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 eIF3mß. By means of the YTH system we also have shown that the new eIF3mß isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mß 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.

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

BACKGROUND: The sequences encoding the yeast RNA polymerase II (RPB) subunits are single copy genes. RESULTS: While 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 hRPB11balpha and hRPB11bbeta 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 hRPB11balpha 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. CONCLUSIONS: The type hRPB11b gene appears to result from recent genomic recombination events in the evolution of primates, involving sequence elements related to the MMR apparatus.

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

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-Delta null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w. A ygr089-Delta 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.

Functional conservation of RNA polymerase II in fission and budding yeasts.

The complementary DNAs of the 12 subunits of fission yeast (Schizosaccharomyces pombe) RNA polymerase II were expressed from strong promoters in Saccharomyces cerevisiae and tested for heterospecific complementation by monitoring their ability to replace in vivo the null mutants of the corresponding host genes. Rpb1 and Rpb2, the two largest subunits and Rpb8, a small subunit shared by all three polymerases, failed to support growth in S. cerevisiae. The remaining nine subunits were all proficient for heterospecific complementation and led in most cases to a wild-type level of growth. The two alpha-like subunits (Rpb3 and Rpb11), however, did not support growth at high (37 degrees C) or low (25 degrees C) temperatures. In the case of Rpb3, growth was restored by increasing the gene dosage of the host Rpb11 or Rpb10 subunits, confirming previous evidence of a close genetic interaction between these three subunits.

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

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.

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.

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

ABC10beta, 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 CX2C. CC that is strictly essential for yeast growth, as shown by site-directed mutagenesis, whereas the rest of the ABC10beta sequence is fairly tolerant to amino acid replacements. ABC10beta has Zn2+ binding properties in vitro, and the CX2C. 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 ABC10beta 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.

Four subunits that are shared by the three classes of RNA polymerase are functionally interchangeable between Homo sapiens and Saccharomyces cerevisiae.

Four cDNAs encoding human polypeptides hRPB7.0, hRPB7.6, hRPB17, and hRPB14.4 (referred to as Hs10 alpha, Hs10 beta, Hs8, and Hs6, respectively), homologous to the ABC10 alpha, ABC10 beta, ABC14.5, and ABC23 RNA polymerase subunits (referred to as Sc10 alpha, Sc10 beta, Sc8, and Sc6, respectively) of Saccharomyces cerevisiae, were cloned and characterized for their ability to complement defective yeast mutants. Hs10 alpha and the corresponding Sp10 alpha of Schizosaccharomyces pombe can complement an S. cerevisiae mutant (rpc10-delta::HIS3) defective in Sc10 alpha. The peptide sequences are highly conserved in their carboxy-terminal halves, with an invariant motif CX2CX12RCX2CGXR corresponding to a canonical zinc-binding domain. Hs10 beta, Sc10 beta, and the N subunit of archaeal RNA polymerase are homologous. An invariant CX2CGXnCCR motif presumably forms an atypical zinc-binding domain. Hs10 beta, but not the archaeal subunit, complemented an S. cerevisiae mutant (rpb10-delta 1::HIS3) lacking Sc10 beta. Hs8 complemented a yeast mutant (rpb8-delta 1::LYS2) defective in the corresponding Sc8 subunit, although with a strong thermosensitive phenotype. Interspecific complementation also occurred with Hs6 and with the corresponding Dm6 cDNA of Drosophila melanogaster. Hs6 cDNA and the Sp6 cDNA of S. pombe are dosage-dependent suppressors of rpo21-4, a mutation generating a slowly growing yeast defective in the largest subunit of RNA polymerase II. Finally, a doubly chimeric S. cerevisiae strain bearing the Sp6 cDNA and the human Hs10 beta cDNA was also viable. No interspecific complementation was observed for the human hRPB25 (Hs5) homolog of the yeast ABC27 (Sc5) subunit.

