PRS3 encoding an essential subunit of yeast proteasomes homologous to mammalian proteasome subunit C5.

We found by computer analysis that a putative yeast proteasome subunit gene named PRS3 that encodes a protein very similar to subunit C5 of rat and human proteasomes is located immediately 3' to the ERD2 gene of Saccharomyces cerevisiae. The similarity of the primary structures of the two suggests that this subunit may have a common function in proteasomes of all eukaryotes. The protein, deduced from the open reading frame of PRS3, consists of 242 amino acid residues with a calculated molecular weight of 27,077. Chromosomal disruption of the PRS3 gene created a recessive lethal mutation. Physical mapping by hybridization to intact S. cerevisiae chromosomal DNA showed that the PRS3 gene is located on chromosome II, unlike two other subunit genes, PRS1 and PRS2, which are located on chromosomes XV and VII, respectively. These findings indicate that the PRS3 protein is a subunit of yeast proteasomes that is essential for cell viability.     

RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth.

RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae. The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit. The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons. The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase. Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures. RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro. The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range.     

An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension.     

Rpa12p, a conserved RNA polymerase I subunit with two functional domains

Rpa12p is a subunit of RNA polymerase I formed of two zinc-binding domains. The N-terminal zinc region (positions 1-60) is poorly conserved from yeast to man. The C-terminal domain contains an invariant Q.RSADE.T.F motif shared with the TFIIS elongation factor of RNA polymerase II and its archaeal counterpart. Deletions removing the N-terminal domain fail to grow at 34 degrees C, are sensitive to nucleotide-depleting drugs and become lethal in rpa14-Delta mutants lacking the non-essential RNA polymerase I subunit Rpa14p. They also strongly alter the immunofluorescent properties of RNA polymerase I in the nucleolus. Finally, they prevent the binding of Rpa12p to immunopurified polymerase I and impair a specific two-hybrid interaction with the second largest subunit. In all these respects, N-terminal deletions behave like full deletions. In contrast, C-terminal deletions retaining only the first N-terminal 60 amino acids are indistinguishable from wild type. Thus, the N-terminal zinc domain of Rpa12p determines its anchoring to RNA polymerase I and is the only critical part of that subunit in vivo.     

Conditional mutations occur predominantly in highly conserved residues of RNA polymerase II subunits.

Conditional mutations in the Saccharomyces cerevisiae RNA polymerase II large subunit, RPB1, were obtained by introducing a mutagenized RPB1 plasmid into yeast cells, selecting for loss of the wild-type RPB1 gene, and screening the cells for heat or cold sensitivity. Sequence analysis of 10 conditional RPB1 mutations and 10 conditional RPB2 mutations revealed that the amino acid residues altered by these distinct mutations are nearly always invariant among eucaryotic RPB1 and RPB2 homologs. These results suggest that RNA polymerase mutants might be obtained in other eucaryotic organisms by alteration of these invariant residues.     

A Mycoplasma protein homologous to mammalian SRP54 recognizes a highly conserved domain of SRP RNA.

A protein homologous to SRP54, a subunit of the mammalian signal recognition particle (SRP), was identified in Mycoplasma mycoides. The mycoplasma protein was expressed in E.coli and purified to near homogeneity. It was shown to bind specifically in vitro to a small mycoplasma RNA with structural features related to the RNA component of SRP. These findings provide evidence of a ribonucleoprotein complex in mycoplasma reminiscent of SRP. A part of the RNA was protected from ribonuclease digestion in the presence of the SRP54 homologue. The protected region contains structural elements that have been highly conserved in SRP RNAs during evolution.     

The amino acid sequence of the human RNA polymerase II 33-kDa subunit hRPB 33 is highly conserved among eukaryotes

We have cloned and sequenced a cDNA of 1766 base pairs in length encoding the 275 amino acids of hRPB 33, the third largest subunit of human RNA polymerase II. The DNA was isolated by screening of a human lambda gt11 cDNA library with oligonucleotides designed on the basis of the amino acid residue analysis of the bovine material. The hRPB 33 amino acid sequence is highly conserved between Saccharomyces cerevisiae and human. Overall, 45% of the amino acid residues are identical with the yeast homologue RPB 3, and 65% of the amino acids are identical in the two major conserved regions at residues 0-103 and 151-197. hRPB 33 is also homologous to yeast RPC 5. The amino acid sequence of hRPB 33 showed no obvious homology with bacterial RNA polymerase or with any of its sigma factors.