Endothelial cell-surface tissue transglutaminase inhibits neutrophil adhesion by binding and releasing nitric oxide.

Nitric oxide (NO) produced by endothelial cells in response to cytokines displays anti-inflammatory activity by preventing the adherence, migration and activation of neutrophils. The molecular mechanism by which NO operates at the blood-endothelium interface to exert anti-inflammatory properties is largely unknown. Here we show that on endothelial surfaces, NO is associated with the sulfhydryl-rich protein tissue transglutaminase (TG2), thereby endowing the membrane surfaces with anti-inflammatory properties. We find that tumor necrosis factor-α-stimulated neutrophil adherence is opposed by TG2 molecules that are bound to the endothelial surface. Alkylation of cysteine residues in TG2 or inhibition of endothelial NO synthesis renders the surface-bound TG2 inactive, whereas specific, high affinity binding of S-nitrosylated TG2 (SNO-TG2) to endothelial surfaces restores the anti-inflammatory properties of the endothelium, and reconstitutes the activity of endothelial-derived NO. We also show that SNO-TG2 is present in healthy tissues and that it forms on the membranes of shear-activated endothelial cells. Thus, the anti-inflammatory mechanism that prevents neutrophils from adhering to endothelial cells is identified with TG2 S-nitrosylation at the endothelial cell-blood interface.

APC-mediated proteolysis of Ase1 and the morphogenesis of the mitotic spindle.

The molecular mechanisms that link cell-cycle controls to the mitotic apparatus are poorly understood. A component of the Saccharomyces cerevisiae spindle, Ase1, was observed to undergo cell cycle-specific degradation mediated by the cyclosome, or anaphase promoting complex (APC). Ase1 was degraded when cells exited from mitosis and entered G1. Inappropriate expression of stable Ase1 during G1 produced a spindle defect that is sensed by the spindle assembly checkpoint. In addition, loss of ASE1 function destabilized telophase spindles, and expression of a nondegradable Ase1 mutant delayed spindle disassembly. APC-mediated proteolysis therefore appears to regulate both spindle assembly and disassembly.

Pathway of promoter melting by Bacillus subtilis RNA polymerase at a stable RNA promoter: effects of temperature, delta protein, and sigma factor mutations.

Bacillus subtilis RNA polymerase (RNAP) contains a catalytic core (beta beta' alpha 2; or E) associated with one of several sigma factors, which determine promoter recognition, and delta protein, which enhances promoter selectivity. We have shown previously that specific mutations in sigma A region 2.3, or addition of delta, decrease the ability of RNAP to melt the ilv-leu promoter. Here we extend these studies to a stable RNA promoter, PtmS, which controls transcription of seven tRNA genes. KMnO4 footprinting was used to visualize DNA melting at PtmS as a function of both temperature and the protein composition of the RNAP holoenzyme. We propose that the pathway leading to productive initiation includes several intermediates: a closed complex (RPc), a complex in which DNA melting has nucleated within the conserved TATA element (RPn), and an open complex in which DNA-melting extends to at least -4 (RPo1). RNAP reconstituted with either of two mutant sigma A proteins, Y189A and W192A, was defective for both the nucleation and propagation of the transcription bubble while a third sigma A mutant, W193A, allows normal nucleation of DNA-melting, but does not efficiently propagate the melted region downstream.

Mutations in sigma factor that affect the temperature dependence of transcription from a promoter, but not from a mismatch bubble in double-stranded DNA.

Specificity of promoter utilization in bacterial RNA polymerases is imparted by a class of proteins referred to as sigma factors. Conserved region 2.3 of these proteins is thought to play a role in the strand separation process that occurs during the formation of an initiation-competent RNA polymerase-promoter complex. We have used a heterologous system consisting of Escherichia coli core RNA polymerase and Bacillus subtilis sigma A to probe the effects of amino acid substitutions in region 2.3. In agreement with previous work [Juang & Helmann (1994) J. Mol. Biol. 235, 1470-1488] we observe that several amino acid substitutions exacerbate the deleterious effect of low temperature on promoter-dependent initiation. On the other hand, no such enhanced cold sensitivity is found with double-stranded templates that contain short "bubbles" of single-stranded DNA, indicating that the DNA-melting defect imposed by these mutant sigma factors can be suppressed by the use of such bubble templates. These results support the involvement of region 2.3 in the strand separation process that accompanies open complex formation at promoters.

The delta subunit of Bacillus subtilis RNA polymerase. An allosteric effector of the initiation and core-recycling phases of transcription.

RNA polymerase purified from Bacillus subtilis contains multiple sigma (sigma) factors and an auxiliary subunit known as delta (delta). We have addressed the roles of the delta polypeptide in a model transcription cycle using the promoter and attenuator of the ilv-leu operon. We demonstrate that delta influences both the promoter selection and core recycling phases of the transcription cycle. The delta protein functions together with sigma as an initiation subunit of RNA polymerase. Remarkably, E sigma delta forms predominantly closed complexes at the P(ilv) promoter even at 40 degrees C, whereas E sigma forms open complexes. The presence of delta inhibits transcription at low temperatures, presumably because delta decreases the rate of open complex formation. In contrast, delta has little or no effect on the overall rate of promoter localization and initiation, rate of elongation, or termination efficiency. Despite the inhibitory effect of delta on DNA-melting, we find that delta stimulates the amount of RNA synthesized from the P(ilv) leader region several-fold in multiple cycle reactions due to an increased rate of enzyme recycling. These results highlight the importance of delta in determining RNA yield during in vitro transcription.

A promoter melting region in the primary sigma factor of Bacillus subtilis. Identification of functionally important aromatic amino acids.

Sigma factor (sigma) is a dissociable subunit of bacterial RNA polymerase that determines promoter recognition. It has been proposed that a cluster of highly conserved aromatic amino acids in bacterial sigma factors (region 2.3) defines a melting motif that functions in strand-separation during open complex formation. We demonstrate that many alterations in region 2.3 of the Bacillus subtilis sigma A protein specifically impair open complex formation. The region 2.3 mutations can be grouped in three classes: (1) mutations that do not significantly affect promoter recognition or melting; (2) mutations that lead to cold-sensitive transcription of linear templates; and (3) mutations that lead to little activity on linear templates but retain activity at high temperatures on supercoiled templates. RNA polymerase holoenzymes containing sigma factor melting mutants (classes 2 and 3) form predominantly closed complexes at 40 degrees C and are defective for RNA synthesis when initiation is rate-limiting. The melting defect of these mutant sigma factors is suppressed by template supercoiling, but further enhanced by inclusion of the auxiliary RNA polymerase subunit delta. Consequently, in the presence of the delta polypeptide, the mutant holoenzymes display cold-sensitive transcription on supercoiled templates: conditions which mimic the in vivo situation. A subset of these mutations also affects promoter selectivity, suggesting that region 2.3 may participate in both -10 recognition and DNA melting.