The vaccinia virus E3L gene product, pE3, is a dsRNA binding protein that prevents activation of the interferon-induced, dsRNA-activated protein kinase, PKR. Activation of PKR, which results in phosphorylation of the translation initiation factor, eIF2alpha, leads to the inhibition of protein synthesis, a process involved in defense against virus infection. The E3L gene product has a conserved dsRNA binding domain (DRBD) in its carboxyl-terminal region and has been shown to function in vitro by sequestration of dsRNA. We have utilized in vitro binding assays and the yeast two-hybrid system to demonstrate direct interactions of pE3 with PKR. By these methods, we demonstrate that pE3 interacts with two distinct regions in PKR, the amino-terminal (amino acids 1-99) located in the regulatory domain and the carboxyl-terminal (amino acids 367-523) located in the catalytic domain. The amino-terminal region of PKR that interacts with pE3 contains a conserved DRBD, suggesting that PKR can form nonfunctional heterodimers with pE3, analogous to those seen with other dsRNA binding proteins. Interaction of pE3 with the amino-terminal region of PKR is enhanced by dsRNA. In contrast, dsRNA reduces the interaction of pE3 with the carboxyl-terminal region of PKR. Competition experiments demonstrate that the carboxyl-terminal region of PKR, to which pE3 binds, overlaps the region with which eIF2alpha and the pseudosubstrate pK3 interact, suggesting that pE3 may also prevent PKR activation by masking the substrate binding domain. Like pE3, the amino-terminal region of PKR also interacts with the carboxyl-terminal domain of PKR. These interactions increase our understanding of the mechanisms by which pE3 downregulates PKR. In addition, the PKR-PKR interactions observed leads us to suggest a novel autoregulatory mechanism for activation of PKR in which dsRNA binding to the DRBD(s) induces a conformational change that results in release of the amino terminal region from the substrate binding domain, allowing access to eIF2alpha and its subsequent phosphorylation.

Overexpression of the RNA polymerase alpha subunit in Bordetella pertussis reduces expression of the virulence factor pertussis toxin. Here we show that this reduction is at the level of transcription, is reversed by overexpression of the transcriptional activator BvgA, and is dependent on the C-terminal domain of alpha.In Bordetella pertussis, expression of virulence factors is regulated by the Bvg two-component signal transduction system, comprising the sensor BvgS and the transcriptional activator BvgA (1, 11). The Bvg system is modulated (with loss of virulence factor expression) by reduced temperature (Ͻ30°C) or the presence of sulfate ions or nicotinic acid in the growth medium (11). Previously we showed that mutant B. pertussis strains with reduced expression of pertussis toxin (Ptx) and adenylate cyclase/hemolysin toxin, but not of other Bvg-regulated virulence factors such as filamentous hemagglutinin (Fha), had mutations upstream of the rpoA gene, which encodes the alpha subunit of RNA polymerase (RNAP) (3). These mutations caused a two-to threefold overexpression of alpha through an increase in translation of the rpoA gene (3). We also showed that inducible overexpression of alpha from a recombinant plasmid in B. pertussis had the same effect (3). The alpha subunit is a common site of interaction of RNAP with transcription activator proteins (6). We therefore hypothesized that the observed effect on virulence factor expression was due to interaction of the excess alpha with BvgA, effectively reducing the level of BvgA present in cells for functional interactions with RNAP. To obtain further evidence that the excess alpha affects BvgA-dependent transcription activation, we first assessed the effect of overexpressing alpha on transcription of both the ptx and fha genes.Overexpression of alpha reduces transcription of both ptx and fha. We introduced a ptx-lac transcriptional fusion (8) into the chromosome of wild-type (Tohama I) and mutant (alphaoverexpressing strains BC75 and RPV3 and the bvg knockout strain Tohama I ⌬bvg) B. pertussis strains as previously described (8). We also introduced a fha-lac transcriptional fusion into the chromosome of the same set of strains, by allelic exchange from the plasmid pSS1581 (kindly provided by Scott Stibitz). The fusion strains were grown at 37°C in SS medium (9) to mid-log phase (nonmodulating conditions that allow full Bvg activity), and then ␤-galactosidase assays (8) were performed on the cultures to determine the level of ptx and fha transcription. As seen in Fig. 1, the level of ptx transcription is significantly reduced in both alpha-overexpressing mutants (approximately twofold in RPV3 and threefold in BC75), but the level of fha transcription is not significantly reduced in these strains. Since there is only a modest (two-to threefold) overexpression of alpha in RPV3 and BC75 (3), we introduced the plasmid pNMD120 (encoding IPTG [isopropyl-␤-D-thiogalactopyranoside]-inducible expression of B. pertussis rpoA) (3), as well as the vector control plasmid pNM...

Multicopy plasmids containing the promoter regions for gdh and mlrA genes from Pyrococcus furiosus were propagated in Haloferax volcanii. High-level expression was detected from gdh promoter sequences, with transcription initiating at the same start-site as that found in P. furiosus. For mlrA, several transcripts were detected, with one initiating at the P. furiosus start-site; removal or disruption of the likely P. furiosus boxA element resulted in the disappearance of this transcript, indicating that these sequences were utilized by the H. volcanii RNA polymerase for initiation. This is the first demonstration of the utilization of promoters from a hyperthermophilic archaeon in a mesophilic haloarchaeon and provides further evidence for the unity of transcription processes in the domain Archaea.