Chapter 1: Introduction 1.1 The Process and Mechanisms of Transcription 1.1.1 Introduction to Transcription and the Transcriptional Machinery Transcription, the process by which RNA is synthesized from a Deoxyribonucleic Acid (DNA) template, is one of the most fundamental processes in a cell. If follows several stages first of which is the assembly of a “preinitiation complex”. This complex drives transcription from the initiation stage to elongation stage where most of the preinitiation complex is released from the active complex. After elongation is complete, post-processing of the RNA product occurs and it is exported from the organelle where it was synthesized (Hahn, 2004). The proteins that comprise a cell’s core transcriptional machinery, the Ribonucleic Acid Polymerase (RNA Polymerase), are strongly conserved within Kingdoms. Indeed, five subunits of RNA polymerase are known to be conserved even between Superkingdoms Prokaryota, Archaea and Eukaryota while the latter two share even an even greater number of conserved subunits. These subunits are classified in Eukaryotic RNA Polymerase II as Rpb1, Rpb2, Rpb3, Rpb6 and Rpb11 (Young, 1991). In Super Kingdoms Prokaryota and Archaea, there exists only one RNA Polymerase while in Super Kingdom Eukaryota, three RNA Polymerases have been identified. RNA Polymerase I transcribes Ribosomal RNA (rRNA) save for 5S rRNA (Russell and Zomerdijk, 2006) which is the purview of RNA Polymerase III. RNA Polymerase III also transcribes Transfer RNA (tRNA) and other Short Nuclear RNAs (snRNA) (Dieci et al., 2007). RNA Polymerase II however is perhaps the widest studied as it transcribes Messenger RNA (mRNA) in the nucleus (Woychik and Hampsey, 2002). Despite the fact that Eukaryotic RNA Polymerase is comprised of far more subunits than that of Prokaryota and Archaea, the majority of subunits share functional if not structural homology and are thought to share the same basic mechanisms of function and regulation (Ebright, 2000). 1.1.2 The Eukaryotic Transcriptional Machinery RNA Polymerase II typically comprises of about a dozen subunits although the exact number of which varies from organism to organism. Of these subunits, designated as “Rpb”s (Repressor of RNA Polymerase B) in modern parlance, Rpb1 and Rpb2 are usually the core catalytic components of RNA Polymerase II (Cramer, 2004). This catalytic core, while necessary for RNA synthesis, is in and of itself insufficient for transcription to proceed. Rather RNA Polymerase II has no actual ability to recognize promoter DNA. It relies on the assembly of various other factors to give it specificity and the ability to bind template DNA. Specifically the Rpb4/7 complex and general transcription factors are also necessary for the initiation of transcription from promoter DNA (Edwards et al., 1991). As DNA in Eukaryotes is found packed in chromatin and thus inaccessible to the transcriptional machinery, gene-specific transcription factors must bind proximally to the site of initiation and recruit the factors necessary to modify the chromatin structure before transcription can begin (Cosma, 2002). Assembly of this “preinitiation complex” marks the beginning of the transcription process and starts when the TBP (TATA-binding protein) component of TFIID binds with a promoter sequence, such as “TATA” in yeast. This is followed by other general transcription factors like TFIIB, TFIIE, TFIIH and TFIIF which is the general transcription factor directly bound to RNA Polymerase II (Reinberg et al., 1998). Despite the fact that the preinitiation complex is fully assembled after this stage, it remains in an inactive state until a conformational change occurs in the template strand to place the coding DNA in the “catalytic cleft” of the RNA polymerase II complex. After the synthesis of the first 30 or so bases, RNA polymerase II is thought to release its contacts with the rest of the transcription machinery and leaves the core promoter region to enter the stage of RNA elongation (Wang et al., 1992). This “release” of the transcriptional machinery by RNA Polymerase II is mediated by the phosphorylation status of its carboxy-terminal domain (CTD). RNA Polymerase II has a repeating Tyr-Ser-Pro-Thr-Ser-Pro-Ser sequence which is unphosphorylated in the initiation stage of transcription. Phosphorylation by a kinase of this CTD signals the disassembly of the preinitiation complex and the progress of transcription into the elongation stage. Phosphatases recycle phosphorylated RNA Polymerase II after termination phase for use in further rounds of transcription (Murray et al., 2001). The phosphorylation status of the RNA Polymerase II CTD also has a role to play