Characterization of RNA polymerase type II from human term placenta

Freund, Erwin; McGuire, Patricia

doi:10.1002/jcp.1041270312

Cited by 6 publications

(4 citation statements)

References 34 publications

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“…Because of this extraordinary level of evolutionary conservation, the plasmid shuffle experiment revealed that the human subunit is also fully functional in S. cerevisiae. Human RNA polymerase II appears have at least nine subunits (16), two of which are large (-200 and -150 kDa) and seven of which are small (<50 kDa). The genes encoding human subunits related to yeast RPB1, RPB3, RPB5, and RPB9 have been isolated (1,10,25,26).…”

Section: Resultsmentioning

confidence: 99%

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

McKune¹,

Woychik²

1994

Mol. Cell. Biol.

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We isolated the cDNA encoding the homolog of the Saccharomyces cerevisiae nuclear RNA polymerase common subunit RPB6 from hamster CHO cells. Alignment of yeast RPB6 with its mammalian counterpart revealed that the subunits have nearly identical carboxy-terminal halves and a short acidic region at the amino terminus. Remarkably, the length and amino acid sequence of the hamster RPB6 are identical to those of the human RPB6 subunit. The conservation in sequence from lower to higher eukaryotes also reflects conservation of function in vivo, since hamster RPB6 supports normal wild-type yeast cell growth in the absence of the essential gene encoding RPB6.The eukaryotic mRNA transcriptional apparatus consists of RNA polymerase II as well as several gene-specific and general transcription factors. As many of the players in this process are becoming better defined genetically and biochemically, the conserved nature of these proteins is becoming increasingly apparent. In the case of one general transcription factor, TATA-binding protein (TBP), the functional relatedness suggested by the evolutionary conservation among TBPs has been verified by using in vitro transcription assays. The TBPs of humans and of the yeast Saccharomyces cerevisiae are functionally interchangeable in their response to yeast acidic activators and for basal transcription in vitro (8,9,15,19,27). However, human TBP is unable to support growth of S. cerevisiae cells in vivo (12,17). In fact, of the few tested, none of the RNA polymerase subunits or basal transcription factors from related eukaryotic species function in S. cerevisiae in vivo.A major component of the eukaryotic transcriptional apparatus is the multisubunit enzyme RNA polymerase II. S. cerevisiae RNA polymerase II comprises 12 known subunits, RPBI1 to RPB12, whose genes have been cloned, sequenced, and characterized (3,20,23,29,(32)(33)(34)(35)(36)(37). Structural and functional studies of yeast RNA polymerase II genes and their products have led to an improved picture of the enzyme. The three largest subunits, RPB1, RPB2, and RPB3, are related to the prokaryotic core RNA polymerase subunits, are essential, and are probably largely responsible for RNA catalysis (reviewed in references 38 and 39). Five additional subunits (RPB5, RPB6, RPB8, RPB1O, and RPB12) are essential components shared by all three nuclear RNA polymerases (32,34,37). The remaining four subunits, RPB4, RPB7, RPB9, and RPB11, are unique to RNA polymerase 11 (23,33,35,36).As part of our approach to understanding the function of each RNA polymerase subunit, we isolated the gene encoding the mammalian homolog of RPB6 from hamster CHO cells (designated hRPB6). The subunit sequence revealed a striking degree of amino acid similarity to S. cerevisiae RPB6. To our astonishment, the hRPB6 is also identical in length and amino acid sequence to the human RPB6 subunit (designated hsRPB6). Using the plasmid shuffle technique to test for in vivo complementation, we demonstrated that expression of the * Corresponding author. Mailing...

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Section: Resultsmentioning

confidence: 99%

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

McKune¹,

Woychik²

1994

Mol. Cell. Biol.

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“…While a number of groups have purified human pol II and associated proteins (26,32,36,45,51; also reviewed in references 57 and 70), it has been clear from a comparison of results that the number, molecular weight, and quantity of copurified proteins are somewhat variable between different groups and starting sources of material, and it was previously uncertain whether a human RPB4 homolog existed. The ϳ27 kDa hsRPB7 species and ϳ18-to 19-kDa native hsRPB4 species described in this work are now shown to be present in affinity-purified pol II, conclusively demonstrating their functionality in mammalian cells.…”

Section: Conservation Of Hsrpb4 Mmrpb4 and Rpb4 Subunitsmentioning

confidence: 99%

Analysis of the Interaction of the Novel RNA Polymerase II (pol II) Subunit hsRPB4 with Its Partner hsRPB7 and with pol II

