Animal DNA‐Dependent RNA Polymerases

Abstract

The binding of purified mammalian RNA polymerases AI and B to Simian virus 40 (SV40) DNA was studied under various conditions (ionic strength, divalent cation, temperature) by two methods, electron microscopy and retention of enzyme * DNA complexes on nitrocellulose filters. Our studies demonstrate that there are multiple binding sites for mammalian RNA polymerases on SV40 DNA form I and that highly stable enzyme -DNA-form-I complexes are formed. However, in contrast to the bacterial RNA polymerase holoenzyme, the purified mammalian enzymes are unable to form a highly stable complex with a linear double-stranded DNA (SV40 DNA form 111). Our results strongly suggest that the formation of highly stable complexes between mammalian RNA polymerases and SV 40 DNA form I is due to their binding to the unpaired regions which are present in the superhelical DNA.Results presented in the accompanying papers have revealed that initiation of RNA synthesis by mammalian RNA polymerases on Simian virus 40 (SV40) DNA is markedly dependent on the configuration of the template and on the nature of the divalent cation present in the reaction medium. These results have also shown that superhelical SV40 DNA-FI contains multiple initiation sites for mammalian RNA polymerases and that both strands of the template are transcribed. On the other hand, Herzberg and Winocour [i] have reported that superhelical SV40 DNA-FI possesses only one single binding site for the mammalian RNApolymerase B. In an attempt to resolve this discrepancy and to get a better insight into the mechanisms regulating the initiation of RNA synthesis by the mammalian enzymes on SV40 DNA-FI, we have studied the binding of purified mammalian RNA polymerases to this DNA. Since such studies can only be adequately performed with highly purified enzymes, we have mainly used purified calf thymus enzymes AI or B. Most of the experiments were carried out with calf thymus enzymes B This paper is part 8 of a series. The binding of mammalian RNA polymerases to SV40 DNA was studied by two complementary methods : electron microscopy and retention of enzyme DNA complexes on nitrocellulose filters. This latter method is very convenient, since it is rapid and since it gives a semi-quantitative picture of the binding of RNA polymerase to DNA and of the effect of some parameters on this binding reaction. Using this method, Hinckle and Chamberlin 113-51 have shown that site selection occurs during binding of Escherichia coli RNA polymerase holoenzyme to T7 bacteriophage DNA and involves the formation of a highly stable holoenzyme * T7 DNA complex at, or near, the T7 promoter site. Formation of this complex requires the cr subunit and most likely involves the opening of the double helix near the promoter site. In contrast, binding of E. coli core RNA polymerase to intact T7 DNA does not lead to the formation of a, highly stable complex.Eur. J. Biochem. 41 (1974)

Hairpin-loop formation by inverted repeats in supercoiled DNA is a local and transmissible property

“…ED underwinding is an energy requiring process either dependent upon the continuous binding of coiling protein (histone) (25) or linkage reduction by an ATP dependent enzyme (gyrase) (26). It seems likely that the cell should expect some return on its investment of energy in this process, and negative supercoiling has been implicated in replication (27), transcription (28)(29)(30)(31)(32)(33) and recombination (34).…”

The interaction of RNA polymerase II from wheat with supercoiled and linear plasmid templates

“…Initiation on double stranded templates has been postulated to occur at nicks (29,30), single strand regions (11) and at regions of high superhelix density (10). We Our demonstrated preference for initiation at single strand regions of DNA, combined with the enhancement of transcription on negatively supercoiled template suggest that the underwinding of the templates provides activation energy for localised 'melting' of DNA which can then serve as an initiation site.…”

“…It is now known that the eukaryotic genome is negatively supercoiled (3)(4)(5), which, taken together with supercoiled bacterial (6) and viral (7) chromosomes, implies that the most common template for RNA polymerase in general is underwound DNA. Whilst theoretical treatments of the interactions of unwinding ligands with negative supercoils have appeared (8,9), the examination of eukaryotic RNA polymerase in this respect is restricted to two investigations (10,11), with conflicting conclusions. A further aspect of eukaryotic transcription which is poorly understood is the nature of gene promoters, and the extent to which concepts of prokaryotic promoter operation may be applied to eukaryotes.…”

“…We Our demonstrated preference for initiation at single strand regions of DNA, combined with the enhancement of transcription on negatively supercoiled template suggest that the underwinding of the templates provides activation energy for localised 'melting' of DNA which can then serve as an initiation site. Chambon (10,32) has previously suggested this for transcription of SV40 Form I DNA by mammalian RNA polymerases, where he also noted (38) lack of selectivity and assymmetry of transcription. Since the linking number (or topological winding number) of the template is fixed, such localised unwinding will result in concomitant lowering of superhelix density.…”

The interaction of RNA polymerase II with non-promoter DNA sites

“…Estimates for half-lives of complexes formed vary widely, from seconds to hours, even when similar enzyme-template combinations are studied (2)(3)(4)(5)(6). These varying estimates might arise from study of different types of complexes or from differences in experimental protocols used to obtain dissociation data.…”