in the processing of mRNA. It is implicated in several phenomena such as 5’ cap addition, 3’ poly-A tail synthesis and the splicing of introns (Proudfoot et al., 2002). Many of the released general transcription factors, termed the Scaffold Complex, remain at the site of initiation; a phenomena which can be used to mark transcriptionally active genes. This allows the cell to circumvent the arduous process of reassembling the initiation complex for subsequent rounds of transcription by using these general transcription factors to aid in the recruitment of the remaining factors necessary to begin the next round of transcription (Yudovsky et al., 2000). The Rpb4/Rpb7 complex is also necessary for proper transcriptional activity. Rather than process RNA or binding to DNA, it is thought that the Rpb4/7 complex’s role in RNA Polymerase II is that of a “clamp” to bind RNA and to contribute to the stability of the RNA Polymerase II complex as a whole (Cramer et al., 2000). However, not all RNA Polymerase II complexes necessarily contain the Rbp4/7 complex, rather subunit composition of the polymerase is dependent on several factors (Kolodziej et al., 1990). These include growth conditions (Choder and Young, 1993) and the transcription factors associated with a particular promoter (Xue and Lehming, 2008). 1.2 The Mediator Complex 1.2.1 Introduction to the Mediator Complex Along with RNA Polymerase II and the general transcription factors, another critical component of the Eukaryotic transcriptional machinery is the Mediator Complex. The first inklings that such a complex existed originated in biochemical experiments that attempted to reconstitute an active transcriptional machinery in vitro. While the general transcription factors and RNA Polymerase II were sufficient to drive promoter-targeted gene expression, they could not replicate a cell’s ability to respond to activators or repressors (Hampsey and Reinberg, 1999). Further experiments identified the Mediator complex in the yeast Saccharomyces cerevisiae (Thompson et al., 1993). Many of these components had already been identified as transcription factors in various genetic screens and thus many of the designations of yeast mediator bear names that are a legacy of this era. For example several yeast mediator components are designated as “Srb”s for “Suppressor of RNA Holoenzyme B”; others were likewise named for the identification methods used (Myers and Kornberg, 2000) or apparent role in cellular regulation such as Gal11 (Carlson, 1997). The human Mediator Complex subcomponents were thus subsequently isolated in various biochemical reactions in vitro by identifying the proteins necessary to restore activator driven transcription of a fully reconstituted RNA Polymerase II and general transcription factors (Sato et al., 2003). Due to this, Mediator is classified as a coactivator of transcription although recent evidence is emerging that challenges this classical view and asserts that Mediator is as fully involved in transcription as a traditional general transcription factor (Taatjes, 2010). Unlike RNA Polymerase II, Mediator is absent from Prokaryotes and Archaea. Furthermore, only eight Mediator subunits are known to be conserved from Saccharomyces cerevisiae to Homo sapiens (Rachez et al., 2001). This is not too large a surprise as the general transcription factors themselves are also absent in Prokaryotes and only a few analogues have been identified in Archaea (Taatjes, 2010). 1.2.2 Structure and Function of the Mediator Complex The Mediator Complex originally isolated in S. cerevisiae has 21 subcomponents while mammalian Mediator has a little over 30 (Tomomori-Sato et al., 2004). These subunits form three distinct modules that undergo significant conformational changes upon binding to RNA Polymerase II or its CTD portion. These have been termed the “head”, “middle” and “tail” modules and each has their own role to play in the function of Mediator (Chadick et al., 2005). The head module is the most evolutionarily conserved and is chiefly concerned with binding RNA Polymerase II. The middle module has two submodules each with a contrary role. The Med9 submodule is involved in repression while the Med10 submodule is involved in the activation of genes. These allow the middle module to respond to signals even after binding to RNA Polymerase II. The tail module mediates binding with DNA-bound transcription factors and thus is the module most associated with Mediator’s classical role in transcription (Woychik, et al., 2002). The Cdk8 submodule is known to transiently associate with the Mediator Complex. It is comprised of Cdk8, Cdk11, Cyclin C, Med12, Med13 and splice variants