Abstract
Changes in near UV circular dichroism (CD) and fluorescence spectra of site-specifically placed pairs of 2-aminopurine residues have been used to probe the roles of the RNA hairpin and the RNA-DNA hybrid in controlling intrinsic termination of transcription. Functional transcription complexes were assembled directly by mixing preformed nucleic acid scaffolds of defined sequence with T7 RNA polymerase (RNAP). Scaffolds containing RNA hairpins immediately upstream of a GC-rich hybrid formed complexes of reduced stability, whereas the same hairpins adjacent to a hybrid of rU-dA base pairs triggered complex dissociation and transcript release. 2-Aminopurine probes at the upstream ends of the hairpin stems show that the hairpins open on RNAP binding and that stem re-formation begins after one or two RNA bases on the downstream side of the stem have emerged from the RNAP exit tunnel. Hairpins directly adjacent to the RNA-DNA hybrid weaken RNAP binding, decrease elongation efficiency, and disrupt the upstream end of the hybrid as well as interfere with the movement of the template base at the RNAP active site. Probing the edges of the DNA transcription bubble demonstrates that termination hairpins prevent translocation of the RNAP, suggesting that they transiently "lock" the polymerase to the nucleic acid scaffold and, thus, hold the RNA-DNA hybrid "in frame." At intrinsic terminators the weak rU-dA hybrid and the adjacent termination hairpin combine to destabilize the elongation complex sufficiently to permit significant transcript release, whereas hairpin-dependent pausing provides time for the process to go to completion.
Highlights
Nascent RNA [2, 3] followed by (ii) a run of 8 –10 nucleotide residues,2 consisting primarily of uridine residues, that is located immediately downstream of the hairpin and at the 3Ј-end of the released RNA [4]
Standard free energy data for the DNA, RNA, and RNADNA base pairs of the nucleic acid framework of the transcription complex were used to calculate that the presence of the weak rU-dA hybrid at intrinsic terminator sites, in combination with the invasion or distortion of the upstream end of the hybrid by the termination hairpin, should result in destabilization of the elongation complex sufficient to bring about significant transcript release and elongation complex dissociation
We have in part been able to bypass these difficulties because the termination hairpin and the weak hybrid work together to bring about intrinsic termination, and these elements do destabilize and otherwise perturb the elongation complex when present together, they do so less for complexes containing only one or the other
Summary
Materials—Unlabeled and 2-AP-labeled DNA oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA) and from Operon (Huntsville, AL). 2-AP-labeled and unlabeled RNA were from Dharmacon (Lafayette, CO). The DNA scaffold constructs containing non-complementary bubble sequences were formed and annealed by heating a solution containing equimolar concentrations of template and non-template strands at 90 °C for 4 min and gradually cooling to room temperature over a period of 2 h. 3 M (equimolar) concentrations of T7 RNAP and nucleic acid framework constructs were used in all experiments. Control spectra measured with identical concentrations of unlabeled oligonucleotides (and RNAP, NTPs, etc.) were subtracted from the probe-containing low energy spectra of the polymerasescaffold complexes to correct for any non-probe-related optical. RNase H Digestion of Free Scaffold Constructs and Scaffold-RNAP Complexes—In these experiments the nucleic acid scaffolds TTGGGGCG T ||||||||n A A AACGGUAACCCCGC ing 5Ј-end-labeled RNA as above) and the scaffold-RNAP complexes (equimolar concentrations) were. RNase H activities of 1 and 0.1 units/l, respectively, were used with the 3 M and 300 nM scaffold-RNAP complexes, and digestions were run for 30 min at 37 °C
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