George V. Shpakovski

Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III.

ABSTRACT Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP..LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP..LW.GKQ provokes a temperature-sensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Δ null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan geneYGR089w. A ygr089-Δ null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.

Mutants in ABC10beta, a conserved subunit shared by all three yeast RNA polymerases, specifically affect RNA polymerase I assembly.

ABC10β, a small polypeptide common to the three yeast RNA polymerases, has close homology to the N subunit of the archaeal enzyme and is remotely related to the smallest subunit of vaccinial RNA polymerase. The eucaryotic, archaeal, and viral polypeptides share an invariant motif CX 2C… CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10β sequence is fairly tolerant to amino acid replacements. ABC10β has Zn2+ binding properties in vitro, and the CX 2C … CC motif may therefore define an atypical metal-chelating site. Hybrid subunits that derive most of their amino acids from the archaeal subunit are functional in yeast, indicating that the archaeal and eucaryotic polypeptides have a largely equivalent role in the organization of their respective transcription complexes. However, all eucaryotic forms of ABC10β harbor a HVDLIEK motif that, when mutated or replaced by its archaeal counterpart, leads to a polymerase I-specific lethal defect in vivo. This is accompanied by a specific lack in the largest subunit of RNA polymerase I (A190) in cell-free extracts, showing that the mutant enzyme is not properly assembled in vivo.

Ancient origin, functional conservation and fast evolution of DNA-dependent RNA polymerase III

RNA polymerase III contains seventeen subunits in yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and in human cells. Twelve of them are akin to the core RNA polymerase I or II. The five other are RNA polymerase III-specific and form the functionally distinct groups Rpc31-Rpc34-Rpc82 and Rpc37-Rpc53. Currently sequenced eukaryotic genomes revealed significant homology to these seventeen subunits in Fungi, Animals, Plants and Amoebozoans. Except for subunit Rpc31, this also extended to the much more distantly related genomes of Alveolates and Excavates, indicating that the complex subunit organization of RNA polymerase III emerged at a very early stage of eukaryotic evolution. The Sch.pombe subunits were expressed in S.cerevisiae null mutants and tested for growth. Ten core subunits showed heterospecific complementation, but the two largest catalytic subunits (Rpc1 and Rpc2) and all five RNA polymerase III-specific subunits (Rpc82, Rpc53, Rpc37, Rpc34 and Rpc31) were non-functional. Three highly conserved RNA polymerase III-specific domains were found in the twelve-subunit core structure. They correspond to the Rpc17-Rpc25 dimer, involved in transcription initiation, to an N-terminal domain of the largest subunit Rpc1 important to anchor Rpc31, Rpc34 and Rpc82, and to a C-terminal domain of Rpc1 that presumably holds Rpc37, Rpc53 and their Rpc11 partner.

Rpc19 and Rpc40, two alpha-like subunits shared by nuclear RNA polymerases I and III, are interchangeable between the fission and budding yeasts.

Abstract The cDNAs and genes encoding the common subunits Rpc19 and Rpc40 of nuclear RNA polymerases I and III of Schizosaccharomyces pombe were isolated from cDNA and genomic libraries of the fission yeast and tested for their ability to substitute for the homologous genes in Saccharomyces cerevisiae by heterospecific complementation of corresponding null alleles and temperature-sensitive mutations. The results obtained indicate that both Sz. pombe genes (rpc19+ and rpc40+) are able to replace their S. cerevisiae counterparts in vivo. The primary structure and general organization of both genes were established: rpc40+ is an intronless gene, while rpc19+ contains three introns (73, 48 and 77 bp long); rpc19+ is situated on the long arm of chromosome I and rpc40+ on the long arm of chromosome II.

Some peculiarities of steroid metabolism in transgenic Nicotiana tabacum plants bearing the CYP11A1 cDNA of cytochrome P450SCC from the bovine adrenal cortex

In the mitochondria of animal steroidogenic tissues, cytochrome P450SCC encoded by the CYP11A1 gene catalyzes the conversion of cholesterol into pregnenolone—the general precursor of all steroid hormones. In this work we study the steroid metabolism in transgenic tobacco plants carrying the CYP11A1 cDNA encoding cytochrome P450SCC from the bovine adrenal cortex. The transgenic plants under investigation markedly surpass the control wild-type plants by size and are characterized by a shortened period of vegetative growth (by rapid flowering); their leaves contain pregnenolone—the product of a reaction catalyzed by cytochrome P450SCC. The level of progesterone in transgenic tobacco leaves is higher than in the control plants of the wild type. The seeds of the transgenic plants contain less (24R)-brassinosteroids than the wild-type tobacco plants. The results obtained indicate that the synthesis of an active P450SCC cytochrome in transgenic Nicotiana tabacum plants has a profound effect on steroid metabolism and is responsible for the specific phenotypic features of transgenic plants bearing CYP11A1 cDNA.

