Electron microscopic mapping of wheat germ RNA polymerase II binding sites on cloned CaMV DNA

Abstract

The binding sites of wheat germ RNA polymerase II were mapped on the cloned CaMV genome by observation of enzyme-linear DNA complexes by electron microscopy. Twelve sites are observed. Three of them are relatively stable in the presence of heparin and are found at positions8-9, 21-23, and 41-44 map units on the physical map of the genome. These positions correspond to ATrich regions of the viral genome which contain potential promoter sites. These results are discussed with reference to current information on the structure and expression of the CaMV genome.

Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers

“…The appearance of a discrete population suggests a cooperative method of binding, in which the presence of bound polymerase facilitates the binding of subsequent molecules. This interpretation is supported by our observation by electron microscopy of clusters of bound molecules at high enzyme : DNA ratios [7]. …”

“…Transcription complex.formation after preincuhation with A T P or GTP. pCa8 DNA (0.5 pg) was preincubated with soybean RNA polymerase at a molar ratio of 0.5 for 5 rnin at 30 "C in the presence of ATP (lanes 1 -5) or GTP (6)(7)(8)(9)(10). Purine NTP concentrations were lOOpM(1,6),50pM(2,7),20pM(3,8),10pM(4,9)and5pM(5,10).…”

“…Lanes 1 and 19 show ethidium bromide staining of BglII-digested pCa8 and pKC7 respectively. Ternary transcription complex formation was primed by CpG (2, lo), ApG (3,11,18), GpG (4, 12), UpG (5,13), CpA (6,14), ApA (7,15), GpA (8; 16, see arrow for insertion) and UpA (9,17) in the presence of ATP (2-9, 18) or GTP (10)(11)(12)(13)(14)(15)(16)(17) in the preincubation medium. Exposure time for preincubations with ATP, 16 h; with GTP, 3 days.…”

“…We have shown that RNA polymerase I1 transcribes CaMV DNA even in the presence of heparin, but that this transcription is initiated principally at the level of the three single-stranded interruptions, which are present on the CaMV genome [8], initiation being more efficient at one of these interruptions [6]. Subsequent electron microscope studies using the entire CaMV genome cloned in the vector pATl53 [7], and thus lacking the single-strand interruptions, showed that RNA polymerase I1 forms stable, binary complexes at several sites on this template, certain of which are relatively resistant to the action of heparin.Most previous studies on transcription in vitro, including our own, have been carried out at relatively high enzyme: DNA ratios and have led to the general conclusion of non-specificity on purified templates. However, several recent reports have indicated that, under certain conditions, yeast RNA polymerase B is capable of selective initiation on the eucaryotic moiety of supercoiled cloned yeast templates at low enzyme:DNA ratios and in the presence [9, 101 or absence [I1 -131 of dinucleotide primers.…”

“…We have shown that RNA polymerase I1 transcribes CaMV DNA even in the presence of heparin, but that this transcription is initiated principally at the level of the three single-stranded interruptions, which are present on the CaMV genome [8], initiation being more efficient at one of these interruptions [6]. Subsequent electron microscope studies using the entire CaMV genome cloned in the vector pATl53 [7], and thus lacking the single-strand interruptions, showed that RNA polymerase I1 forms stable, binary complexes at several sites on this template, certain of which are relatively resistant to the action of heparin.…”