of the latter two Mediator subunits (Sun et al., 1998). Similar to the middle Mediator module, the Cdk8 submodule can act to both repress and activate transcription. Initial studies indicated that Cdk8containing Mediator could not initiate transcription because Cdk8 can negatively affect Mediator’s ability to both recruit RNA Polymerase II to the promoter and activate RNA synthesis (Knuesel et al., 2009). Along with its global effect on transcription, Cdk8 is also implicated in gene-specific repression. The Cdk8 submodule, particularly the Med12 subunit, activates the G9a histone methyltransferase. This catalyses methylation of H3K9 which typically leads to restricted access to chromatin. However this only represses a particular subset of neuronal genes rather than a general decrease in transcription (Ding et al., 2008). The Cdk8 submodule’s positive role in transcription follows a similar pattern. Cdk8 can function as an H3S10 kinase to create a permissive chromatin environment for the assembly of transcriptional activity (Cheung et al., 2000). A link to cancer has recently been established as Cdk8 is a positive factor for the activation of certain p53 regulated genes such as p21 and HDM2 (Donner et al., 2007). In contrast to the general transcription factors, Mediator lacks the ability to bind to template or promoter DNA. As was alluded to in how it was originally characterized, Mediator has a high affinity for DNA-binding transcription factors and is thus recruited to various regulatory sites in the genome during transcription. Indeed, there is evidence in yeast that Mediator subunits localize to promoters on a genome-wide scale (Zhu et al., 2006). This is the origin of its name as Mediator was proposed to be the bridge that mediated binding between promoter-specific transcription factors and the general transcription factors (Ryu et al., 1999). 1.3 The Ubiquitin Proteosome System 1.3.1 The Discovery of the Ubiquitin Proteosome System With the discovery of the lysosome in the 1950s, contemporary scientists believed that proteolysis of cellular proteins were localized in this organelle (de Duve, 1953). It was logically consistent that proteases were not only sequestered from their substrates by a membrane but that they required a radically different environment from the cytoplasm in order to function. Otherwise a protease would indiscriminately degrade its substrates. In addition the ability of the lysosome to perform autophagy (Ashford et al., 1962) on smaller vesicles further strengthened the notion that while proteins were in a dynamic state of synthesis and degradation (Simpson, 1953), these activities were confined to very particular organelles specifically designed for such purposes (Mortimore et al., 1987). This view held for a time but with increasingly sophisticated tools for investigation into cellular phenomena, various observations could not be accounted for solely by the activity of the lysosome. For one, cellular proteins have wildly different half-lives and the stability of a given protein can be altered due to the status of the cell (Goldberg et al., 1976). This is patently inconsistent with the notion that the lysosome was supposed to be the organelle that indiscriminately degraded proteins through autophagy, a process that did not allow for selective proteolysis. 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Mol Cell 22, 169-178 Websites Swiss-Prot Protein Knowledgebase (http://www.expasy.org/sprot/) Saccharomyces Genome Database (http://www.yeastgenome.org) National Centre for Biotechnology Information Database (http://www.ncbi.nlm.nih.gov) ImageJ (http://rsbweb.nih.gov/ij/) 197 Appendix Common Stock Solutions Luria Bertani (LB) Broth 10g tryptone 5g yeast extract 10g Sodium Chloride litre distilled water DNA Agarose Gel 1g Agarose 100ml 1x TBE Buffer 1μl ethidum bromide Agarose was dissoved in TBE buffer and microwaved for minutes and then allowed to cool before ethidium bromide was added. Mixture was poured in a gel tray and allowed to set. LB agar 15g of granulated agar was dissolved in one litre of LD broth and autoclaved for 15 minutes at 125 degrees Celsius. Any antibiotics required are added after autoclaving to prevent degradation. Publications Ang K, Ee G, Ang E, Koh E, Siew WL, Lehming, N (2011) Mediator Acts Upstream of the Transcriptional Activator Gal4. Mechanisms of Eukaryotic Transcription August 30 – September (Poster) Ang K, Ee G, Siew WL, Chan YM, Nur S, Ang E, Tan YS, Lehming, N (2011) Mediator Acts Upstream of the Transcriptional Activator Gal4. PLOS Biology (PBIOLOGY-D-11-02778R3) Manuscript accepted and under copyediting at time of theis publication 198 [...]