Different intracellular distribution of avian reovirus core protein sigmaA in cells of avian and mammalian origin

A comparative analysis of the intracellular distribution of avian reovirus (ARV) core protein sigmaA in cells of avian and mammalian origin revealed that, whereas the viral protein accumulates in the cytoplasm and nucleolus of avian cells, most sigmaA concentrates in the nucleoplasm of mammalian cells in tight association with the insoluble nuclear matrix fraction. Our results further showed that sigmaA becomes arrested in the nucleoplasm of mammalian cells via association with mammalian cell-specific factors and that this association prevents nucleolar targeting. Inhibition of RNA polymerase II activity, but not of RNA polymerase I activity, in infected mammalian cells induces nucleus-to-cytoplasm sigmaA translocation through a CRM1- and RanGTP-dependent mechanism, yet a heterokaryon assay suggests that sigmaA does not shuttle between the nucleus and cytoplasm. The scarcity of sigmaA in cytoplasmic viral factories of infected mammalian cells could be one of the factors contributing to limited ARV replication in mammalian cells.     

Streptomyces aureofaciens sporulation-specific sigma factor sigma(rpoZ) directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds.

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, sigma(RpoZ), of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P(ren24), P(ren57), and P(ren71), contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P(ren71) was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P(ren71) promoter is recognized by an RNA polymerase holoenzyme containing sigma(RpoZ) in S. aureofaciens. Sequence analysis of the region directed by P(ren71) revealed a gene, ren71, encoding a protein of 358 amino acids with an Mr 37,770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.     

Molecular anatomy of the β' subunit of the E. coli RNA polymerase: identification of regions involved in polymerase assembly

Background : The E. coli RNA polymerase is a multisubunit enzyme, which is present in two different forms: the catalytic competent core enzyme (α₂ββ ') and the promoter selective holoenzyme (α₂ββ 'σ). Correct assembly of individual subunits into core or holoenzyme is essential for the function of this enzyme.
Results : Mutant β ' proteins truncated near the centre or at the C-terminus were able to form stable core enzyme-like complexes under reconstitution conditions. Mutant β ' proteins lacking the region between amino acids 201–477 failed to form holoenzyme complexes while retaining the ability to form core enzyme complexes. Furthermore, free β ' subunit interacted with free σ subunit to form a stable β 'σ subassembly. Removal of amino acids 201–477 from the β ' subunit strongly interfered with this interaction.
Conclusion : Our results suggest that the N-terminal region of the β ' subunit is involved in the assembly of core enzyme. The region between amino acids 201 and 477 on β ' may be directly or indirectly involved in the interaction between the β ' subunit and the σ subunit.     

Streptomyces aureofaciens sporulation-specific sigma factor σ directs expression of a gene encoding protein similar to hydrolases involved in degradation of the lignin-related biphenyl compounds

A previously established method for the identification of promoters recognized by a heterologous RNA polymerase holoenzyme containing a particular sigma factor was used to identify promoters dependent upon a sporulation specific sigma factor, σ^RpoZ, of Streptomyces aureofaciens. Three new positive DNA fragments were identified, and these putative rpoZ-dependent promoters, P_ren24, P_ren57, and P_ren71, contained sequences similar to the consensus sequence of flagellar and chemotaxis promoters. However, only P_ren71 was active in S. aureofaciens. The promoter was induced at the time of aerial mycelium formation, and was inactive in an S. aureofaciens strain with an rpoZ-disrupted gene. The results suggest that the P_ren71 promoter is recognized by an RNA polymerase holoenzyme containing σ^RpoZ in S. aureofaciens. Sequence analysis of the region directed by P_ren71 revealed a gene, ren71, encoding a protein of 358 amino acids with an M_r 37 770. The deduced protein product showed end-to-end sequence similarity to the meta-cleavage compound hydrolase of Sphingomonas paucimobilis.     

Hyper-processive and slower DNA chain elongation catalysed by DNA polymerase III holoenzyme purified from the dnaE173 mutator mutant of Escherichia coli

BACKGROUND: A strong mutator mutation, dnaE173, leads to a Glu612 --> Lys amino acid change in the alpha subunit of Escherichia coli DNA polymerase III (PolIII) holoenzyme and abolishes the proofreading function of the replicative enzyme without affecting the 3' --> 5' exonuclease activity of the epsilon subunit. The dnaE173 mutator is unique in its ability to induce sequence-substitution mutations, suggesting that an unknown function of the alpha subunit is hampered by the dnaE173 mutation. RESULTS: A PolIII holoenzyme reconstituted from dnaE173 PolIII* (DNA polymerase III holoenzyme lacking the beta clamp subunit) and the beta subunit showed a strong resistance to replication-pausing on the template DNA and readily promoted strand-displacement DNA synthesis. Unlike wild-type PolIII*, dnaE173 PolIII* was able to catalyse highly processive DNA synthesis without the aid of the beta-clamp subunit. The rate of chain elongation by the dnaE173 holoenzyme was reduced to one-third of that determined for the wild-type enzyme. In contrast, an exonuclease-deficient PolIII holoenzyme was vastly prone to pausing, but had the same rate of chain elongation as the wild-type. CONCLUSIONS: The hyper-processivity and slower DNA chain elongation rate of the dnaE173 holoenzyme are distinct effects caused by the dnaE173 mutation and are likely to be involved in the sequence-substitution mutagenesis. A link between the proofreading and chain elongation processes was suggested.