RNA polymerase II

RNA polymerase II is typically distinguished from RNA polymerases I and III by its sensitivity to low concentrations of the toxin a-amanitin (Roeder, 1976). This enzyme is a complex protein composed of ten polypeptides (Table 2). The subunit structure is remarkably conserved in divergent species, suggesting essential roles for each polypeptide. In fact, the subunit structure of all three eukaryotic RNA polymerases is similar, and three polypeptides are thought to be held in common. This suggests that the three types of polymerases evolved from a single enzyme and share common constraints in structure (Huet et ah, 1982). 

Polypeptide Structure of RNA Polymerase II from Various Eukaryotic 

Chemical modification of enzymes is frequently correlated with changes in levels of activity. Mammalian RNA polymerase II subunits are phosphorylated in vivo (Bell et al., 1977 Dahmus, 1981a). Labeling of HeLa cells with results in phosphate incorporation into pol II polypeptides of 240,000, 214,000, and 20,500 daltons (Dahmus, 1981a). Purified pol II is a substrate for both casein kinase I and II and for the cyclic AMP independent nuclear protein kinase NIL The 

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The general transcription factor TFllD is believed to be the key link between specific transcription factors and the general preinitiation complex. However, the purification and molecular characterization of TFllD from higher eucaryotes have been hampered by its instability and heterogeneity. All preparations of TFllD contain the TATA box-binding protein in combination with a variety of different proteins called TBP-associated factors, TAFs. When the preinitiation complex has been assembled, strand separation of the DNA duplex occurs at the transcription start site, and RNA polymerase II is released from the promoter to initiate transcription. However, TFIID can remain bound to the core promoter and support rapid reinitiation of transcription by recruiting another molecule of RNA polymerase. 

Buratowski, S. The basics of basal transcription by RNA Polymerase II. Cell 77 1-3, 1994. 

Due to the large amount of DNA present within the nucleus it must be carefully packaged. In the resting cell DNA is tightly compacted around basic histone proteins, excluding the binding of the enzyme RNA polymerase II, which activates the formation of mRNA. This conformation of the chromatin structure... 

Peroxisome Proliferator-Activated Receptors. Figure 3 Transcription of PPAR target genes. A schematic representation of the transcription of PPAR-regulated genes in the absence (a) and presence (b) of PPAR ligand. Abbreviations PPAR-RE, peroxisome proliferator-activated receptor-response element RNA Pol II, RNA polymerase II TATA-BP, TATA-binding protein. 

Although we will stick to the IL-6 gene, it should be mentioned at the side that two other RNA polymerases exist in mammalian cells responsible for the synthesis of RNA molecules, which are not translated into proteins ribosomal (rRNA), transfer (tRNA), small nuclear (snRNA), small nucleolar (snoRNA), and some of the recently discovered microRNAs and piRNAs. These RNA molecules act in the process of translation and mRNA turnover. Micro and piRNAs are probably extremely important in the definition of stem cells and of differentiation programs. Some of them are synthesized by RNA polymerase II. 

TBP-RNA polymerase) (ii) they are potent histone acetyl transferases inducing chromatin remodeling (loosening). 

Phosphorylation of HSF substantially enhances the transcriptional activity of HS gene expression which may be up to 100-fold of basal levels after HSFl binds to the promoter element. Heat shock will increase the C-terminal-domain-kinase activity in cell extracts, and this action may enhance the activity of RNA polymerase II that is bound to HS genes (Legagneux et al., 1990). Whether this kinase activity also affects HSFl phosphorylation is not known, but increased HS gene expression appears to occur as long as HSFl is bound to the promoter region. The CTD kinase complex contains multiple proteins, and it is quite possible that one or more of these proteins is also regulated by stress. 

Gilmour, D.S. Lis, J.T. (1986). RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene Drosophila melanogaster cells. Mol. Gen. Genet. 6, 3984-3989. 

Legagneux, V., Morange, M., Bensaude, O. (1990). Heat-shock and related stress enhance RNA polymerase II C-terminal-domain kinase activity in HeLa cell extracts. Eur. J. Biochemistry 193, 121-126. 

Dickinson, L.A., J.W. Trauger, E.E. Baird, P. Ghazal, P. B. Dervan, and J.M. Gottesfeld. Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides. Biochemistry 1999, 38, 10801-10807. 