The fission yeast Schizosaccharomyces pombe rpb6 gene encodes the common phosphorylated subunit of RNA polymerase and complements a mutation in the corresponding gene of Saccharomyces cerevisiae.

A single-copy gene, homologous to the RPB6 gene from Saccharomyces cerevisiae, encoding a small phosphorylated subunit common to all three forms of nuclear DNA-dependent RNA polymerase was isolated from the fission yeast Schizosaccharomyces pombe. Its cDNA copy consists of an open reading frame of 142 codons and encodes an acidic protein (predicted pI 4.1) with a M(r) of 15,730. The genomic copy of Sz. pombe rpb6 contains an intron (219 nucleotides) located at codon 92, a position which does not correspond to the single intron of the S. cerevisiae gene. The sequencing of both genomic and cDNA copies of rpb6 allowed us to determine the probable positions of the start and stop of rpb6 transcription and to identify a putative TATA box. The primary structures of the Sz. pombe and S. cerevisiae Rpb6 proteins have 60.7% identity, with the same general organization: a highly acidic N-terminal region followed by a short basic region and a C terminus featuring a putative heptad Leu repeat. The C-terminal half of the sequence is particularly well conserved and, therefore, probably contains the most important functional domain. Moreover, a heterospecific complementation test showed that rpb6 from Sz. pombe fully complements a complete deletion of its S. cerevisiae homologue.

Sequence analysis of closely related retrotransposon families from fission yeast.

Two families of retrotransposons, Tf1 and Tf2, have been isolated from the fission yeast, Schizosaccharomyces pombe. We report here the nucleotide (nt) sequence of a Tf2 element, the only retrotransposon family known from the commonly used laboratory strains, 972 and 975, and their derivatives. The total nt sequence of Tf2 was derived from the complete sequence of the coding region and 3' long terminal repeat (LTR) of randomly cloned element Tf2-1, and from a full 5' LTR and approximately one-third of the open reading frame (ORF) of Tf2-43, a Tf2 element found in the head-to-head orientation adjacent to the Sz. pombe rpb6 gene. The two Tf2 sequences are nearly identical and both of them contain a single ORF encoding a protein with regions of sequence similar to protease, reverse transcriptase, RNase H (RH) and integrase from other retrotransposons and retroviruses. Sequence comparisons between Tf1 and Tf2 indicate an extreme divergence of the putative capsid protein-encoding regions of these two elements, as well as divergence of a segment of the LTR, but otherwise virtually identical sequence.

Lambda plac10 transducing bacteriophage: DNA primary structure of the region of the abnormal excision.

In studying molecular mechanisms of the formation of transducing bacteriophages, we have elucidated the primary structure of the phage-bacterial DNA junction which resulted from the abnormal excision of the lambda plac10 phage. The process is structurally similar to the excision of the lambda plac5 phage and involves, in both cases, highly homological DNA stretches approximately 20 bp long, one of them being a part of the Z-Y spacer of the lac operon and possessing a developed secondary structure. The conception of regioselective recombination as a type of illegitimate recombinational process with a certain degree of site-specificity is suggested.

Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.

Site-specificity of abnormal excision: the mechanism of formation of a specialized transducing bacteriophage lambda plac5.

Molecular mechanism of the specialized transducing bacteriophage lambda plac5 formation has been studied. Phage-bacterial DNA junctions in lambda plac5 DNA are localized and primary structure of regions of the abnormal excisional recombination leading to the phage formation is elucidated; the crossover region proved to be comparable with the central part of attP and attB sites (the core and the adjacent tetranucleotide) in length and degree of homology. Bacterial insert in lambda plac5 DNA is shown to end immediately after Z-Y spacer, the DNA not containing lacY gene segments. The data obtained led to the conclusion of site-specific (homologous) character of abnormal excision upon formation of lambda transducing bacteriophages. Possible mechanisms of the excision are discussed.