Direct evidence for the preferential binding of Escherichia coli RNA polymerase holoenzyme to the ends of deoxyribonucleic acid restriction fragments.
Escherichia coli RNA polymerase holoenzyme has been observed to form a variety of nonpromoter complexes with DNA restriction fragments in experiments performed with the nitrocellulose filter assay [Melançon, P., Burgess, R. R., & Record, M. T., Jr. (1982) Biochemistry 21, 4318-4331]. Here we report the use of this assay to investigate aspects of the weak (heparin-sensitive) interactions of RNA polymerase core and holoenzyme with a 1600 base pair (bp) fragment of T7 DNA which contains no promoters or TB (tight binding; heparin-resistant) sites. Under the ionic conditions investigated [50 mM NaCl/10 mM MgCl2/10 mM sodium N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid (pH 7.7)], both core and holoenzyme bind to the linear DNA fragment and cause comparable levels of filter retention. When the DNA fragment is self-ligated into a circular molecule (nonsupercoiled), the extent of binding of holoenzyme (but not that of core) is dramatically reduced. This directly proves our previous hypotheses that holoenzyme recognizes and preferentially binds to the ends of DNA fragments and that this mode of binding is responsible for most of the heparin-sensitive filter retention of nonpromoter fragments. The residual mode of binding of holoenzyme detected with the circular DNAs was considered in determining the amount of protein bound at ends only. To calculate end-binding constants (KE), the amount of protein bound nonspecifically (which does not appear to cause efficient filter retention) was also taken into consideration. At 0 degrees C, we obtain a value for KE of (2.1 +/- 0.5) X 10(8) M-1, in good agreement with that determined earlier.(ABSTRACT TRUNCATED AT 250 WORDS)
D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011485:Protein Binding
D004270:DNA, Circular; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases