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.     

Single-cell landscape of the ecosystem in early-relapse hepatocellular carcinoma

1. Download : Download high-res image (233KB)
2. Download : Download full-size image

     

Expression of tracheal differentiated functions in serum-free hormone-supplemented medium

Most dissociated airway epithelial cells in culture express few of their in vivo functions and only to a limited degree. In this report, we demonstrate that hamster tracheal epithelial (HTE) cells cultured on a collagen gel substratum in a serum-free hormone-supplemented medium differentiate to cilia-beating and mucus-secreting cell types. The medium is Ham's F-12 supplemented with insulin, epidermal growth factor, transferrin, hydrocortisone, cholera toxin, bovine hypothalamus extract, and vitamin A. Under these culture conditions, HTE cells exhibit a growth rate of 24 h/population doubling and reach confluency, at a density of 2-5 X 10(4) cells/cm2, within 2 weeks. Both the collagen gel substratum and vitamin A of this culture system are important to the growth and differentiation of HTE cells in vitro. Evidence of HTE cell differentiation has been obtained at both the ultrastructural and the histochemical levels. In addition, a variety of biochemical studies (gel filtration, ion exchange column chromatography, enzyme digestion, nitrous acid treatment, and composition analysis) indicate the production of mucin-like glycoprotein in the HTE cultures. The levels of mucin-like glycoprotein were found to closely correlate with the histochemically quantitated levels of the mucous cell type. Kinetic studies demonstrate that HTE cells rapidly lose their differentiated features during the attachment stage of primary culture but redifferentiation occurs after the cultures reach confluency. The ability of HTE cells to grow and differentiate in this serum-free culture system in the absence of other cell types should greatly facilitate the study of mucociliary functions in vitro.     

Glucoraphasatin: Chemistry, occurrence, and biological properties

Glucoraphasatin is an atypical glucosinolate mainly found in Raphanus sativus roots and sprouts. This review focuses on the chemistry, the occurrence, and the biological properties of glucoraphasatin.     

Immunological studies of the relationship between the antigenic change of Ascaris oocytes and the junctional fluid of the fertilization chamber

Antigenic change on the surface of immature oocytes of Ascaris occurred during passage through the oviduct. Using immunodiffusion and immunoelectrophoretic techniques we examined the possible relationship between the antigenic change of immature oocytes and the junctional fluid (JF) which is present in the fertilization chamber. From the immunodiffusion it was found that the anti-immature oocyte serum (A-I serum) had more immunoprecipitation arcs than did the anti-mature oocyte serum (A-M serum) when reacted against the junctional fluid. This indicated that A-I serum contained more immunoglobulins that reacted with junctional fluid than did the A-M serum. This result was substantiated by using immunoelectrophoretic analysis and two dimension crossed immunoelectrophoresis. Our results suggest that during the migration toward the oviduct-uterine junction the immature oocytes might shed surface proteins into the luminal fluid. Alternatively, the membrane surface of mature oocytes may be altered by the interaction with the substances present in the junctional fluid.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.