Mechanistic aspects of promoter binding and chain initiation by RNA polymerase

Summary The physicochemical properties of the interactions of RNA polymerase (RPase) with promoter and nonspecific DNA sequences have been investigated. These show that nonspecific binding is principally an ionic interaction and that promoter binding is more complex, involving nonionic interactions. Nonspecific binding has been shown to be very important in the promoter search, and one-dimensional diffusion can account for the rate at which RPase finds the promoter. Significant differences have been reported in the binding process for various promoters and in the effects of regulatory proteins. Further investigation of these differences will lead to a better understanding of the selectivity and regulation of the initiation process.The pathways of the initiation process have been outlined, by recent studies and considerable progress has been made in determining the rates of interconversion of the intermediate states. A number of questions remain about the detail of initiation and the effects of various parameters on the reactions. Of particular importance is the identification of the point at which the enzyme becomes truly processive. In addition, the step which is rate limiting has not been identified in either the productive or nonproductive process. The mechanistic features of the steps after bond formation are just beginning to yield to investigation.Use of substrate analogs with RPase has led to a picture of the polymerization site according to the ability of the enzyme to incorporate analogs. Base specificity appears to be determined primarily by interaction with the template rather than the enzyme, but the ribose moiety must interact with the site quite specifically. The orientation of the phosphate residues has been determined by NMR, which has also proved to be a valuable probe of the initiation site. At this site base specificity is resident in the enzyme and expressed through the interaction of the base and intrinsic metal, as shown by studies with the Cobalt substituted enzyme. In both initiation and polymerization, the reaction has been shown to proceed by inversion of configuration. Techniques similar to those used for initiation will probably be applied to the polymerization reaction as well, which has not recently received as much attention with respect to mechanism. Functional phenomena such as pausing make the polymerization process particularly promising for producing insight into RPase reactions.     

A synopsis of the family Lissocarpaceae

A species erroneously described as aDiospyros is here transferred toLissocarpa, asL. tetramera (Rusby) P. E. Berry. This gives a total of five species currently recognized in Lissocarpaceae. A key to the species and a discussion of the individual taxa are presented.     

Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

Intracellular mitochondrial membrane potential as an indicator of hepatocyte energy metabolism: further evidence for thermodynamic control of metabolism

The lipophilic triphenylmethylphosphonium cation (TPMP+) has been employed to measure delta psi m, the electrical potential across the inner membrane of the mitochondria of intact hepatocytes. The present studies have examined the validity of this technique in hepatocytes exposed to graded concentrations of inhibitors of mitochondrial energy transduction. Under these conditions, TPMP+ uptake allows a reliable measure of delta psi m in intracellular mitochondria, provided that the ratio [TPMP+]i/[TPMP+]e is greater than 50:1 and that at the end of the incubation more than 80% of the hepatocytes exclude Trypan blue. Hepatocytes, staining with Trypan blue, incubated in the presence of Ca2+, do not concentrate TPMP+. The relationships between delta psi m and two other indicators of cellular energy state, delta GPc and Eh, or between delta psi m and J0, were examined in hepatocytes from fasted rats by titration with graded concentrations of inhibitors of mitochondrial energy transduction. Linear relationships were generally observed between delta psi m and delta GPc, Eh or J0 over the delta psi m range of 120-160 mV, except in the presence of carboxyatractyloside or oligomycin, where delta psi m remained constant. Both the magnitude and the direction of the slope of the observed relationships depended upon the nature of the inhibitor. Hepatocytes from fasted rats synthesized glucose from lactate or fructose, and urea from ammonia, at rates which were generally linear functions of the magnitude of delta psi m, except in the presence of oligomycin or carboxyatractyloside. Linear relationships were also observed between delta psi m and the rate of formation of lactate in cells incubated with fructose and in hepatocytes from fed rats. The linear property of these force-flow relationships is taken as evidence for the operation of thermodynamic regulatory mechanisms within hepatocytes.     

Lung protein leaks in ventilated lambs: effects of gestational age

To study the protein permeability properties of the ventilated premature lung, we delivered groups of eight lambs at 122 and 135 days gestational age and ventilated the lambs equivalently. The lambs at 122 days gestational age had been treated with natural sheep surfactant at birth, and both groups of lambs had similar pH and blood gas values to 3 h of age. Three groups of lambs at 146 days gestational age also were studied for comparison; four lambs were ventilated to normalized PCO2 values, four lambs were ventilated equivalently to the premature lambs with supplemental CO2 used to normalize PCO2 values, and four lambs were treated with natural surfactant and ventilated similarly to the preterm lambs. The percent recovery into an alveolar wash and lung tissue of 131I-albumin given by intravascular injection and of 125I-albumin given into the airways was measured in each animal after killing at 3 h of age. Full-term lambs had a small bidirectional leak of albumin to and from the alveoli and lung tissue. The recovery of intravascular 131I-albumin in the alveolar wash was 5.8- and 4.1-fold higher in lambs at 122 and 135 days gestational age, respectively, than in full-term lambs. The loss of 125I-albumin from the airways and alveoli also increased as gestational age decreased. The bidirectional flux of albumin to and from the alveoli increased as gestational age decreased in the prematurely delivered and ventilated lambs.