Billingsley, Daniel Jeffrey (2012) Convergent transcription and nested gene models studied by AFM. PhD thesis, University of Leeds.
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Genomic DNA is organised in complex spatial arrangements, and a given stretch of DNA may encode more than one gene. In some cases one gene may be entirely contained within a region of the DNA already occupied by another larger gene. The presence of these nested genes, often situated in introns and in the opposite orientation, poses important implications with regards to gene expression, function and regulation. A consequence of the nested gene arrangement is convergent transcription, occurring when two promoters on opposite DNA strands are active. Elucidating the mechanics of multiple interacting proteins on single DNA templates requires single molecule methods such as atomic force microscopy (AFM). AFM can accurately determine the relative positions of enzymes, such as RNA polymerase (RNAP), on individual DNA templates. The central aim of this thesis is to use AFM to study the outcomes of convergent transcription, using linear DNA templates that function as models for nested genes. Fundamental aspects of imaging DNA on mica with AFM were investigated, with a view to optimising sample preparation. The main processes involved with preparing DNA samples, ready for scanning, were examined in turn. Effective binding was achieved by introducing divalent cations into a deposition buffer. Mica ion exchanged with Ni(II) usually gave rise to kinetically-trapped DNA molecules, however short linear fragments (< 800bp) were seen to deviate from the expected behaviour, indicating that ion-exchanged mica is heterogeneous, and contains patches or domains. These findings can be used to more readily control binding of DNA to substrates. The outcomes of varying the relative humidity while imaging biomolecular systems are largely unexplored to date. Various DNA samples were imaged in conditions of varying humidity. In particular when supercoiled plasmids are scanned at very high relative humidity (> 90% RH), localised DNA backbone motions or conformational changes were observed. Humidity controlled AFM will be a useful technique for probing DNA topology without some of the drawbacks of imaging under bulk solution. Initial studies of transcription utilised templates containing two promoter sites and E. Coli RNAP. Two promoter arrangements were studied: a convergent template containing the promoter sites on opposite strands directed towards each other, and a tandem template containing the promoters in the same direction, on the same stand. It was shown that collisions between RNAPs led to similar outcomes in both cases: RNAPs are unable to pass each other and remain stalled against each other. In the convergent case, it was observed that after collision one RNAP could cause another to backtrack along the template. By end-labelling double-stranded (ds) DNA templates with a single-stranded DNA loop, the polarity of the molecules can be established in the AFM. It allowed better discrimination between outcomes of collision events on single DNA molecules and importantly, it enabled a quantitative comparison of the relative frequencies of the outcomes. The most common outcome is a collision between an actively transcribing elongation complex (EC) and a “sitting duck” (SD), which is a stalled RNAP or open promoter complex (OPC). In collisions initiated from OPCs, the most likely outcome, a collision between an EC and an SD occurs ~74% of the time. This causes sizeable back-tracking of the inactive RNAP, on average 59 nm upstream of the promoter. A significant fraction of the collisions (~15%) are between actively transcribing RNAP while the remainder (~11%) are undetermined. End-labelling of dsDNA using nucleic acid structures did not interfere with AFM sample preparation and can be used as a generic approach to studying interactions of multiple proteins on DNA templates at the single molecule level.
|Item Type:||Thesis (PhD)|
|Academic Units:||The University of Leeds > Faculty of Maths and Physical Sciences (Leeds) > School of Physics and Astronomy (Leeds)|
|Depositing User:||Repository Administrator|
|Date Deposited:||03 Dec 2012 14:06|
|Last Modified:||08 Aug 2013 08:51|