Studies on T3 induced ribonucleic acid polymerase. III. Purification and characterization of the T3 induced ribonucleic acid polymerase from bacteriophage T3 infected Escherichia coli cells

P. R. Chakraborty, P. Sarkar, H. H. Huang, U. Maitra

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Abstract

A procedure for the isolation and purification of bacteriophage T3 induced RNA polymerase from T3 infected Escherichia coli cells is presented. The procedure described leads to the isolation of polymerase preparation that displays a single protein band by polyacrylamide gel electrophoresis and is free of detectable ribonuclease and deoxyribonuclease activities. Other enzyme activities absent from such preparations include inorganic pyrophosphatase, nucleoside diphosphokinase, DNA polymerase, E. coli RNA polymerase, nucleoside triphosphatase, polynucleotide phosphorylase, polyriboadenylate polymerase, and polyphosphate kinase. T3 induced RNA polymerase was characterized with regard to various biochemical parameters. The enzyme is a single polypeptide chain of molecular weight 105,000 ± 5,000. The enzyme activity is completely dependent on the presence of Mg2+, all 4 nucleoside triphosphates, and T3 DNA. T7 DNA shows only 2 to 5% of activity as that obtained with T3 DNA, while other native DNA preparations examined are inactive. The T3 RNA polymerase is highly sensitive to salt concentrations above 0.03 M or to reagents which react with sulfhydryl groups such as p hydroxymercuribenzoate and N ethylmaleimide. The enzyme catalyzes a T3 DNA dependent 32PP(i) exchange into nucleoside triphosphates. However, no pyrophosphorolysis of free RNA can be demonstrated. The 32PP(i) exchange reaction can occur with GTP alone. The exchange reaction with the other 3 nucleoside triphosphates, present either alone or in combination, does not occur except when GTP is included in the reaction. The effect of E. coli RNA chain termination factor, rho, on the T3 RNA polymerase reaction was studied. It was found that rho factor had no effect on the rate, yield, or size of RNA formed in the T3 RNA polymerase catalyzed reactions.

Original languageEnglish (US)
Pages (from-to)6637-6646
Number of pages10
JournalJournal of Biological Chemistry
Volume248
Issue number19
StatePublished - 1973

Fingerprint

Bacteriophage T3
Bacteriophages
Escherichia coli
Purification
RNA
Rho Factor
Nucleosides
DNA
Enzyme activity
Enzymes
DNA-Directed RNA Polymerases
Guanosine Triphosphate
Polyribonucleotide Nucleotidyltransferase
Nucleoside-Triphosphatase
Nucleoside-Diphosphate Kinase
Inorganic Pyrophosphatase
Ethylmaleimide
Deoxyribonucleases
DNA-Directed DNA Polymerase
Ribonucleases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Studies on T3 induced ribonucleic acid polymerase. III. Purification and characterization of the T3 induced ribonucleic acid polymerase from bacteriophage T3 infected Escherichia coli cells. / Chakraborty, P. R.; Sarkar, P.; Huang, H. H.; Maitra, U.

In: Journal of Biological Chemistry, Vol. 248, No. 19, 1973, p. 6637-6646.

Research output: Contribution to journalArticle

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abstract = "A procedure for the isolation and purification of bacteriophage T3 induced RNA polymerase from T3 infected Escherichia coli cells is presented. The procedure described leads to the isolation of polymerase preparation that displays a single protein band by polyacrylamide gel electrophoresis and is free of detectable ribonuclease and deoxyribonuclease activities. Other enzyme activities absent from such preparations include inorganic pyrophosphatase, nucleoside diphosphokinase, DNA polymerase, E. coli RNA polymerase, nucleoside triphosphatase, polynucleotide phosphorylase, polyriboadenylate polymerase, and polyphosphate kinase. T3 induced RNA polymerase was characterized with regard to various biochemical parameters. The enzyme is a single polypeptide chain of molecular weight 105,000 ± 5,000. The enzyme activity is completely dependent on the presence of Mg2+, all 4 nucleoside triphosphates, and T3 DNA. T7 DNA shows only 2 to 5{\%} of activity as that obtained with T3 DNA, while other native DNA preparations examined are inactive. The T3 RNA polymerase is highly sensitive to salt concentrations above 0.03 M or to reagents which react with sulfhydryl groups such as p hydroxymercuribenzoate and N ethylmaleimide. The enzyme catalyzes a T3 DNA dependent 32PP(i) exchange into nucleoside triphosphates. However, no pyrophosphorolysis of free RNA can be demonstrated. The 32PP(i) exchange reaction can occur with GTP alone. The exchange reaction with the other 3 nucleoside triphosphates, present either alone or in combination, does not occur except when GTP is included in the reaction. The effect of E. coli RNA chain termination factor, rho, on the T3 RNA polymerase reaction was studied. It was found that rho factor had no effect on the rate, yield, or size of RNA formed in the T3 RNA polymerase catalyzed reactions.",
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