ABSTRACT
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.
Subject(s)
Eukaryotic Initiation Factor-3/metabolism , RNA Polymerase II/metabolism , Eukaryotic Initiation Factor-3/genetics , Humans , Protein Binding , RNA Polymerase II/genetics , Two-Hybrid System TechniquesABSTRACT
The elongation complex, which involves RNA polymerase, DNA template and nascent RNA, is a central intermediate in transcription cycle. It is elongation complex that represents the main target for the action of different regulatory factors. Over the past several years, many structural and biochemical data have been obtained that shed light upon the molecular details of RNA polymerase function. Cooperation between RNA polymerase elongation complex and translating ribosome was established recently. Here we discuss the mechanisms of the regulation of bacterial transcription elongation.
Subject(s)
DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation, Bacterial , Ribosomes/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Adenosine Triphosphatases/metabolism , Catalysis , DNA-Directed RNA Polymerases/genetics , Nucleotides/metabolism , Protein Biosynthesis/genetics , Transcription Factors/geneticsABSTRACT
Site-directed mutagenesis was used to study the leader region of the Bacillus subtilis pbuE gene, which codes for adenine-specific sensor RNA. Two nucleotide substitutions, 70U --> C and A100 --> G, in the conserved region (A box) of the leader changed the specificity of the sensor RNA in vivo: guanine acted as a positive transcription effector in place of adenine.
Subject(s)
Bacillus subtilis/genetics , Genes, Bacterial , Mutation , RNA, Bacterial/genetics , Base Sequence , Molecular Sequence Data , Nucleic Acid ConformationABSTRACT
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.
Subject(s)
Genetic Complementation Test , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Saccharomyces cerevisiae/enzymology , Schizosaccharomyces/enzymology , Conserved Sequence/genetics , Conserved Sequence/physiology , Fungal Proteins/chemistry , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Deletion , Gene Dosage , Genes, Fungal/genetics , Genes, Fungal/physiology , Humans , RNA Polymerase II/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Schizosaccharomyces/genetics , Species Specificity , Suppression, Genetic/genetics , TemperatureABSTRACT
A full-length cDNA of the rpb8+ gene encoding a common subunit Rpb8 of nuclear RNA polymerases I-III only specific for Eucarya was isolated from an expression library of the fission yeast Schizosaccharomyces pombe. The primary structure of the corresponding fragment of the Sz. pombe genome was also established. The rpb8+ gene contains two short introns, 59 and 48 bp long. Only short segments of homology were found upon comparing the Rpb8 subunit homologs from various eukaryotic species, and substantial differences exist between the corresponding proteins of unicellular and multicellular organisms. Subunit Rpb8 of Sz. pombe proved to be the smallest one among the known related proteins: it lacks the 21-aa fragment corresponding to amino acids residues 68-88 of the central part of the homologous subunit ABC14.5 of Saccharomyces cerevisiae. Accordingly, subunit Rpb8 of the fission yeast was not capable of substituting in vivo subunit ABC14.5 in nuclear RNA polymerases of the baker's yeast.
