Extension of RNA Molecules with DNA An

Abstract

Sequences in DNA, which act as signals for the termination of transcription, may lie wholly or partially beyond the distal end of the DNA region actually transcribed into RNA. In a hybrid formed between an intact RNA molecule and its DNA complement, any such untranscribed signal should lie in the region of single-stranded DNA close to the 3'-hydroxyl terminus of the RNA. We show here that hybrids of this type, formed on circular bacteriophage DNA, act as efficient primers for Escherichia coli DNA polymerase I and T4-induced DNA polymerase, under the normal conditions for :DNA synthesis in vitro. The DNA product is covalently linked to the 3'-end of the RNA, and co-purifies with it in CsCl equilibrium centrifugation. Priming by RNA is readily detectable even when DNA primers are also present. These and other features of the reaction suggest that it might form the basis of a general method for the study of DNA sequences beyond the termini of RNA chains.For the controlled expression of genetic information, the transcription of RNA from DNA must be initiated and terminated a t defined loci. There is evidence that RNA chain extension ceases when a termination signal encoded in the DNA is reached and recognised by the RNA-polymerising enzyme [i]. I n some, but not all cases this recognition seems to require the co-operation of accessory proteins, such as rho-factor in Escherichia coli [a].To understand the mechanism of RNA termination, it seems desirable to study the base sequence of termination signals. If these signals are fully transcribed from the DNA into the distal, 3'-ends of the RNA molecules involved, it should be comparatively easy to discover their nature through sequencing Enzymes. RNA polymerase or nucleoside triphosphate : RNA nucleotidyltransferase (EC 2.7.7.6) ; DNA polymerase or deoxynucleoside triphosphate : DNA nucleotidyltransferase (EC 2.7.7.7) ; pancreatic deoxyribonuclease or deoxyribonucleate oligonucleotide hydrolase (EC 3.1.4.5) ; ribonuclease T, or ribonucleate guaninenucleotide-2'-transferase (EC 2.7.7.26) ; alkaline phosphat,ase or orthophosphoric monoester phosphohydrolase (EC 3.1.3.1). studies on the 3'-regions of appropriate RNA species [3].However, it is quite conceivable that termination signals lie wholly or partially beyond the distal end of that portion of the DNA which is actually transscribed. Novel techniques would then be necessary for the study of terminator sequences. The present work was motivated by the desire to develop such a technique .If one imagines an intact RNA molecule hybridised with the DNA strand to which it is complementary, the presumptive untranscribed termination signal should lie in the region of single-stranded DNA following the 3'-hydroxyl terminus of the RNA, and presumably not far beyond it. A DNA polymerase capable of using this RNA terminus as a primer would synthesise a polydeoxyribonucleotide, the initial portion of which should be complementary to the sequence of interest on the DNA template strand. The sequence of the synthetic polynucleotide could...

Dinucleotide Sequences in the Regions of T7 DNA Coding for Termination of Early Transcription

“…After centrifugation the supernatants were neutrdlised, and subjected to electrophoresis as described previously [3], but using Whatman no. 52 paper.…”

“…52 paper. After autoradiography, some tracks were analysed by scintillation counting [3]. Relevant spots were eluted with 30 triethylamine carbonate, and further characterised by ascending chromatography on Whatman 3MM paper, using 66 % isobutyric acid, 0.2 mM EDTA, adjusted to pH 3.7 with NH,OH.…”

“…This drug prevents the initiation of RNA chains, while permitting completion of nascent molecules [16]. The incorporation of radioisotope into acidprecipitable form was monitored as described previously [3]. The amount of [3H]RNA formed was estimated from the incorporation data, assuming the base composition to be 25 % uracil.…”

“…DNA synthesis reactions (0.3 ml or 0.6 ml) were prepared as described previously [3], using fresh DNA .RNA hybrids (0.8-3.6 pg [3H]RNA) as templates, and 2 units of DNA polymerase I per 0.3 ml. Where specified, deoxyribonucleoside [a-32P]triphosphates were included (3 -10 Ci/mmol).…”

“…Equally, it might overlap the RNA terminus, or lie a short way beyond the end of the RNA-coding unit. We have previously described a method especially designed for the determination of sequences of the latter type [3]. The basic idea is to (a) hybridise a specific RNA transcript to the complementary template DNA strand; (b) use the 3'-hydroxyl end of the RNA as a primer for DNA synthesis by, for example, E.coli DNA polymerase I, with Enz,vrnes.…”