... means of regulating the 32 ubiquitin signal independent of the conventional ubiquitination pathway save for the ubiquitin ligase itself For example Eps15 can recruit the mono-ubiquitinated species of NEDD4 and the Parkin E3 ligases to affect its own mono-ubiquitination (Fallon et al., 2006) 1.4.6 Ubiquitin s Role in DNA Repair One of the important roles that ubiquitination as a signal plays in the cell... synthetic mutant with an isoleucine 44 mutation or the deletion of Vps9 itself As Vps9 possesses the ubiquitin- binding CUE domain, one possibility is that this UBD mediates intracellular interactions with a conjugated ubiquitin molecule on the same protein and gains sorting activity for ubiquitinated cargo upon release of this interaction (Donaldson et al., 20 03) 1.4.5 Ubiquitin in the Regulation of. .. endocytosis within the signaling cell and reception of the Notch signal in the receiving cell (Lai et al., 2005) Further investigation into the mechanisms behind the recognition of ubiquitin by proteins functioning as endocytotic adaptors found that some of these proteins can interact with more than one ubiquitin molecule at a time either through possessing more than one ubiquitin- binding domain or by the auspices... ubiquitin from the E2 to the target substrate It is the E3 that confers specificity to the ubiquitination process and catalyses the formation of an isopeptide bond between the ε-amino group of one of the substrate’s internal lysines and the C-terminal glycine residue of ubiquitin (Hochstrasser, 1996) 16 The ratio of these enzymes is not unlike a pyramid; in mammals there is only one gene encoding the. .. conjugating enzyme that has already bound a molecule of ubiquitin in order to facilitate ubiquitination by an E3 ubiquitin ligase (Di Fiore et al., 20 03) It is thought that this mono-ubiquitination serves to regulate the activity or the binding affinity of proteins possessing UBDs to either free ubiquitin in the cell or to other ubiquitinated proteins One such example where UBDs and the protein-protein interactions... out the components of the ubiquitin ligase Isolation of the E1 Ubiquitin Activating Enzyme, E2 Ubiquitin Conjugating Enzyme and the E3 Ubiquitin Ligase resulted in the Nobel Prize for Chemistry being awarded to Ciechanover and colleagues in 2004 (Hershko, et al., 19 83) The notion that it is the ubiquitin moiety that signals the destruction of proteins answered the key criticisms leveled against the. .. about by mono-ubiquitinated proteins tends to be rather nonspecific and exhibit low affinity This can be remedied by the formation of multimeric complexes of ubiquitin binding proteins In some cases the ubiquitin binding proteins can have additional interactions with the mono-ubiquitinated target through other domains to achieve a similar result (Di Fiore et al., 20 03) Another means of conferring specificity... through the use of the structural differences inherent in the types of ubiquitin chains K-48 chains tend to be more “kinked” in structure while the K- 63 chains have a more “extended” conformation It is the dynamic between these interactions of ubiquitinated and ubiquitin- binding proteins that gives rise to an ubiquitin network” in the cell (Kanayama et al., 2004) It has been posited that 28 ubiquitinated... the endosomes by certain ubiquitin- binding endocytotic regulators and adaptors The ubiquitin- binding domains have been implicated in the ubiquitination of their parent proteins as well Many proteins possessing UBDs are mono-ubiquitinated and their UBDs are required for this mono-ubiquitination to occur (Polo et al., 2002) One hypothesis is that these UBDs are necessary to recruit an E2 ubiquitin conjugating... for a polyubiquitin chain A polyubiquitin chain propagated at lysine 48 (K48) on ubiquitin is the classical degradation signal but a similar chain propagated at lysine 63 (K 63) on ubiquitin (Hicke et al., 2005) instead has other physiological roles; one such role is the regulation of NF-κB 1.4.2 Ubiquitin as a Signaling Molecule in the Regulation of Transcription Factors NF-κB is among a well characterized . 2005). 1 .3. 4 E4 Ubiquitin Ligases in the Elongation of Polyubiquitin Chains While the E3 ubiquitin ligases mediate the initial ubiquitination of the substrate, a new class of ubiquitin ligases. protein to fish out the components of the ubiquitin ligase. Isolation of the E1 Ubiquitin Activating Enzyme, E2 Ubiquitin Conjugating Enzyme and the E3 Ubiquitin Ligase resulted in the Nobel. antigen presentation (Kloetzel et al., 1999). 1 .3. 3 The Machinery and Process of Ubiquitination in Brief The ubiquitination of a single protein requires the concerted action of three separate