On the Nature of DNA Promoter Conformations

Abstract

Low concentrations (up to 20 "/, v/v) of glycerol and dimethylsulphoxide stimulate RNA synthesis in vitro, the extent of stimulation being dependent on the nature of the DNA template. This stimulation results mainly from an increase in initiation. The target of these compounds is the DNA template rather than RNA polymerase. Ribosomal RNA synthesis is affected in a temperaturedependent manner, 20 % glycerol lowering the transition temperature between the open and closed forms of the promoter by 4-5 "C. Thus in a manner analogous to its effect on gal mRNA synthesis in vitro glycerol partially releases rRNA synthesis from the constraints essential for control.The first step in the synthesis of an RNA molecule is the interaction of RNA polymerase with a particular site on the DNA template. It is presumably this interaction which determines the specificity of transcription. Consequently we might expect that alterations in the structure of either the polymerase or the template could result in changes in specificity. Among the factors which can directly affect the ability of a given DNA molecule to act as a template are the temperature [l -41, the superhelicity of a closed DNA duplex [5 -71 and the presence of ethylene glycol [8].Under certain conditions temperature operationally defines two states of a specific promoter, that for ribosomal RNA synthesis; above 36 OC the promoter is open, below 35 "C it is closed [4]. The transition temperature between these two states can be altered by molecules which interact directly with the template. It is, for example, raised by a DNA-binding protein, H I protein [9].In this paper I show that two rather dissimilar compounds, glycerol and dimethylsulphoxide, have very similar effects on transcription in vitro. At low concentrations (up to 20 "/, v/v) both stimulate total RNA synthesis, the extent of stimulation depending on the type of DNA template. This stimulation results mainly from an increase in initiation and is a consequence of the action of the compounds on the DNA template. Ribosomal RNA synthesis is affected such that the transition temperature between the two states of the promoter is lowered by 4-5 "C in the presence Enzymt,. Nucleosidetriphosphate : RNA nucleotidyltransferase or R N A polyiiierase (EC 2.7.7.6).of 20% (v/v) glycerol. Since the characteristics of the control of ribosomal RNA synthesis in vivo are paralleled most closely in vitro from the closed state to rhe promoter [lo], the effect of glycerol is thus partially to release rRNA synthesis in vitro from the constraints essential for control. A striking analogy to this effect exists in the lactose system where the requirement for cup protein for gal mRNA synthesis in vitro is abolished by glycerol [ll]. MATERIALS AND METHODS Prepuration of R N A Polymerase and E . coli D N ARNA polymerase holoenzyme was prepared from E. coli CA 285 by the method of Burgess and Travers [12]. The enzyme preparation was > 95 % pure as judged by electrophoresis in 7.5 % polyacrylamide gels in 0.1 % sodium dodecylsulphate. The ...

Template selection by E. Coli RNA polymerase holoenzyme

“…T2 DNA and ¢80 da rrnB ÷ ilv ÷ su+7 DNA were prepared by gentle phenol extraction of the corresponding purified phage followed by dialysis against 0.01 M Tris-HCl pH 7.9, 0.1 M potassium chloride, 0.0001 M EDTA. E. coli DNA was prepared as described by Travers [5]. The experiments were repeated with two preparations each of¢80 da rrnB ÷ ilv ÷ su÷7 DNA and E. coli DNA and three of RNA polymerase.…”

Transcription of DNAs of Known Sequence after Injection into the Eggs and Oocytes of Xenopus laevis

“…Most samples of DNA were also tested for transcriptional activity in the presence of E. coli RNA polymerase. The assay contained 85 pl energy mix [25], 5 pl E. coli polymerase (1.4 pgjml), 2 p1 DNA (400 pg/ml) and 2 pl [4-14C]UTP. Assays were carried out at 37 "C for 15 min.…”

DNA-dependent RNA polymerase from the extremely halophilic archaebacterium Halococcus morrhuae

“…morrhuur RNA polymerase activity by glycerol and MezSO is probably at least in part due to facilitating strand separation, which is required during initiation of transcription [17,18]. This is, however, not the entire cause, since some stimulation was observed on singlestranded DNA.…”