Abstract
Escherichia coli Rho factor is a ring-shaped, homohexameric protein that terminates synthesis of RNA through interactions with the nascent RNA transcript. Because its mechanism of action may involve translocation of the RNA transcript through the hole in its ring structure, its action could depend on the availability of a free 5' terminus. To determine whether Rho's activity is 5'-end-dependent, its ability to bind to and function on a circular derivative of lambda cro mRNA was investigated. The circular derivative was made in vitro by action of RNA ligase on a derivative of lambda cro RNA containing an extra 10-nucleotide sequence near the 5'-end that was complementary to a sequence located near the 3'-end. Rho bound nearly as tightly to the circular derivative RNA as to the standard cro transcript. Rho was also able to readily dissociate a DNA oligonucleotide from its helical complex with the circular RNA in an ATP-dependent reaction. Thus, the action of Rho on a transcript does not depend on the availability of a free 5' terminus.
Highlights
The circular derivative was made in vitro by action of RNA ligase on a derivative of cro RNA containing an extra 10-nucleotide sequence near the 5-end that was complementary to a sequence located near the 3-end
Rho factor is believed to function in vivo as a hexamer consisting of six polypeptide subunits arranged in a ring-shaped structure [6, 7]
To test the model in which Rho requires a free end of RNA to thread onto in order to function as a terminator, we investigated Rho’s ability to utilize its ATP-dependent helicase activity on an RNA lacking a free end
Summary
Materials—All restriction enzymes, T4 RNA ligase, T4 polynucleotide kinase, and calf intestinal alkaline phosphatase were purchased from New England Biolabs, Inc. Circularization of the Linear pBcCro RNA Transcript—The 5Ј-terminal triphosphate of the transcript was removed by treatment of 20 g of RNA with 2 units of calf intestinal alkaline phosphatase in 50 l of a solution containing 20 mM Tris-HCl, pH 8.0, 50 mM KCl, 1 mM dithiothreitol, and 40 units of RNasin for 2 h at 23 °C [16]. The 5Ј-end labeled RNA was circularized by treatment with 25 units of T4 RNA ligase in a 50-l solution containing 25 mM Tris-HCl, pH 7.6, 10 mM dithiothreitol, 5 mM MgCl2, 5 mM ATP, and 0.25 mg/ml acetylated bovine serum albumin, and 40 units RNasin for 15 h at 17 °C. The reactions contained 32 mM Hepes-KOH buffer, pH 8.0, 50 mM KCl, 1 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 40 units of RNasin, 0.2 mg/ml acetylated bovine serum albumin, and 5 nM wild-type Rho hexamer. Where F is the fraction RNA-DNA hybrid remaining after time t (t ϭ 0 s was normalized to 1), A1 and k1 are the amplitude and rate constant (units sϪ1), respectively, for the first fast burst (exponential) decay phase, and A2 and k2 are the intercept and slope, respectively, of the second slower linear-decay phase
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