Khazak

Estojak

Cho

et al. 1998

Molecular and Cellular Biology

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Under conditions of environmental stress, prokaryotes and lower eukaryotes such as the yeast Saccharomyces cerevisiae selectively utilize particular subunits of RNA polymerase II (pol II) to alter transcription to patterns favoring survival. In S. cerevisiae, a complex of two such subunits, RPB4 and RPB7, preferentially associates with pol II during stationary phase; of these two subunits, RPB4 is specifically required for survival under nonoptimal growth conditions. Previously, we have shown that RPB7 possesses an evolutionarily conserved human homolog, hsRPB7, which was capable of partially interacting with RPB4 and the yeast transcriptional apparatus. Using this as a probe in a two-hybrid screen, we have now established that hsRPB4 is also conserved in higher eukaryotes. In contrast to hsRPB7, hsRPB4 has diverged so that it no longer interacts with yeast RPB7, although it partially complements rpb4 ؊ phenotypes in yeast. However, hsRPB4 associates strongly and specifically with hsRPB7 when expressed in yeast or in mammalian cells and copurifies with intact pol II. hsRPB4 expression in humans parallels that of hsRPB7, supporting the idea that the two proteins may possess associated functions. Structure-function studies of hsRPB4-hsRPB7 are used to establish the interaction interface between the two proteins. This identification completes the set of human homologs for RNA pol II subunits defined in yeast and should provide the basis for subsequent structural and functional characterization of the pol II holoenzyme.Selective control of mRNA transcription in response to intracellular and extracellular signals occurs at multiple levels, with targets for regulation including gene-specific transcription factors, general transcription factors, and the RNA polymerase II holoenzyme (15,18,38,55,63). This last mechanism of regulation, involving modification of core RNA polymerase II (pol II) structural composition by altering incorporation of subunits or regulated phosphorylation, has been well documented in prokaryotes and in yeast (17,31,59,62,70). In higher eukaryotes, the majority of transcriptional control studies have focused on characterizing the expression and modification of gene-specific and general transcription factors. However, a growing body of work on mammalian transcriptional control has demonstrated that mammalian pol II is also subject to modification by phosphorylation of the largest subunit, presumably as a means of regulation (10,24,41,42,49). In contrast, the issue of subunit variation has not been actively investigated.Studies of eukaryotic pol II function have depended heavily on paradigms developed through detailed characterization of the yeast Saccharomyces cerevisiae pol II (reviewed in reference 70). Yeast pol II contains 12 subunits (RPB1-12), all of which have been cloned and sequenced and many of which have been subjected to genetic and biochemical functional analysis. Five of these subunits, the common subunits (RPB5, RPB6, RPB8, RPB10, and RPB12), are also incorporated into RNA polymeras...

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“…Upon biochemical purification, human pol II appears have at least nine subunits (8), two large (ϳ200-and 150-kDa) and seven small (Ͻ50-kDa) subunits. However, human pol II is likely have at least as many subunits as the 12-subunit yeast S. cerevisiae enzyme since all 12 human genes related to the yeast subunits have now been identified.…”

Section: Resultsmentioning

confidence: 99%

“…It is still unclear whether these 12 human pol II subunits represent the complete set of subunits. Human pol II is relatively poorly defined at the biochemical level (8). Therefore, the precise number of human RNA polymerase subunits that copurify with in vitro transcription activity is not known.…”

Section: Resultsmentioning

confidence: 99%

Six Human RNA Polymerase Subunits Functionally Substitute for Their Yeast Counterparts

McKune

Moore

Hull

et al. 1995

Molecular and Cellular Biology

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To assess functional relatedness of individual components of the eukaryotic transcription apparatus, three human subunits (hsRPB5, hsRPB8, and hsRPB10) were tested for their ability to support yeast cell growth in the absence of their essential yeast homologs. Two of the three subunits, hsRPB8 and hsRPB10, supported normal yeast cell growth at moderate temperatures. A fourth human subunit, hsRPB9, is a homolog of the nonessential yeast subunit RPB9. Yeast cells lacking RPB9 are unable to grow at high and low temperatures and are defective in mRNA start site selection. We tested the ability of hsRPB9 to correct the growth and start site selection defect seen in the absence of RPB9. Expression of hsRPB9 on a high-copy-number plasmid, but not a low-copy-number plasmid, restored growth at high temperatures. Recombinant human hsRPB9 was also able to completely correct the start site selection defect seen at the CYC1 promoter in vitro as effectively as the yeast RPB9 subunit. Immunoprecipitation of the cell extracts from yeast cells containing either of the human subunits that function in place of their yeast counterparts in vivo suggested that they assemble with the complete set of yeast RNA polymerase II subunits. Overall, a total of six of the seven human subunits tested previously or in this study are able to substitute for their yeast counterparts in vivo, underscoring the remarkable similarities between the transcriptional machineries of lower and higher eukaryotes.The eukaryotic mRNA transcription apparatus comprises RNA polymerase II, general transcription factors and their associated factors, and gene-specific factors (reviewed in references 6, 13, and 23). RNA polymerase II (pol II) is a multisubunit enzyme with ϳ12 to 15 subunits, depending on the organism, that plays a major role in mRNA synthesis since it possesses the catalytic machinery for the formation of the 3Ј-5Ј phosphodiester bonds between ribonucleoside triphosphates and presumably responds to signals from the multiple factors involved in regulating its function during initiation and elongation of mRNA synthesis.Yeast Saccharomyces cerevisiae pol II has been a useful paradigm for enzyme function since its subunit structure and amino acid sequences are strikingly similar to pol II subunits from a variety of eukaryotes and even archaebacteria. S. cerevisiae pol II has 12 subunits, designated RPB1-RPB12, ranging in size from ϳ190 to ϳ8 kDa (reviewed in references 26 and 27). Five of these subunits (RPB5, RPB6, RPB8, RPB10, and RPB12) are also present in RNA polymerases I and III and are usually referred to as the common subunits.Since the functions of most pol II subunits are unclear, one approach to understanding their role in transcription has been to isolate the genes encoding human RNA polymerase subunits and test for functional similarities by determining if the human subunit can function in place of its yeast homolog. Only a few of the human subunit genes have been tested for heterocomplementation in S. cerevisiae. hsRPB6 has a highly conserve...

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Characterization of RNA polymerase type II from human term placenta

Cited by 6 publications

References 34 publications

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

Functional substitution of an essential yeast RNA polymerase subunit by a highly conserved mammalian counterpart.

Analysis of the Interaction of the Novel RNA Polymerase II (pol II) Subunit hsRPB4 with Its Partner hsRPB7 and with pol II

Six Human RNA Polymerase Subunits Functionally Substitute for Their Yeast Counterparts

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