Nuclear RNA polymerase activity

Shah GM, Bhattacharya RK. 1989. Alteration in hepatic nuclear RNA polymerase activity following benzo[a]pyrene administration in rat. In Vivo 3(2) 125-127. 

Eukaryotic RNA polymerases are less well-characterized than the bacterial enzyme. Biochemical studies have so far identified three distinct nuclear RNA polymerase activities in eukaryotic cell extracts prepared from both yeast and human cells. These RNA polymerases, referred to as RNA pol I, RNA pol II, and RNA pol HI, each contain a large number of subunits, some of which appeared to be shared between the different RNA polymerase subtypes. As shown in Table 24.1, RNA pol I transcribes ribosomal RNA genes, RNA pol II transcribes the majority of protein-coding genes, into mRNA, and RNA pol III transcribes tRNA, small nuclear RNAs (snRNAs), and ribosomal 5S genes. Note also that the mitochondrial genome is transcribed by a nuclear-encoded mitochondrial RNA polymerase. The yeast mitochondrial RNA polymerase holoenzyme consists of a 140-kd catalytic subunit and a 43-kd promoter-recognition protein similar to the bacterial s factor. 

Just as biochemical analyses had identified three separate nuclear RNA polymerase activities, molecular ge-... 

Yu FL, Bender W, Fang Q, Ludeke A, Welch B. Prevention of chemical carcinogen DNA binding and inhibition of nuclear RNA polymerase activity by organosulfur compounds as the possible mechanisms for their anticancer initiation and proliferation effects. Cancer Detect Prev 2003 27 370-379. 

Nuclear extracts can be fractionated by chromatography on DEAE-cellulose to give three peaks of RNA polymerase activity (the use of column chromatography is explained in chapter 6). These three peaks correspond to three different RNA polymerases (I, II, and III), which differ in relative amount, cellular location, type of RNA synthesized, subunit structure, response to salt and divalent cation concentrations, and sensitivity to the mushroom-derived toxin a-amanitin. The three polymerases and some of their properties are summarized in table 28.4. 

Majumdar, A. P., Effects of fasting and tryptophan force-feeding on the activity of hepatic nuclear RNA polymerases in rats, Scand.. Clin. Lab. Invest., 39[1], 61, 1979. 

Italian scientists focussed their attention on the growth-promoting action of orotic acid in vitamin Bi2-deficient rats and chicks [24, 25]. Orotic acid and vitamin B12 have similar effects on the metabolism of the Cj-unit, whereby orotic acid increases the concentration of folate derivatives and influences the enzymes involved in the synthesis and utilization of folate intermediates [26,27]. Orotic acid also results in an increase in liver RNA concomitant with the stimulation of nuclear DNA-dependent RNA polymerase activity [28,29]. These findings led to the presumption that orotic acid increases messenger RNA synthesis. There are a number of further reports, mainly from Italy [3(M7], dealing with the relationship between orotic acid and vitamin deficiency, distribution and function [48 1]. 

The stimulation of the synthesis of poly (A) containing mRNA was obtained when the Artemia salina embryos development was stimulated. It was demonstrated that the proportion of mRNA containing poly (A) is increased in the nuclear RNA and in the soluble fraction of the cytoplasmic RNA (Christy and Jayaraman, 1975). At the same time the increasing of the RNA-polymerase activity was registered (Renart and Sebastian, 1976 D Alession and Bagshaw, 1977). 

Differences were found in in vitro and in vivo transcriptional activity of chromatin from various parts of the embryo (Flickinger et al., 1965). These differences can be explained by differences in permeability of animal and vegetative halves of the embryo. These differences might also be explained by a reduction of the amount of nuclear RNA-polymerase in the chromatin preparation from late... 

Activity of eukaryotic RNA polymerase may also be modulated by a phosphorylation-dephosphorylation modification. For example, in vitro studies with ovarian nuclear RNA polymerase and a cAMP-dependent protein kinase led to marked stimulation of the polymerase activity. A growing body of data supports the intriguing contention that cAMP mediates the translocation of the cytoplasmic protein kinases into the nucleus. This would then be the means by which the information first received at the cell membrane is communicated to the genome (Jungmann and Russell, 1977 Jungmann and Kranias, 1977). 

Thus, the concept that estrogens may exert their stimulatory action on the uterus by activation of RNA synthesis has been developed by several lines of biochemical investigation. The evidence includes the observation that estrogen stimulation leads quickly to an increase in the rate of incorporation of precursors into RNA with the initial increase noted in the nuclear fraction of RNA, that RNA polymerase activity in uterine cells is enhanced by estrogen stimulation, that Rie nucleotide sequence of RNA synthesized by uterine nuclei is altered by estrogen, and that specific inhibitors of DNA-dependent RNA synthesis prevent the stimulatory action of estrogen. 

Fig. 6.2. Proposed mechanisms of action of pure antiestrogens (fulvestrant). 1 Fulvestrant (ICI) binds to estrogen receptor (ER). 2 Fulvestrant binding to ER accelerates receptor degradation ( ER down-regulator ). 3 Rate of dimerization and nuclear localization of fulvestrant-ER complex is reduced. 4 Reduced binding of fulvestrant-ER to ERE. 5 No transcription of estrogen-responsive genes since AF-1 and AF-2 are inactive, no coactivators are recruited and the activity of RNA polymerase II is not activated (or inhibited) (Wakeling 2000)...

While bound to its cognate response element, the liganded/dimerized receptor recruits co-activator proteins that link with additional transcription factors, often leading to acetylation of histones, which opens up the nucleo-some to admit RNA polymerase II to the transcription start site. As would be expected, given that there is a sizeable superfamily of nuclear receptors and numerous interacting proteins, this simplified central theme is subject to many variations and complexities that allow subtle fine-tuning of regulatory responses. 

Fig. 7. Model for activation by coactivators (A) and inhibition by corepressors (B) of transcription. Abbreviations CBP/p300, cAMP response element binding protein SRC-1, steroid receptor coactivator 1 TBP, TATA-binding protein TAF, TBP-associated factor pol II, RNA polymerase II N-CoR, nuclear receptor corepressor SMRT, silencing mediator of retinoic and thyroid hormone receptors.

As mentioned above, one consequence of stalled RNA polymerase II at a DNA adduct is activation of transcription-coupled repair [27], This effect may depend on the type of polymerase, however, since the removal of some types of DNA damage is slower from RNA-polymerase I transcribed ribosomal DNA than from a nuclear gene [160], The lower level of repair in the nucleolus could also reflect the influence of other transcription factors, such as the HMG-domain protein UBF, which bind to cisplatin-mod-ified DNA [145]. When HeLa cells were exposed to cisplatin at concentrations which did not seem to affect nuclear transcription, inhibition of rDNA gene expression was associated with the redistribution of UBF, along with other factors responsible for rRNA transcription [138], These observations indicate how cisplatin might exert a combination of effects. Transcription is stopped due to titration of essential factors by the platinum-DNA adducts, and the same proteins could shield the lesions from the repair activity. 

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