In Vitro Transcription in E. Coli Crude Lysates Prepared on Cellophane Discs

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1978 Jul 1

PMID 27767

Authors

S Valla

B H Lindqvist

Affiliations

Soon will be listed here.

Abstract

An in vitro RNA-synthesizing system consisting of gently lysed E. coli cells on cellophane discs is described. The system has been optimalized with respect to total RNA synthesis. Under certain standard conditions DNA dependent RNA polymerase (EC 2.7.7.6) is responsible for the majority of the RNA synthesis. The extensive rifampicin sensitivity of the synthesis indicates that most of the transcripts are initiated in vitro. The RNA synthesizing system described here has been developed with the aim of studying phage transcription in vitro. We show here that lysates of a P4 infected P2 lysogen support initiation and propagation of transcription from the P2 prophage.

References

Six E . The helper dependence of satellite bacteriophage P4: which gene functions of bacteriophage P2 are needed by P4?. Virology. 1975; 67(1):249-63. DOI: 10.1016/0042-6822(75)90422-5. View

Giorno R, Stamato T, Lydersen B, Pettijohn D . Transcription in vitro of DNA in isolated bacterial nucleoids. J Mol Biol. 1975; 96(2):217-37. DOI: 10.1016/0022-2836(75)90344-7. View

Ohasa S, Tsugita A . Purification and characterization of a new ribopolynucleotide synthesizing enzyme from Escherichia coli. J Mol Biol. 1976; 105(4):545-65. DOI: 10.1016/0022-2836(76)90234-5. View

Rabussay D, Geiduschek E . Phage T4-modified RNA polymerase transcribes T4 late genes in vitro. Proc Natl Acad Sci U S A. 1977; 74(12):5305-9. PMC: 431695. DOI: 10.1073/pnas.74.12.5305. View

Lindqvist B, BOVRE K . Asymmetric transcription of the coliphage P2 genome during infection. Virology. 1972; 49(3):690-9. DOI: 10.1016/0042-6822(72)90526-0. View

Schaller H, Otto B, Nusslein V, Huf J, Herrmann R, BONHOEFFER F . Deoxyribonucleic acid replication in vitro. J Mol Biol. 1972; 63(2):183-200. DOI: 10.1016/0022-2836(72)90369-5. View

Six E, Klug C . Bacteriophage P4: a satellite virus depending on a helper such as prophage P2. Virology. 1973; 51(2):327-44. DOI: 10.1016/0042-6822(73)90432-7. View

Otto B, BONHOEFFER F, Schaller H . Purification and properties of DNA polymerase 3. Eur J Biochem. 1973; 34(3):440-7. DOI: 10.1111/j.1432-1033.1973.tb02777.x. View

Lindqvist B . Expression of phage transcription in P2 lysogens infected with helper-dependent coliphage P4. Proc Natl Acad Sci U S A. 1974; 71(7):2752-5. PMC: 388548. DOI: 10.1073/pnas.71.7.2752. View

10.

Fuchse , MILLETTE R, Zillig W, Walter G . Influence of salts on RNA synthesis by DNA-dependent RNA-polymerase from Escherichia coli. Eur J Biochem. 1967; 3(2):183-93. DOI: 10.1111/j.1432-1033.1967.tb19514.x. View

11.

Reiner A . Characterization of polynucleotide phosphorylase mutants of Escherichia coli. J Bacteriol. 1969; 97(3):1437-43. PMC: 249866. DOI: 10.1128/jb.97.3.1437-1443.1969. View

12.

Sasaki I, Bertani G . Growth abnormalities in Hfr derivatives of Escherichia coli strain C. J Gen Microbiol. 1965; 40(3):365-76. DOI: 10.1099/00221287-40-3-365. View

13.

COHEN S . The inhibition of polynucleotide phosphorylase by certain substrate analogues. Biochim Biophys Acta. 1966; 123(3):471-7. DOI: 10.1016/0005-2787(66)90215-2. View

14.

Zimmerman S, Sandeen D . The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal Biochem. 1966; 14(2):269-77. DOI: 10.1016/0003-2697(66)90137-0. View

15.

Ishii S, Imamoto F . In vitro transcription of the tryptophan operon in isolated bacterial nucleoids. Mol Gen Genet. 1976; 148(3):295-305. DOI: 10.1007/BF00332904. View