Effect of template conversion from the B to the Z conformation on RNA polymerase activity

Butzow, J. J.; Shin, Y. A.; Eichhorn, G.

doi:10.1021/bi00316a004

Cited by 35 publications

(21 citation statements)

References 23 publications

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“…The reported ratio is 3.2 for Z-form and 8.5 for B-form poly(dG-dC) (1). It had been previously shown that under the experimental conditions employed here there is little if any aggregation of Z-poly(dG-dC) (15). Indeed the ultraviolet absorbance and circular dichroism spectra were those expected for the Z-form of poly(dG-dC) with no absorbance at wavelengths greater than 300 nm nor evidence of scattered uv light due to aggregation (16).…”

Section: Methodssupporting

confidence: 50%

Interactions of water soluble porphyrins with Z-poly(dG-dC)

Pasternack¹,

Sidney²,

Hunt³

et al. 1986

Nucl Acids Res

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The water soluble porphyrin tetrakis(4-N-methylpyridyl)porphine (H2TMpyP) and its copper(II) derivative (CuTMpyP) convert Z-poly(dG-dC) to the B-form. For H2TMpyP, the fraction Z character (fr-Z) is given by fr-Z = 1.0 - 21 rO and for CuTMpyP, fr-Z = .94 - 12 rO where rO identical to [Porphyrin]O/[DNA]O. Neither the manganese(III) derivative of of this porphyrin (MnTMpyP) nor tetrakis(2-N-methylpyridyl)porphine (H2TMpyP-2) is nearly as effective at causing the conversion. The former two porphyrins have been shown to intercalate into B-poly(dG-dC) whereas the latter two porphyrins do not. The kinetics of the Z----B conversion are independent of porphyrin or poly(dG-dC) concentration for 1/rO greater than 6. At smaller values of 1/rO, the conversion rate is greatly increased for H2TMpyP and CuTMpyP. The interaction of these porphyrins with Z-poly(dG-dC) follows simple first order kinetics in this latter concentration range. It is proposed that for small values of 1/rO the sequence of events begins with a porphyrin-unassisted distortion of the Z-duplex (with a rate constant of 0.6 s-1) followed by a rapid uptake of porphyrin in what may be an intercalative mode. The porphyrin thus located in Z-regions brings about rapid conversion to the B-form. Binding of H2TMpyP or CuTMpyP to B-regions of a predominantly Z-strand leads to conversion of Z to B. However, this conversion process is considerably slower than when the porphyrins bind directly to Z-regions.

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Section: Methodssupporting

confidence: 50%

Interactions of water soluble porphyrins with Z-poly(dG-dC)

Pasternack¹,

Sidney²,

Hunt³

et al. 1986

Nucl Acids Res

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“…Consistent with this idea, the Z-conformation has been shown to provide a poor template for transcriptional activity with Escherichia coli RNA polymerase (27), and further, the d(C-G)16 sequence in supercoiled plasmids causes a severe block to transcription in vitro (28). This notion is consistent also with the location of alternating purine-pyrimidine tracts in simian virus 40 DNA regulatory sequences that bind anti-Z-DNA antibodies (29), of the d(G-T), tracts at the ends of yeast chromosomes (30) and of the alternating purine-pyrimidine long terminal repeats in mouse mammary tumor virus (31).…”

Section: -64 T-c-c-g-c-g-c-a-c-a-tmentioning

confidence: 82%

Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris(diphenylphenanthroline)cobalt(III).

Barton¹,

Raphael²

1985

Proc. Natl. Acad. Sci. U.S.A.

