No abstract

The binding of the benzodioxolo-benzoquinolizine alkaloid, berberine chloride to natural and synthetic DNAs has been studied by intrinsic and extrinsic circular dichroic measurements. Binding of berberine causes changes in the circular dichroism spectrum of DNA as shown by the increase of molar ellipticity of the 270nm band, but with very little change of the 240nm band. The molar ellipticity at the saturation depends strongly on the base composition of DNA and also on salt concentration, but always larger for the AT rich DNA than the GC rich DNA. The features in the circular dichroic spectral changes of berberine-synthetic DNA complexes were similar to that of native DNA, but depends on the sequence of base pairs. On binding to DNA and polynucleotides, the alkaloid becomes optically active. The extrinsic circular dichroism developed in the visible absorption region (300-500nm) for the berberine-DNA complexes shows two broad spectral bands in the regions 425-440nm and 340-360nm with the maximum varying depending on base composition and sequence of DNA. While the 425nm band shows less variation on the binding ratio, the 360nm band is remarkably dependent on the DNA/alkaloid ratio. The generation of the alkaloid associated extrinsic circular dichroic bands is not dependent on the base composition or sequence of base pairs, but the nature and magnitude of the bands are very much dependent on these two factors and also on the salt concentration. The interpretation of the results with respect to the modes of the alkaloid binding to DNA are presented.

No abstract

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin-resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position 2 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (2103 to 2108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at 296 position relative to galP1. b-galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its 210 region result in a substantial reduction in the gal promoter activity.Keywords: RNA polymerase; E. coli; promoter; galactose operon; transcription.Gene expression in bacteria involves a series of events, in which the first step is the initiation of transcription. During the initiation process, RNA polymerase holoenzyme (RNPol) recognizes and binds to specific sites on DNA, known as promoters. The initial recognition is followed by the formation of a`closed complex', which, through the steps of isomerization, leads to an`open complex' where a portion of the DNA is melted, and transcription starts [1]. Promoter recognition is a highly specific phenomenon, and the strength of promoters are largely influenced by the hexanucleotide sequences present at the 210 and 235 regions of the coding strand (with the transcription start point designated 11), as well as by the spacing between these regions [2,3]. However, the correspondence of sequences at these sites is not the sole determinant of promoter strength [1,4,5].Control of gene expression can occur at many levels. One of the important points of control that predominates in many bacterial systems is found to occur at the stage of transcription initiation [1]. This control is exercised through the action of negative or positive regulatory proteins [6], as well as through the topology of DNA [7±9]. A large number of promoters that employ these means of regulation have been characterized in E. coli. Thus, the control region consists of the site of interaction of RNA polymerase holoenzyme (promoter) as well as the binding sites for repressor proteins (operator) and for activator proteins. A variety of relative organizations of these sites are found in different operons of the E. coli bacterium.In the galactose operon of E. coli, two overlapping promoters P2 and P1 have been found to be responsible for initiation of transcripts at 25 and at 11, respectively, corresponding to the epimerase-transferase-galactokinase genes [10]. The cyclic AMP r...