265(13):7552-7558.
Synthesis of accurately initiated transcripts has been reconstituted with RNA polymerase II and a set of five transcription factors purified from rat liver. In addition to three previously identified factors α, βγ, and δ (Conaway, R. C, and Conaway, J. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7356–7360), transcription in the reconstituted liver system requires two novel factors designated τ and ε. These five transcription factors comprise two functional classes: (i) promoter recognition factors (τ and ε), which interact with template DNA to facilitate formation of a stable initial complex that is subsequently recognized and bound by RNA polymerase II, and (ii) RNA chain initiation factors (α, βγ, and δ), which do not participate in formation of the initial complex, but which are essential for transcription initiation.
PMCID: PMC3380428  PMID: 2332442
5.  The RNA polymerase II general elongation factors 
Trends in Biochemical Sciences  1996;21(9):351-355.
Synthesis of eukaryotic messenger RNA by RNA polymerase II is governed by the concerted action of a set of general transcription factors that control the activity of polymerase during both the initiation and elongation stages of transcription. To date, five general elongation factors [P-TEFb, SII, TFIIF, Elongin (SIII) and ELL] have been defined biochemically. Here, we discuss these transcription factors and their roles in controlling the activity of the RNA polymerase II elongation complex.
PMCID: PMC3374595  PMID: 8870500
6.  Mechanism and regulation of transcriptional elongation by RNA polymerase II 
Current Opinion in Cell Biology  1999;11(3):342-346.
Over the past few years, biochemical and genetic studies have shed considerable light on the structure and function of the RNA polymerase II (pol II) elongation complex and the transcription factors that control it. Novel elongation factors have been identified and their mechanisms of action characterized in increasing detail; new insights into the biological roles of elongation factors have been gained from genetic studies of the regulation of mRNA synthesis in yeast; and intriguing links between the pol II elongation machinery and the pathways of DNA repair and recombination have emerged.
PMCID: PMC3371606  PMID: 10395562
7.  Function and Regulation of the Mediator Complex 
Over the past few years, advances in biochemical and genetic studies of the structure and function of the Mediator complex have shed new light on its subunit architecture and its mechanism of action in transcription by RNA polymerase II (pol II). The development of improved methods for reconstitution of recombinant Mediator subassemblies is enabling more in-depth analyses of basic features of the mechanisms by which Mediator interacts with and controls the activity of pol II and the general initiation factors. The discovery and characterization of multiple, functionally distinct forms of Mediator characterized by the presence or absence of the Cdk8 kinase module have led to new insights into how Mediator functions in both Pol II transcription activation and repression. Finally, progress in studies of the mechanisms by which the transcriptional activation domains (ADs) of DNA binding transcription factors target Mediator have brought to light unexpected complexities in the way Mediator participates in signal transduction.
PMCID: PMC3086004  PMID: 21330129
8.  Both BC-Box Motifs of Adenovirus Protein E4orf6 Are Required To Efficiently Assemble an E3 Ligase Complex That Degrades p53 
Molecular and Cellular Biology  2004;24(21):9619-9629.
Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.
PMCID: PMC522240  PMID: 15485928
9.  In Vivo Requirement of the RNA Polymerase II Elongation Factor Elongin A for Proper Gene Expression and Development 
Molecular and Cellular Biology  2004;24(22):9911-9919.
A number of transcription factors that increase the catalytic rate of mRNA synthesis by RNA polymerase II (Pol II) have been purified from higher eukaryotes. Among these are the ELL family, DSIF, and the heterotrimeric elongin complex. Elongin A, the largest subunit of the elongin complex, is the transcriptionally active subunit, while the smaller elongin B and C subunits appear to act as regulatory subunits. While much is known about the in vitro properties of elongin A and other members of this class of elongation factors, the physiological role(s) of these proteins remain largely unclear. To elucidate in vivo functions of elongin A, we have characterized its Drosophila homologue (dEloA). dEloA associates with transcriptionally active puff sites within Drosophila polytene chromosomes and exhibits many of the expected biochemical and cytological properties consistent with a Pol II-associated elongation factor. RNA interference-mediated depletion of dEloA demonstrated that elongin A is an essential factor that is required for proper metamorphosis. Consistent with this observation, dEloA expression peaks during the larval stages of development, suggesting that this factor may be important for proper regulation of developmental events during these stages. The discovery of the role of elongin A in an in vivo model system defines the novel contribution played by RNA polymerase II elongation machinery in regulation of gene expression that is required for proper development.
PMCID: PMC525478  PMID: 15509793
10.  Synthetic peptides define critical contacts between elongin C, elongin B, and the von Hippel-Lindau protein 
Journal of Clinical Investigation  1999;104(11):1583-1591.
The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-inducible mRNAs such as the mRNA encoding vascular endothelial growth factor (VEGF). This activity has been linked to its ability to form multimeric complexes that contain elongin C, elongin B, and Cul2. To understand this process in greater detail, we performed a series of in vitro binding assays using pVHL, elongin B, and elongin C variants as well as synthetic peptide competitors derived from pVHL or elongin C. A subdomain of elongin C (residues 17–50) was necessary and sufficient for detectable binding to elongin B. In contrast, elongin B residues required for binding to elongin C were not confined to a discrete colinear domain. We found that the pVHL (residues 157–171) is necessary and sufficient for binding to elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly bind to elongin C and/or alter the conformation of pVHL such that binding to elongin C is at least partially diminished. These results are consistent with the view that diminished binding of pVHL to the elongins plays a causal role in VHL disease.
J. Clin. Invest. 104:1583–1591 (1999).
PMCID: PMC481054  PMID: 10587522
11.  Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes Containing Elongins B/C and Cul2 
Molecular and Cellular Biology  1998;18(2):732-741.
The von Hippel-Lindau tumor suppressor protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter proteins form complexes that target specific proteins for ubiquitin-dependent proteolysis.
PMCID: PMC108784  PMID: 9447969

Results 1-11 (11)