A human RNA polymerase II subunit is encoded by a recently generated multigene family

BackgroundThe sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes.ResultsWhile those characterized so far for the human (h) RPB are also unique, we show that hRPB subunit 11 (hRPB11) is encoded by a multigene family, mapping on chromosome 7 at loci p12, q11.23 and q22. We focused on two members of this family, hRPB11a and hRPB11b: the first encodes subunit hRPB11a, which represents the major RPB11 component of the mammalian RPB complex ; the second generates polypeptides hRPB11bα and hRPB11bβ through differential splicing of its transcript and shares homologies with components of the hPMS2L multigene family related to genes involved in mismatch-repair functions (MMR). Both hRPB11a and b genes are transcribed in all human tissues tested. Using an inter-species complementation assay, we show that only hRPB11bα is functional in yeast. In marked contrast, we found that the unique murine homolog of RPB11 gene maps on chromosome 5 (band G), and encodes a single polypeptide which is identical to subunit hRPB11a.ConclusionsThe type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

Interactions between the full complement of human RNA polymerase II subunits

As an approach to elucidating the rules governing the assembly of human RNA polymerase II (hRPB), interactions between its subunits have been systematically analyzed. Eleven of the 12 expected hRPB subunits have previously been tested for reciprocal interactions (J. Biol. Chem. 272 (1997) 16815–16821). We now report the results obtained for the last subunit (hRPB4; Mol. Cell. Biol. 18 (1998) 1935–1945) and propose an essentially complete picture of the potential interactions occurring within hRPB. Finally, complementation experiments in yeast indicated that hRPB4 expression efficiently cured both heat and cold‐sensitivity of RPB4‐lacking strains, supporting the existence of conserved functional subunit interactions.

A minor isoform of the human RNA polymerase II subunit hRPB11 (POLR2J) interacts with several components of the translation initiation factor eIF3

Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mβ. By means of the YTH system we also have shown that the new eIF3mβ isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mβ isoform is detected predominantly in the cell nucleus. The discovered interactions of the hRPB11cα isoform with several hEIF3 subunits demonstrate a new type coordination between transcription and the following (downstream) stages of gene expression (such as mRNA transport from nucleus to the active ribosomes in cytoplasm) in Homo sapiens and point out the possibility of existence of nuclear hEIF3 subcomplexes.

i-Clamp phenoxazine for the fine tuning of DNA i-motif stability

Abstract Non-canonical DNA structures are widely used for regulation of gene expression, in DNA nanotechnology and for the development of new DNA-based sensors. I-motifs (iMs) are two intercalated parallel duplexes that are held together by hemiprotonated C-C base pairs. Previously, iMs were used as an accurate sensor for intracellular pH measurements. However, iM stability is moderate, which in turn limits its in vivo applications. Here, we report the rational design of a new substituted phenoxazine 2′-deoxynucleotide (i-clamp) for iM stabilization. This residue contains a C8-aminopropyl tether that interacts with the phosphate group within the neighboring chain without compromising base pairing. We studied the influence of i-clamp on pH-dependent stability for intra- and intermolecular iM structures and found the optimal positions for modification. Two i-clamps on opposite strands provide thermal stabilization up to 10–11°C at a pH of 5.8. Thus, we developed a new modification that shows significant iM-stabilizing effect both at strongly and mildly acidic pH and increases iM transition pH values. i-Clamp can be used for tuning iM-based pH probes or assembling extra stable iM structures for various applications.

PMS2 and POLR2J gene families as molecular markers of the higher primates evolution

We have studied the molecular evolution of two gene families specific for primates: POLR2J of the transcription system and PMS2 of the mismatch repair (MMR) system. The appearance of these families and upgrading of their genetic structures was shown to neatly correlate with the main stages of the biological evolution of higher primates. Our results indicate that the PMS2 and POLR2J genes can serve as helpful and reliable molecular markers of anthropogenesis.