Four Subunits That Are Shared by the Three Classes of RNA Polymerase Are Functionally Interchangeable between Homo sapiens and Saccharomyces cerevisiae

Abstract

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10␣, Hs10␤, Hs8, and Hs6, respectively), homologous to the ABC10␣, ABC10␤, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10␣, Sc10␤, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Eukaryotic mRNAs are synthesized by large transcription complexes formed by RNA polymerase II and a number of protein cofactors controlling the selectivity and efficiency of transcriptional initiation, elongation, and termination (14, 36). Purified preparations of RNA polymerase II were obtained for several eukaryotes (references 26, 37, 38, and 54 and references therein) and were found to consist of at least 10 distinct polypeptides ranging from 220 to less than 10 kDa. Their subunit structure is thus much more complex than is that of the three-component bacterial core enzyme ␣ 2 ␤␤Ј. Archaeal RNA polymerases also contain a large number of polypeptides, and most of them are related to eukaryotic subunits (21,(23)(24)(25)38).The genes encoding the 12 subunits of the yeast enzyme have all been cloned and sequenced (see Table 1). The three largest subunits, Sc1, Sc2, and Sc3, are homologous to the ␤Ј, ␤, and ␣ components of the bacterial core enzyme (28, 38, 54). Sc11 (50) is homologous to AC19, a subunit which is shared by yeast RNA polymerases I and III (15). Sc7 is similar to what is most probably the C25 subunit of RNA polymerase III (39). Sc7 was initially believed to be nonessential for mRNA synthesis in vivo (54), but further studies indicated that deletion of the corresponding gene is lethal (29). Five small subunits, Sc5, Sc6, Sc8, Sc10␣, and Sc10␤ (22,44,49,52), are present in all three nuclear RNA polymerases (11,12,37,46). These 10 specific or common subunits are essential components of the transcription apparatus, as strains carrying the corresponding null alleles are nonviable. In contrast, the deletion of the genes encoding the RNA polymerase II-specific Sc4 and Sc9 subunits leads to slowly growing but viable mutants (48, 51).The human RNA polymerase II, although less extensively characterized, contains at least 10 distinct subunits (19,26). The yeast and animal enzymes are closely related antigenically (18), indicating a strong evolutionary conservation. This was directly established by cloning and sequencing seven human cDNAs encoding RNA polymerase II subunits Hs1, Hs2, Hs3, Hs5, Hs6, Hs9,[32][33][34]47), which all showed significant homology to the corresponding yeast subunits (see Table 1). However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the human enzyme (26) failed to reveal small polypeptides of less than 10 kDa that would correspond to the Sc10␣ and Sc10␤ subunits shared by all three yeast RNA polymerases (12).It was recently demonstrated that the common subunit Sc6 of Saccharomyces cerevisiae can be functionally replaced in vivo by homologs from Schizosaccharomyces pombe and ha...

The Rpb9 Subunit of RNA Polymerase II Binds Transcription Factor TFIIE and Interferes with the SAGA and Elongator Histone Acetyltransferases

“…Plasmids are listed in Table I. pGEN-13MYC was constructed by directional cloning of the 552 nt BamHI-EcoRI fragment from pFA6 -13MYC (25) in the yeast expression vector pGEN (30). pVV101 is a pGEN-13MYC derivative obtained by PCR cloning of the TFA1 coding sequence at the BamHI site upstream of the 13MYC epitope.…”

Molecular assembly of RNA polymerase II from the fission yeast Schizosaccharomyces pombe: subunit–subunit contact network involving Rpb5

“…On the other hand, purified RNA polymerase II from S. pombe contains 10 polypeptides (Sakurai et al 1996) and up to the present time, we have cloned and sequenced the genes encoding three large core subunits, Rpb1, Rpb2 and Rpb3 (Azuma et al 1991;Kawagishi et al 1993;Azuma et al 1993) and five small subunits, Rpb5, Rpb7, Rpb8, Rpb10, and Rpb11 (this paper, H. Sakurai & A. Ishihama, in preparation). Shpakovski (1994) and Shpakovski et al (1995) isolated the genes encoding Rpb6 and Rpb12.…”

Identification of an autonomously initiating RNA polymerase III holoenzyme containing a novel factor that is selectively inactivated during protein synthesis inhibition

“…Despite these differences, some mechanistic similarities are apparent. The three RNA polymerases share five common subunits and other subunits that are highly related (Woychik et al 1990;McKune et al 1995;Shpakovski et al 1995), whereas the TATA-binding protein (TBP) is shared by three RNA polymerase type-specific accessory factors (Struhl 1994). Studies with isolated components have shown ordered pathways for the assembly of RNA polymerases and cognate initiation factors into active preinitiation complexes (for review, see Zawel and Reinberg 1995;Roeder 1996a).…”