104

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The chiral complex tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), A-Co(DiP)3+, binds to and, with photoactivation, cleaves left-handed DNA helices, thereby providing a unique molecular probe for local DNA conformation. We have mapped the specific left-handed sites where A-Co(DiP)3+ cleaves in the plasmids pBR322 and pLP32, which is the derivative ofpBR322 containing a Z-form d(C-G)16 insert. For pLP32, a primary cleavage is at the insert; for native pBR322, cleavage occurs at four disctte sites: 1.45, 2.3, 3.3, and 4.2 kilobase pairs. These sites correspond to segments of alternating purine-pyrimidines. Moreover, these positions map to the ends of the three distinct coding regions in pBR322: the tetracycline-resistance gene, the origin of replication, and either end of the ampicillin-resistance (.3-lactamase) gene. The locations of these left-handed segments suggest to us that Z-DNA might serve as a conformational punctuation mark to demarcate the ends of genes.Numerous spectroscopic and x-ray crystallographic studies have shown that DNA may adopt a range of conformations, from the right-handed A-and B-forms to the striking lefthanded Z-DNA helix (1-7). Regions of conformational heterogeneity along the strand, such as cruciform structures, single-stranded loops, and left-handed segments, have been detected by using DNA enzymes (8-10), and it has been suggested that local DNA conformation might play some role in regulating gene expression. Chiral metal complexes that can intercalate into the helix are particularly advantageous in probing local DNA conformation (11)(12)(13)(14). Tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) complexes [Ru(DiP)"+] provide a spectroscopic probe for helix handedness; the A isomer, which does not bind B-DNA owing to steric constraints, binds avidly to Z-DNA (13). Upon photoactivation, the analogous cobalt isomers, Co(DiP)3+, furthermore cleave DNA stereospecifically, providing a sensitive assay for local regions in the Z form (14). In this report we describe the specific cleavage by A-Co(DiP)31 of discrete sites in the plasmid pLP32 (15), which contains a well-defined Z-DNA segment, and in pBR322 (16, 17). The chiral complex serves as a conformation-specific tool to map these left-handed DNA helical segments. [Co(DiP)3](tartrate)3 (10 ttM) was added to plasmid DNA (100 jkM nucleotides) in 50 mM Tris acetate buffer containing 18 mM NaCl (pH 7.0). The 20-td sample was then irradiated at 315 nm for 90 sec and ethanol-precipitated. The ethanol wash removes unreacted Co(DiP)3+ and the metal and ligand products of the reaction. After resuspension in Tris acetate buffer containing 50 mM NaCl and 10 mM MgCl2 (pH 7.0), restriction enzyme was added (EcoRI, BamHI, Ava I, or Nde I) in at least a 3-fold excess to ensure complete linearization. This was incubated at 370C for 45 min. The pH of the reaction mixture was then lowered to 5.0, and 10 mM Zn(NO3)2 was added along with 4 units of nuclease S1, and the samples were incubated for 5 min at 370C. This step causes cleavage of the...

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“…Previous studies from this and other laboratories have shown that synthetic Z DNA helices are relatively poor substrates for Escherichia coli RNA polymerase (van de Sande and Jovin, 1982;Butzow et al, 1984). Furthermore, in experiments cited in Rich et al (1984), d(G-C)n tracts within a transcribed gene (in negatively supercoiled plasmid molecules) lead to termination of transcription.…”

Section: Advantages Of High Resolution Darkfield Em Of Native Dna-iggmentioning

confidence: 90%

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

Révet¹,

Zarling²,

Jovin

et al. 1984

The EMBO Journal

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The specific interaction between left‐handed Z DNA sequences in negatively supercoiled bacteriophage phi X174 replicative form I (RFI) DNA and anti‐Z DNA immunoglobulin G (IgG) was investigated by high resolution darkfield immuno‐electron microscopy. DNA‐antibody complexes were formed and maintained under optimal binding conditions, purified by column chromatography, and visualized after uranyl acetate staining without using aldehyde fixation, shadowing, or second antibody. Bivalent anti‐Z DNA IgGs bound to RFI molecules, thus forming intramolecular bridges. They could also oligomerize separate molecules by intermolecular linking of Z DNA sequences. At relatively low ionic strength and low temperature, high affinity anti‐Z IgG was retained at certain loci even after restriction endonuclease cleavage of the DNA. In these cleaved molecules some superhelices could be visualized in the loops generated by the bivalent IgG. To our knowledge this is the first example of polypeptide stabilization of local superhelical strain in a cut molecule. Z DNA sequences in phi X174 RFI DNA were mapped. Alternating tracts of purines and pyrimidines starting at nucleotides 763, 1027, 1714, 2146, 2363, 3504, 4161, 4911 and 5345 occur within the nine different anti‐Z IgG binding sites which were expressed with varying frequencies (53‐3%) on the molecules. Usually, a limited number of sites (generally less than or equal to 2) exists on any one molecule. The formation of multiple Z sites (at the extracted superhelix density) in a given molecule is probably non‐cooperative due to relaxation of torsional stress by the B–‐Z transition. Z sites occur in several different genes, including regions where transcription is attenuated and, in one case, in front of a promoter of transcription.

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Effect of template conversion from the B to the Z conformation on RNA polymerase activity

Cited by 35 publications

References 23 publications

Interactions of water soluble porphyrins with Z-poly(dG-dC)

Interactions of water soluble porphyrins with Z-poly(dG-dC)

Site-specific cleavage of left-handed DNA in pBR322 by lambda-tris(diphenylphenanthroline)cobalt(III).

Different Z DNA forming sequences are revealed in phi X174 RFI by high resolution darkfield immuno-electron microscopy.

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