Length measurements of RNA synthesized in vitro by Escherichia coli RNA polymerase

Abstract

A method for visualizing in vitro synthesized RNA in extended form still attached to the DNA template is described. Bacteriophage T4 gene 32 product is attached to the RNA and after fixation with glutaraldehyde the transcription complexes are prepared for electron microscopy by the Kleinschmidt technique. Secondary structure caused by partial complementarity of the RNA can be stretched out by the attachment of bacteriophage T4 gene 32 protein as is shown in the case of bacteriophage R17 RNA. The possibilities of the method are exemplified using T7 as a template for Escherichia coli RNA polymerase. Data for the position of the promoter and the extent of transcription in the region of early T7 messenger RNA are confirmed. In addition, we have demonstrated the presence of a weak promotor at the right-hand end of the DNA; as in the early region, the direction of RNA synthesis is from left to right. Using SV40 viral DNA as a template, up to six RNA chains can be synthesized in the presence of rifampicin. After synthesis for 15 minutes at 37 °C, the length of the RNA chains shows that the polymerase has transcribed at least four times around the DNA circle.