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

Abstract

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...

Characterization of Human RNA Polymerase III Identifies Orthologues for Saccharomyces cerevisiae RNA Polymerase III Subunits

“…1B correspond to cytoskeletal and other known abundant proteins and were not characterized further. The bands labeled with a triangle correspond to previously characterized RNA polymerase III subunits [RPC1/RPC155 (50), RPC3/RPC62, RPC6/RPC39, and RPC7/RPC32 (58), RPC4/HsRPC53 (26,27,50), and RPABC1, RPABC2, RPABC3, and RPABC5 (8,38,40,51)]. The bands labeled with asterisks correspond to either known [RPC9/HsCGRP (Homo sapiens calcitonin gene-related peptide) receptor component (18,33), RPC10 (7), and RPAC1/RPA39 (12)] or novel (RPC2, RPC5, RPC8, and RPAC2) polypeptides whose function has not been analyzed in the context of human RNA polymerase III transcription but that show different degrees of similarity to S. cerevisiae RNA polymerase III subunits.…”

Interaction with general transcription factor IIF (TFIIF) is required for the suppression of activated transcription by RPB5-mediating protein (RMP)

“…With the exception of RPB5, all of the common subunits in human RNA polymerases I, II, and III can substitute for their yeast homologues, although RPB5 is highly conserved among humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, and is essential in yeast [2,31,32]. Yeast RPB5 has been reported to interact with RPB3 in vitro, and both RPB5 and RPB3 are present in two molar amounts in RNA polymerase II and seem to play an important role in subunit assembly, as does human RPB5.…”

Cloning of a novel human RNA polymerase II subunit downregulated by doxorubicin: new potential mechanisms of drug related toxicity

“…Eucaryotic RNA polymerases are multisubunit enzymatic complexes consisting of 10-12 subunits and RPB11 is a component of the RNA polymerase II multisubunit complex [11,12]. In yeast this protein is associated with 11 other subunits, is not shared among RNA polymerases I and III, and is also essential for cell viability [6,12,13]. The identity between yeast RPBll and the human homologue discussed in this paper is very high, demonstrating that this subunit is well conserved among eucaryotes as it has been previously shown for other subunits [12][13][14].…”

“…In yeast this protein is associated with 11 other subunits, is not shared among RNA polymerases I and III, and is also essential for cell viability [6,12,13]. The identity between yeast RPBll and the human homologue discussed in this paper is very high, demonstrating that this subunit is well conserved among eucaryotes as it has been previously shown for other subunits [12][13][14]. Interestingly, while this manuscript 51 was in progress Pati [6] described the cloning of human RNA polymerase II subunit hRPB14.…”