Disease relevance of RPO21

• A dot matrix comparison of the deduced amino acid sequence of subunit A190 with Escherichia coli beta' and cognate subunits B220 and C160 from yeast RNA polymerases B and C showed a conserved pattern of homology regions (I-VI) [1].
• In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU [2].

High impact information on RPO21

• All strains containing suppressor mutations in RPB1 and RPB2 have reduced transcription of the INO1 gene and an inositol requirement [3].
• We have determined the nucleotide sequence of two yeast RNA polymerase genes, RPO21 and RPO31, which encode the largest subunits of RNA polymerases II and III, respectively [4].
• Export ceases in either the presence of the RNA synthesis inhibitor thiolutin or in a temperature-sensitive RNA polymerase (rpb1) mutant [5].
• RPB9 appears to play a unique role in transcription initiation, as the defects revealed in its absence are distinct from those seen with mutants in RNA polymerase subunit RPB1 and factor e (TFIIB), two other yeast proteins also involved in start site selection [6].
• The diverse functions of Saccharomyces cerevisiae RNA polymerase II are partitioned among its 12 subunits, designated RPB1-RPB12 [6].

Chemical compound and disease context of RPO21

• Mutations in the Saccharomyces cerevisiae RPB1 Gene Conferring Hypersensitivity to 6-Azauracil [2].

Biological context of RPO21

• We have used a yeast genomic library to isolate plasmids that can suppress a temperature-sensitive mutation in RPO21 (rpo21-4), with the goal of identifying gene products that interact with the largest subunit of RNAPII [7].
• However, scs32 was unable to suppress the ts phenotype of mutant alleles of RPO21, or result in accumulation of the unstable rpo21-4p [8].
• Synthetic lethality was associated with double sua7 sua8 suppressor mutations, and recessive sua7 mutants failed to fully complement recessive sua8 mutants in heterozygous diploids (nonallelic noncomplementation) [9].
• The sua8 suppressors of Saccharomyces cerevisiae encode replacements of conserved residues within the largest subunit of RNA polymerase II and affect transcription start site selection similarly to sua7 (TFIIB) mutations [9].
• Shorter or longer RPO21 C-terminal domains enhanced or partially suppressed, respectively, the effects of deletions in the transcriptional-activation domains of GAL4 [10].

Anatomical context of RPO21

• The data show that B220 inhibits neutrophil release of reactive oxygen species, as well as intracellular generation of reactive oxygen species [11].
• In addition we show here that the levels of expression of this same RNA polymerase II subunit directly affect cellular differentiation, reducing the rate of cell proliferation, clonogenicity and increasing the expression of E-cadherin, a marker of epithelial cell differentiation [12].

Associations of RPO21 with chemical compounds

• A screen for mutations in RPO21 that confer 6AU sensitivity identified seven mutations that had been generated by either linker-insertion or random chemical mutagenesis [13].
• Underproduction of RNAPII was achieved by expressing the gene (RPO21), encoding the largest subunit of the enzyme, from the LEU2 promoter or a weaker derivative of it, two promoters that can be repressed by the addition of leucine to the growth medium [14].
• We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect [15].
• Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and ubiquitination lie within the region required for in vivo function [16].
• Treatment of yeast and human cells with DNA-damaging agents elicits lysine 48-linked polyubiquitylation of Rpb1, the largest subunit of RNA polymerase II (Pol II), which targets Pol II for proteasomal degradation [17].

Physical interactions of RPO21

• These data indicate that RPB2 and RPB3 form a complex that subsequently interacts with RPB1 during the assembly of RNA polymerase II [18].
• Rpb5 was found to interact with any fragment of Rpb1 that contained the region H, which is conserved among the subunit 1 homologues of all RNA polymerases, including the beta' subunit of prokaryotic RNA polymerases [19].

Enzymatic interactions of RPO21

• Third, the association of SET2 with CTD phosphorylated RPO21 remained in the absence of ssn3 [20].
• Fusion of the Rpb1 carboxyl-terminal domain to another protein also causes that protein to be ubiquitinated by Rsp5 [21].

Regulatory relationships of RPO21

• We have changed the number of heptapeptide repeats in this yeast RPO21 C-terminal domain and have expressed these mutant RNA polymerase II polypeptides in yeast cells containing either wild-type or defective GAL4 proteins [10].
• We found that cells that underproduced RPO21 were unable to derepress fully the expression of a reporter gene under the control of the INO1 UAS [14].
• We found that the 6AU sensitivity of the rpo21 mutants can be suppressed by increasing the dosage of the wild-type PPR2 gene, presumably as a result of overexpression of TFIIS [13].
• Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II [22].

Other interactions of RPO21

• These results support the idea that the RPO26 and RPO21 gene products interact [7].
• Analysis of cyc1 transcripts from sua8 mutants revealed that suppression is a consequence of diminished transcription initiation at the normal start sites in favor of initiation at downstream sites, including a site between the aberrant and normal ATG start codons [9].
• A potential DNA-binding site (zinc-binding motif) is conserved in the N-terminal region I. Remarkably, the A190 subunit does not harbor the heptapeptide repeated sequence present in the B220 subunit [1].
• RNA polymerase II subunit composition, stoichiometry, and phosphorylation were investigated in Saccharomyces cerevisiae by attaching an epitope coding sequence to a well-characterized RNA polymerase II subunit gene (RPB3) and by immunoprecipitating the product of this gene with its associated polypeptides [23].
• The same RPO21 mutations also affected transcriptional activation by a GAL4-GCN4 chimera [10].

Analytical, diagnostic and therapeutic context of RPO21

• The utility of HisUb as a method for purification of proteins ubiquitinated in vivo was demonstrated by metal chelation chromatography of yeast extracts expressing HisUb and immunoblotting for Rpb1, the largest subunit of RNA polymerase II [24].

References