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Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Billingsley DJ, Crampton N, Kirkham J, Thomson NH, Bonass WA - Nucleic Acids Res. (2012)

Bottom Line: A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM.Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase.This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, Leeds Dental Institute, University of Leeds, Woodhouse Lane, Leeds, West Yorkshire, LS2 9JT, UK.

ABSTRACT
Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

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Montage of single-molecule software zooms depicting OPCs. The RNAPs are identified as two globular features, much wider than the chain width, and separated by a distance consistent with the inter-promoter length. Also visible is the loop feature at one end of the template. Scale bars: 50 nm.
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gks276-F8: Montage of single-molecule software zooms depicting OPCs. The RNAPs are identified as two globular features, much wider than the chain width, and separated by a distance consistent with the inter-promoter length. Also visible is the loop feature at one end of the template. Scale bars: 50 nm.

Mentions: Of the complexes containing two RNAPs, the holoenzymes were identified as being two equally sized globular structures situated in the central region of the template. A montage of labelled DNA molecules containing two RNAPs, characteristic of OPCs is shown in Figure 8. These contain RNAPs bound in the position expected of the promoter. Such complexes also display the same globular feature at just a single end as seen previously. This shows that the presence of a loop structure at one end does not affect a proteins ability to locate and form interactions with its binding site.Figure 8.


Single-stranded loops as end-label polarity markers for double-stranded linear DNA templates in atomic force microscopy.

Billingsley DJ, Crampton N, Kirkham J, Thomson NH, Bonass WA - Nucleic Acids Res. (2012)

Montage of single-molecule software zooms depicting OPCs. The RNAPs are identified as two globular features, much wider than the chain width, and separated by a distance consistent with the inter-promoter length. Also visible is the loop feature at one end of the template. Scale bars: 50 nm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401461&req=5

gks276-F8: Montage of single-molecule software zooms depicting OPCs. The RNAPs are identified as two globular features, much wider than the chain width, and separated by a distance consistent with the inter-promoter length. Also visible is the loop feature at one end of the template. Scale bars: 50 nm.
Mentions: Of the complexes containing two RNAPs, the holoenzymes were identified as being two equally sized globular structures situated in the central region of the template. A montage of labelled DNA molecules containing two RNAPs, characteristic of OPCs is shown in Figure 8. These contain RNAPs bound in the position expected of the promoter. Such complexes also display the same globular feature at just a single end as seen previously. This shows that the presence of a loop structure at one end does not affect a proteins ability to locate and form interactions with its binding site.Figure 8.

Bottom Line: A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM.Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase.This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

View Article: PubMed Central - PubMed

Affiliation: School of Physics and Astronomy, Leeds Dental Institute, University of Leeds, Woodhouse Lane, Leeds, West Yorkshire, LS2 9JT, UK.

ABSTRACT
Visualization of DNA-protein interactions by atomic force microscopy (AFM) has deepened our understanding of molecular processes such as DNA transcription. Interpretation of systems where more than one protein acts on a single template, however, is complicated by protein molecules migrating along the DNA. Single-molecule AFM imaging experiments can reveal more information if the polarity of the template can be determined. A nucleic acid-based approach to end-labelling is desirable because it does not compromise the sample preparation procedures for biomolecular AFM. Here, we report a method involving oligonucleotide loop-primed synthesis for the end labelling of double-stranded DNA to discriminate the polarity of linear templates at the single-molecule level. Single-stranded oligonucleotide primers were designed to allow loop formation while retaining 3'-single-strand extensions to facilitate primer annealing to the template. Following a DNA polymerase extension, the labelled templates were shown to have the ability to form open promoter complexes on a model nested gene template using two Escherichia coli RNA polymerases in a convergent transcription arrangement. Analysis of the AFM images indicates that the added loops have no effect on the ability of the promoters to recruit RNA polymerase. This labelling strategy is proposed as a generic methodology for end-labelling linear DNA for studying DNA-protein interactions by AFM.

Show MeSH
Related in: MedlinePlus