http://nova.newcastle.edu.au/vital/access/services/Feed ${session.getAttribute("locale")} 5 http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:11074 The synthesis of RNA is highly regulated at all stages by transcription factors. As an essential transcription elongation factor, NusA has been studied biochemically for more than 30 years. However, until now no structural information has been available on NusA-RNAP complexes and the NusA interaction site on RNAP was a point of speculation. Determination of the structure of RNA polymerase in complex with NusA is helping us understand how NusA regulates transcription. The resulting model of RNA polymerase in complex with NusA has shed light on the transition from the initiation to elongation stages of transcription, and how NusA functions in promoting regulatory pausing and termination. 2012-07-12T03:30:03.672Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:10634 There are three stages of transcription: initiation, elongation and termination, and traditionally there has been a clear distinction between the stages. The specificity factor sigma is completely released from bacterial RNA polymerase after initiation, and then recycled for another round of transcription. Elongation factors then associate with the polymerase followed by termination factors (where necessary). These factors dissociate prior to initiation of a new round of transcription. However, there is growing evidence suggesting that sigma factors can be retained in the elongation complex. The structure of bacterial RNAP in complex with an essential elongation factor NusA has recently been published, which suggested rather than competing for the major σ binding site, NusA binds to a discrete region on RNAP. A model was proposed to help explain the way in which both factors could be associated with RNAP during the transition from transcription initiation to elongation. 2012-04-12T05:59:55.195Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:8345 There are three stages of transcribing DNA into RNA. These stages are initiation, elongation and termination, and they are well-understood biochemically. However, despite the plethora of structural information made available on RNA polymerase in the last decade, little is available for RNA polymerase in complex with transcription elongation factors. To understand the mechanisms of transcriptional regulation, we describe the first structure, to our knowledge, for a bacterial RNA polymerase in complex with an essential transcription elongation factor. The resulting structure formed between the RNA polymerase and NusA from Bacillus subtilis provides important insights into the transition from an initiation complex to an elongation complex, and how NusA is able to modulate transcription elongation and termination. 2011-07-20T00:10:08.906Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:171 Correlative physiological evidence suggests that membrane transport into storage parenchyma cells is a key step in determining hexose levels accumulated in tomato (Lycopersicon esculentum Mill.) fruit (Ruan et al. 1997). Expression of three previously identified hexose transporter genes (LeHT1, 2 and 3) demonstrated that LeHT3, and to a lesser extent LeHT1, are the predominant transporters expressed in young fruit (10 d after anthesis; DAA). Expression of both transporters dropped sharply until 24 DAA, after which only LeHT3 expression remained at detectable levels through to fruit ripening. LeHT2 was not expressed substantially until the onset of fruit ripening. For fruit at both 10 and 30 DAA, LeHT3 transcripts were detected in storage parenchyma cells of the outer pericarp tissue, but not in vascular bundles or the first layer of parenchyma cells surrounding these bundles. In contrast to LeHT gene expression, hexose transporter protein levels were maximal between 20 and 30 DAA, which corresponded to the period of highest hexose accumulation. The delayed appearance of transporter protein is consistent with some form of post-transcriptional regulation. Based on these analyses, LeHT3 appears to be responsible for the rapid hexose accumulation in developing tomato fruit. © CSIRO 2005. 2010-04-27T05:57:41.894Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5034 Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4 °C) used to isolate protein complexes. In addition, a further vector incorporates a GST moiety in place of the 3 × FLAG of the SPA tag which provides an additional tagging option for situations where SPA binding may be inefficient. The vectors are all compatible with previously constructed fluorescent protein fusion vectors enabling construction of a suite of affinity and fluorescently tagged genes using a single PCR product. 2010-04-27T04:50:53.964Z ]]> http://nova.newcastle.edu.au/vital/access/manager/Repository/uon:5015 We describe a vector-based system for the production of recombinant Bacillus subtilis RNA polymerase. The recombinant enzyme is C-terminally tagged with nine consecutive histidine residues resulting in about 90% pure enzyme in a single nickel-affinity purification step. The vectors permitted production of recombinant enzyme lacking an ω subunit or containing either the ω₁ (YkzG) or ω₂ (YloH) subunits. In transcription time-course assays all of the recombinant enzymes exhibited identical activity to native RNAP. The modular assembly of the artificial RNA polymerase operon permits ready mutation of any subunit and incorporation into the recombinant enzyme, which will enable new functional/structural studies with this enzyme. 2010-04-27T04:45:10.012Z ]]>