Figure 36-4. Illustration of the tight correlation between the presence of RNA polymerase II and RNA synthesis. A number of genes are activated when Chirono-mus tentans larvae are subjected to heat shock (39 °C for 30 minutes). A Distribution of RNA polymerase II (also called type B) in isolated chromosome IV from the salivary gland (at arrows). The enzyme was detected by immunofluorescence using an antibody directed against the polymerase. The 5C and BR3 are specific bands of chromosome IV, and the arrows indicate puffs. B Autoradiogram of a chromosome IV that was incubated in H-uridine to label the RNA. Note the correspondence of the immunofluorescence and presence of the radioactive RNA (black dots). Bar = 7 pm. (Reproduced, with permission, from Sass H RNA polymerase B in polytene chromosomes. Cell 1982 28 274. Copyright 1982 by the Massachusetts Institute of Technology.)...

The moderately repetitive sequences, which are defined as being present in numbers of less than 10 copies per haploid genome, are not clustered but are interspersed with unique sequences. In many cases, these long interspersed repeats are transcribed by RNA polymerase II and contain caps indistinguishable from those on mRNA. 

The primary transcripts generated by RNA polymerase II—one of three distinct nuclear DNA-depen-dent RNA polymerases in eukaryotes—are promptly capped by 7-methylguanosine triphosphate caps (Figure 35-10) that persist and eventually appear on the 5 end of mature cytoplasmic mRNA. These caps are necessary for the subsequent processing of the primary transcript to mRNA, for the translation of the mRNA, and for protection of the mRNA against exonucleolytic attack. 

A small number of genes lack a TATA box. In such instances, two additional cis elements, an initiator sequence (Inr) and the so-called downstream promoter element (DPE), direct RNA polymerase II to the promoter and in so doing provide basal transcription starting from the correct site. The Inr element spans the start... 

Figure 37-7. Transcription elements and binding factors in the herpes simplex virus thymidine kinase ffW gene. DNA-dependent RNA polymerase II binds to the region of the TATA box (which is bound by transcription factor TEND) to form a multicomponent preinitiation complex capable of initiating transcription at a single nucleotide (+1).The frequency of this event is increased by the presence of upstream c/s-acting elements (the GC and CAAT boxes). These elements bind frans-acting transcription factors, in this example Spl and CTF (also called C/EBP, NF1, NFY). These cis elements can function independently of orientation (arrows).

Sequences farther upstream from the start site determine how frequently the transcription event occurs. Mutations in these regions reduce the frequency of transcriptional starts tenfold to twentyfold. Typical of these DNA elements are the GC and CAAT boxes, so named because of the DNA sequences involved. As illustrated in Figure 37—7, each of these boxes binds a protein, Spl in the case of the GC box and CTF (or C/EPB,NF1,NFY) by the CAAT box both bind through their distinct DNA binding domains (DBDs). The frequency of transcription initiation is a consequence of these protein-DNA interactions and complex interactions between particular domains of the transcription factors (distinct from the DBD domains—so-called activation domains ADs) of these proteins and the rest of the transcription machinery (RNA polymerase II and the basal factors TFIIA, B, D, E, F). (See... 

Figure 37-9. The eukaryotic basal transcription complex. Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions. The entire complex spans DNA from position -30 to +30 relative to the initiation site (+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and ofTBP bound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 A resolution. The structure of TFIID complexes have been determined by electron microscopy at 30 A resolution. Thus, the molecular structures of the transcription machinery are beginning to be elucidated. Much of this structural information is consistent with the models presented here.

Pol II associates with other proteins to form a holoenzyme complex. In yeast, at least nine gene products—called Srb (for suppressor of RNA polymerase B)—bind to the CTD. The Srb proteins—or mediators, as they are also called—are essential for pol II transcription, though their exact role in this process has not been defined. Related proteins comprising even more complex forms of RNA polymerase II have been described in human cells. 

Table 37-3. Some of the transcription control elements, their consensus sequences, and the factors that bind to them which are found in mammalian genes transcribed by RNA polymerase II. A complete list would include dozens of examples. The asterisks mean that there are several members of this family.

Cramer P, Bushnell DA, Kornberg R Structural basis of transcription RNA polymerase II at 2.8 angstrom resolution. Science 2001 292 1863. 

Hirose Y, Manley JL RNA polymerase II and the integration of nuclear events. Genes Dev 2000 l4 l4l5. 

Woychik NA, Hampsey M The RNA polymerase II machinery structure illuminates function. Cell 2002 108 453. 

Ebright RH RNA polymerase structural similarities between bacterial RNA polymerase and eukaryotic RNA polymerase II. J Mol Biol 2000 304 S87. 

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