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The in vitro transcription units of bacteriophage φX174. I. Characterization of synthetic parameters and measurement of transcript molecular weights

Smith, L. H. and Sinsheimer, R. L. (1976) The in vitro transcription units of bacteriophage φX174. I. Characterization of synthetic parameters and measurement of transcript molecular weights. Journal of Molecular Biology, 103 (4). pp. 681-697. ISSN 0022-2836. https://resolver.caltech.edu/CaltechAUTHORS:20160805-103218662

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Abstract

In vitro transcription of bacteriophage replicative φX174 form I DNA by Escherichia coli RNA polymerase holoenzyme is studied under a variety of ionic conditions. Uniformly ^(32)P-labeled product RNA is fractionated by electrophoresis on 2% acrylamide, 0.5% agarose or 7.5 mm-CH_3HgOH-containing, 1.5% agarose slab gels. A large fraction (65 to 70%) of the RNA made during 30 minutes synthesis at 37 °C is present as a heterodisperse population of molecules with a mean molecular weight of 2 to 3 × 10^6. From 30 to 35% of the product RNA is present in several discretely sized bands of molecular weights 1.55, 1.95, 2.35, 3.70, 4.0, 4.15 and approximately 5 × 10^6. All components except the first arise from more than one circumtranscription of φX replicative form I template. Thus specific termination is detected under the conditions studied, but apparently is not totally efficient. A method allowing (α-^(32)P)-labeling of a short (10 to 80 nucleotides) 5′ proximal region of φX174 in vitro RNA is described. Fractionation of 5′ proximally labeled in vitro φX RNA resolves all of the discrete components detected by the uniform labeling technique and, in addition, several smaller components. Approximately 50% of the molecules synthesized are both initiated and terminated specifically; the remaining 50% of the molecules synthesized are present as heterodisperse material presumably arising from either non-specific initiation or termination (or both).


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1016/0022-2836(76)90203-5DOIArticle
http://www.sciencedirect.com/science/article/pii/0022283676902035PublisherArticle
Additional Information:© 1976 Elsevier Ltd. Received 30 June 1975, and in revised form 15 December 1975. We are indebted to Harumi Kasamatsu for help with the RNA polymerase preparation. We are grateful to Drs J. Bailey and N. Davidson for providing the details of the methyl mercury/agarose electrophoresis technique. J. B. Clements and A. Szalay are acknowledged for their contributions to the preliminary part of this work. This research was supported by Grant no. GM 13554 from the National Institute of Health.
Funders:
Funding AgencyGrant Number
U.S. Public Health Service (USPHS)GM 13554
Issue or Number:4
Record Number:CaltechAUTHORS:20160805-103218662
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160805-103218662
Official Citation:L.H. Smith, R.L. Sinsheimer, The in vitro transcription units of bacteriophage φX174: I. Characterization of synthetic parameters and measurement of transcript molecular weights, Journal of Molecular Biology, Volume 103, Issue 4, 5 June 1976, Pages 681-697, ISSN 0022-2836, http://dx.doi.org/10.1016/0022-2836(76)90203-5. (http://www.sciencedirect.com/science/article/pii/0022283676902035)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:69471
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:05 Aug 2016 18:00
Last Modified:03 Oct 2019 10:22

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