A regulator that inhibits transcription by targeting an intersubunit interaction of the RNA polymerase holoenzyme.

The structures of the bacterial RNA polymerase holoenzyme have provided detailed information about the intersubunit interactions within the holoenzyme. Functional analysis indicates that one of these is critical in enabling the holoenzyme to recognize the major class of bacterial promoters. It has been suggested that this interaction, involving the flap domain of the beta subunit and conserved region 4 of the sigma subunit, is a potential target for regulation. Here we provide genetic and biochemical evidence that the sigma region 4/beta-flap interaction is targeted by the transcription factor AsiA. Specifically, we show that AsiA competes directly with the beta-flap for binding to sigma region 4, thereby inhibiting transcription initiation by disrupting the sigma region 4/beta-flap interaction.

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17.

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.

Mitral valve replacement: techniques to eliminate myocardial rupture and prevent valvular disruption.

Twenty fresh canine hearts were used to compare the peak left ventricular pressures required to disrupt prosthetic mitral valves sutured in place with horizontal mattress sutures using either subannular or supraannular placed pledgets. Separate groups were developed to determine the effect of leaving the whole mitral valve apparatus or only the posterior leaflet apparatus intact and what effect, if any, each had on the ventricular pressure required to disrupt the implanted prosthetic mitral valve. Group 1 consisted of 10 hearts with the entire mitral apparatus left in place (5 valves implanted with supraannular pledgets and 5 with subannular pledgets). Group 2 consisted of 10 hearts with only the posterior leaflet apparatus left in place (5 valves implanted with supraannular pledgets and 5 with subannular pledgets). A 29-mm Medtronic mitral valve was secured in the mitral position with a fixed number of ten pledgeted sutures in each annulus. The aorta was cannulated and normal saline solution infused into the left ventricle until end-point rupture occurred. The peak pressure and mechanism of any disruption were then noted. No specimen exhibited subannular myocardial rupture or left atrial wall dissection. Similar protection was provided by leaving the posterior leaflet only or the entire mitral valve. In each case peak left ventricular pressure resulted in only paravalvular leaking around the limited number of sutures as the end point. In each of these four groups the peak left ventricular pressures required for end-point rupture were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Correction of condylar displacement following intraoral vertical ramus osteotomy.

Fifteen patients who demonstrated condylar sag after intraoral vertical ramus osteotomy for the correction of mandibular prognathism were treated nonsurgically to establish the desired postoperative occlusion. A mean inferior displacement of 3.33 mm and anterior displacement of 2.18 mm were observed tomographically after surgery. Postoperatively, a geometric splint was constructed to compensate for the magnitude of condylar displacement and was used to replace the original splint to hold the distal segment in an overcorrected position. Skeletal fixation was maintained for 5 to 6 weeks. Tomographic evaluation of the temporomandibular joint (TMJ) during maxillomandibular fixation showed a slight superior (1.03 mm) and posterior (0.51 mm) movement of the condyle in the fossa. After release of fixation and removal of splint, a further superior (2.05 mm) and posterior (1.01 mm) repositioning of the condyle was observed. This later movement correlated with the placement of light class III elastic traction to seat the condyles into the glenoid fossae and establish a class I occlusion. Temporomandibular joint tomograms confirmed complete seating of the condyles in the fossa and lateral cephalograms demonstrated a corresponding change in the position of the mandible to the desired postoperative position. This technique has been effective in preventing postoperative malocclusion resulting from condylar sag.