BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows single and multiround transcription at the T7A1-tR′ and galP-tR′ promoters. Two transcriptionally competent open model promoters from different classes, −10/−35 class (T7A1) and extended—10 class (galP1), attached to a rho-independent terminator were used to qualitatively visualize efficiency of run-off transcription. Reaction 1, in 20 μl of transcription buffer (30 mM tris-HCl, pH 8.0, 10 mM MgC12, 40 mM KCl, 1 mM β-mercaptoethanol), contained core enzyme, sigma (σ) and Ys18. Reaction 1 was incubated at 65° C. (for T. th) and 37° C. (for E. coli) for 10 minutes, followed by the addition of promoter DNA fragments. Reaction 2, in 10 μl of transcription buffer, contained core enzyme and sigma, and, in parallel, Ys18 and a promoter DNA fragment. Reaction 2 was incubated at 65° C. (for T. th) and 37° C. (for E. coli) for 10 minutes and then mixed together. For both reactions 1 and 2, after 10 minutes of incubation at the same temperatures, 200 μM ATP, CTP, UTP, 20 μM GTP and 10 μCi of [α-32P] GTP were added, the reactions were incubated for the next 10 minutes, and terminated by an equal volume of 9 M urea loading buffer. In E. coli, if during the transcription reaction heparin is not added with the nucleotides, the RNA polymerase is able to initiate transcription from the promoter repeatedly (multi-round transcription). If heparin is added, the RNA polymerase is only able to transcribe once (single-round transcription). The open complexes formed by T. th RNA polymerase at the promoters used are very sensitive to heparin so single-round transcription cannot be performed.
FIG. 1A shows multi-round transcriptional inhibition by Ys18 of both T7A1-tR′ and galP-tR′ with the T. th RNA polymerase core and σA. With both promoters, increasing amounts of Ys18 (triangle) repressed transcription in a dose dependent manner (run-off bands) when the components were mixed as in Reaction 1 (order 1). When the components were mixed as in Reaction 2, Ys18 did not significantly affect transcription of promoter T7A1-tR′, but repressed transcription of promoter galP-tR′. The similar change in band intensity of run-off bands and terminator tR′ bands indicated that Ys18 did not affect elongation or termination.
FIG. 1B shows single-round (+heparin) and multi-round (−heparin) transcriptional inhibition by Ys18 of both T7A1-tR′ and galP-tR′ with the E. coli RNA polymerase core and σ70. With both promoters, increasing amounts of Ys18 (triangle) repressed transcription in a dose dependent manner (run-off bands). In the presence of heparin, when the components were mixed as in Reaction 1 (order 1), Ys18 was not as active in repressing transcription as when the components were mixed as in Reaction 2 (order2). In the absence of heparin, Ys18 was more active in repressing transcription when the components were mixed as in Reaction 2 (order 2) with the T7A1-tR′ promoter. There was only a slight difference in repressing activity of Ys18 in the absence of heparin between the two reaction mixtures. The similar change in band intensity of run-off bands and terminator tR′ bands indicated that Ys18 did not affect elongation or termination.
FIG. 1C shows the relative transcriptional activity of the run-off assay in graphical form. Transcriptional activity without Ys18 present is 100% (dark bar). The addition of Ys18 in incremental amounts represses transcriptional activity in a dose dependent manner (light bar). The amount of transcriptional repression by Ys18 presences differs with amount of Ys18 added, promoter type, RNA polymerase core, reaction mixture and presence or absence of heparin.
FIG. 2 shows native binding experiments with histidine-tagged phage protein Ys18 and primary sigma factors from T. thermophilus and E. coli. Reactions, containing corresponding proteins in 20 μl of binding buffer (20 mM tris HCl, pH8.0, 0.5 M NaCl, 2 mM imidazole, 5% v/v glycerol), were preincubated for 10′ at 65° C. (for T. th σA) and 37° C. (E. coli σ70). The binding mixtures were then added to Ni—NTA agarose beads equilibrated in the binding buffer. Reactions were incubated for 10′ at room temperature. The agarose beads were pelleted by quick centrifugation and the unbound proteins were withdrawn. The beads were washed 3 times with the binding buffer containing 20 mM imidazole, and the bound proteins were eluted with the binding buffer containing 200 mM imidazole. Fractions were resolved by SDS-PAGE and stained by Coomassie (L=proteins loaded, U=proteins unbound, B=proteins bound to Ni—NTA agarose).
FIG. 2A shows Ys18HIS bound to the primary sigma factor from T. thermophilus (σA). With both Ys18HIS and σA present in the sample (+lane, L), σA was detected in the unbound (+lane, U) and the bound fractions (+lane, B). In the absence of Ys18HIS (−lane, L), σA was exclusively observed in the unbound fraction (−lane, U). σA cannot bind to the Ni—NTA agarose beads without Ys18HIS, indicating Ys18HIS was capable of binding to σA.
FIG. 2B shows Ys18HIS bound to the primary sigma factor from E. coli (σ70). When both Ys18HIS and σ70 were present in the sample (+lane, L), σ70 was detected in the unbound (+lane, U) and the bound fractions (+lane, B). In the absence of Ys18HIS (−lane, L), σ70 was exclusively observed in the unbound fraction (−lane, U). σ70 cannot bind to the Ni—NTA agarose beads without Ys18HIS, indicating Ys18HIS was capable of binding to σ70.
FIG. 2C shows Ys18HIS bound to the primary sigma factor from E. coli lacking region 4 (σ701-549). When both Ys18HIS and σ701-549 were present in the sample (+lane, L), σ701-549 was detected in the unbound (+lane, U) and the bound fractions (+lane, B). In the absence of Ys18HIS (−lane, L), σ701-549 was exclusively observed in the unbound fraction (−lane, U). σ701-549 cannot bind to the Ni—NTA agarose beads without Ys18HIS, indicating Ys18HIS was capable of binding to σ701-549 in a region other than region 4.
Patent Application
20070202508
