Ire1 is an ancient transmembrane sensor of ER stress with dual protein kinase and ribonuclease activities. In response to ER stress, Ire1 catalyzes the splicing of target mRNAs in a spliceosome-independent manner. We have determined the crystal structure of the dual catalytic region of Ire1at 2.4 A resolution, revealing the fusion of a domain, which we term the KEN domain, to the protein kinase domain. Dimerization of the kinase domain composes a large catalytic surface on the KEN domain which carries out ribonuclease function. We further show that signal induced trans-autophosphorylation of the kinase domain permits unfettered binding of nucleotide, which in turn promotes dimerization to compose the ribonuclease active site. Comparison of Ire1 to a topologically disparate ribonuclease reveals the convergent evolution of their catalytic mechanism. These findings provide a basis for understanding the mechanism of action of RNaseL and other pseudokinases, which represent 10% of the human kinome.

The antiviral protein kinase PKR inhibits protein synthesis by phosphorylating the translation initiation factor eIF2alpha on Ser51. Binding of double-stranded RNA to the regulatory domains of PKR promotes dimerization, autophosphorylation, and the functional activation of the kinase. Herein, we identify mutations that activate PKR in the absence of its regulatory domains and map the mutations to a recently identified dimerization surface on the kinase catalytic domain. Mutations of other residues on this surface block PKR autophosphorylation and eIF2alpha phosphorylation, while mutating Thr446, an autophosphorylation site within the catalytic-domain activation segment, impairs eIF2alpha phosphorylation and viral pseudosubstrate binding. Mutational analysis of catalytic-domain residues preferentially conserved in the eIF2alpha kinase family identifies helix alphaG as critical for the specific recognition of eIF2alpha. We propose an ordered mechanism of PKR activation in which catalytic-domain dimerization triggers Thr446 autophosphorylation and specific eIF2alpha substrate recognition.

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The bacterial cell division protein FtsZ assembles into the cytokinetic Z ring that directs cytokinesis in prokaryotes. In Escherichia coli the formation of the Z ring is prevented by induction of the cell division inhibitor SulA (SfiA), a component of the SOS response. Here we show that a MalE-SulA fusion that retains this inhibitory function in vivo inhibits the GTPase activity and polymerization of FtsZ in vitro. MalE-SulA10, which does not block Z ring formation in vivo, is unable to inhibit the GTPase activity and polymerization in vitro. Furthermore, FtsZ114, which is refractory to SulA in vivo, is not inhibited by MalE-SulA. These results indicate that SulA blocks Z ring formation by blocking FtsZ polymerization.

The interaction of FtsZ with itself, GTP, and FtsA was examined by analyzing the sensitivity of FtsZ to proteolysis and by using the yeast two-hybrid system. The N-terminal conserved domain consisting of 320 amino acids bound GTP, and a central region of FtsZ, encompassing slightly more than half of the protein, was cross-linked to GTP. Site-directed mutagenesis revealed that none of six highly conserved aspartic acid and asparagine residues were required for GTP binding. These results indicate that the specificity determinants for GTP binding are different than those for the GTPase superfamily. The N-terminal conserved domain of FtsZ contained a site for self-interaction that is conserved between FtsZ proteins from distantly related bacterial species. FtsZ 320 , which was truncated at the end of the conserved domain, was a potent inhibitor of division although it expressed normal GTPase activity and could polymerize. FtsZ was also found to interact directly with FtsA, and this interaction could also be observed between these proteins from distantly related bacterial species.

The interaction between inhibitors of cell division and FtsZ were assessed by using the yeast two-hybrid system. An interaction was observed between FtsZ and SulA, a component of the SOS response, and the interacting regions were mapped to their conserved domains. This interaction was reduced by mutations in sulA and by most mutations in ftsZ that make cell refractory to sulA. No interaction was detected between FtsZ and MinCD, an inhibitory component of the site selection system. However, interactions were observed among various members of the Min system, and MinE was found to reduce the interaction between MinC and MinD. The implications of these findings for cell division are discussed.Cell division in bacteria is a complex process involving spatial and temporal regulation of the formation of the septum (reviewed in reference 11). The earliest defined step is the assembly of FtsZ, an essential cell division protein (8), into a ring at the future division site (4). The FtsZ protein has GTPase activity (10, 32, 37) and can assemble in vitro to form protein filaments (7,15,33). It has been suggested that the assembly of the FtsZ ring occurs through self-assembly involving a GTP cycle and is a key regulated step during cell division (25). Several inhibitors of cell division, SulA, a component of the SOS response, and MinCD, involved in selection of the division site, prevent formation of the FtsZ ring (6).As a component of the SOS response, SulA is induced following damage to DNA (21). It is an unstable protein and is degraded primarily by a protease encoded by the lon gene (31). As a result, lon mutants are hypersensitive to DNA damage, as the induction of SulA leads to a lethal filamentation (19). In wild-type cells, induction of SulA by DNA damage leads to filamentation which is readily reversed after the inducing signal dissipates. This inhibition of division by SulA is readily reversible, even in the absence of protein synthesis, arguing that SulA does not irreversibly inactivate the cell division machinery (29).MinCD is a more complex inhibitor of cell division, as its activity is spatially regulated by a third component, MinE (12). MinC is thought to be the component of the inhibitor that interacts with the division machinery, since overproduction of MinC alone can lead to division inhibition (14) and MinC can combine with another partner, DicB, to become an efficient inhibitor (13,23,35). Since MinE can regulate MinCD but not MinC overproduction or the MinCDicB combination, it is thought that it acts through MinD (14).There is genetic and biochemical evidence suggesting that FtsZ is the target of SulA and MinCD. Overproduction of FtsZ suppresses the lethal filamentation caused by SulA or MinCD (13, 26), and mutations, selected as resistant to SulA, map to the ftsZ gene (2, 24). These mutations, particularly ftsZ2, also show increased resistance to MinCD (3). At least two of these mutations, ftsZ114 and ftsZ9, result in an increased degradation rate of SulA that has been interpreted as a decreased interact...

SummaryThe localization of FtsN in Escherichia coli was investigated by immunofluorescence microscopy. FtsN is an essential cell division protein with a simple bitopic topology, a short N-terminal cytoplasmic segment fused to a large carboxy periplasmic domain through a single transmembrane domain.

Translation initiation is typically restricted to AUG codons, and scanning eukaryotic ribosomes inefficiently recognize near-cognate codons. We show that queuing of scanning ribosomes behind a paused elongating ribosome promotes initiation at upstream weak start sites. Ribosomal profiling reveals polyamine-dependent pausing of elongating ribosomes on a conserved Pro-Pro-Trp (PPW) motif in an inhibitory non-AUG-initiated upstream conserved coding region (uCC) of the antizyme inhibitor 1 (AZIN1) mRNA, encoding a regulator of cellular polyamine synthesis. Mutation of the PPW motif impairs initiation at the uCC's upstream near-cognate AUU start site and derepresses AZIN1 synthesis, whereas substitution of alternate elongation pause sequences restores uCC translation. Impairing ribosome loading reduces uCC translation and paradoxically derepresses AZIN1 synthesis. Finally, we identify the translation factor eIF5A as a sensor and effector for polyamine control of uCC translation. We propose that stalling of elongating ribosomes triggers queuing of scanning ribosomes and promotes initiation by positioning a ribosome near the start codon.