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Copyright © 2009 Elsevier Inc. All rights reserved.
Molecular Cell, Volume 35, Issue 2, 191-205, 31 July 2009
Add/View Comments (0)Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes
Hideaki Saeki1 and Jesper Q. Svejstrup1, ,
Corresponding authorSummary
- Multiple RNA polymerase II (RNAPII) molecules can transcribe a gene simultaneously, but what happens when such polymerases collide—for example due to polymerase pausing or DNA damage? Here, RNAPII collision was characterized using a reconstituted system for simultaneous transcription by two polymerases. When progression of leading polymerase is obstructed, rear-end collision entails a transient state in which the elongation complexes interact, followed by substantial backtracking of trailing polymerase. Elongation complexes remain stable on DNA, with their activity and the integrity of transcription bubbles remaining intact. Subsequent TFIIS-stimulated transcript cleavage allows resumed forward translocation, resulting in trailing polymerase oscillating at the obstruction. Conversely, if leading polymerase is merely stalled at a pause site, collision and TFIIS cooperate to drive it through. We propose that dynamic interactions between RNAPII elongation complexes help regulate polymerase traffic and that their conformational flexibility buffers the effect of collisions with objects on DNA, thereby maintaining stability in the face of obstacles to transcription.
RNA polymerase II (RNAPII) is responsible for transcription of all protein-encoding genes, as well as for the generation of numerous noncoding RNAs. The movement of RNAPII on DNA induces changes in the structure of chromatin and involves dynamic interaction with other objects on DNA, such as those relevant for replication, repair, and transcription itself.
Structural analysis, single-molecule approaches, and biochemical characterization of RNAPII activity in reconstituted transcription systems have shed light on processes such as transcriptional translocation, pausing, arrest, and RNAPII backtracking at the level of individual polymerases (Arndt and Kane, 2003,Herbert et al., 2008,Kornberg, 2007). On the other hand, techniques such as chromatin immunoprecipitation (ChIP) and live cell imaging have provided insight into the average behavior of large populations of polymerases on active genes in vivo (Saunders et al., 2006,Singer et al., 2005,Struhl, 2007). However, the gap of knowledge between these extremes, including how RNAPII elongation complexes dynamically interact, has not yet been filled.
Biochemical analyses of transcription support the idea that transcript elongation involves frequent pausing and even arrest, which has to be overcome in order for overall transcription to be efficient (Arndt and Kane, 2003,Conaway et al., 2000,Shilatifard et al., 2003). A defining response to all transcription obstacles that have been studied is retrograde motion of RNAPII, so-called backtracking, during which RNAPII loses contact with the 3′ end of the RNA and often needs the help of general elongation factor TFIIS (also called SII) to recover. TFIIS stimulates transcript cleavage by the RNAPII active site so that a new RNA end in correct register with the active site is generated (Izban and Luse, 1992,Reines, 1992).
Equally important for our insight into fundamental transcription principles has been the progress in understanding overall RNAPII traffic on active genes. ChIP has been used to generate extensive maps of RNAPII location across genes and genomes and recently provided compelling evidence for widespread promoter-proximal pausing (Margaritis and Holstege, 2008), but this technique provides little or no information on the dynamics of RNAPII movement. Recent developments in the analysis of overall RNAPII dynamics by cell imaging have uncovered evidence for transcription occurring in bursts with pulses of high polymerases density (Chubb et al., 2006) and support the idea that RNAPII frequently stops, also during processive elongation. Indeed, although it is generally accepted that transcript elongation occurs at an average rate of 20–30 nt/s, evidence for a rate of up to 70 nt/s, interrupted by periods of pausing, was reported (Darzacq et al., 2007).
In general, the speed of transcript elongation, combined with the high likelihood of transcriptional pausing/arrest, would be expected to result in frequent polymerase rear-end collisions. Interestingly, collision between bacteriophage T7 RNA polymerases results in the leading polymerase falling off the DNA (Zhou and Martin, 2006), while bacterial RNA polymerases seem to cooperate during elongation (Epshtein and Nudler, 2003). However, results of studies aimed at characterizing the consequences of transcriptional collision by eukaryotic RNAPII have not been reported, and the basis for the stability and dynamic interactions of colliding elongation complexes in general remains poorly understood.
In this study, we used an oligonucleotide-based RNAPII transcription system to study colliding RNAPII elongation complexes transcribing the same DNA strand. Our data suggest that dynamic interactions between conformationally elastic elongation complexes may make significant and fundamental contributions to transcript elongation.
The Anatomy of an RNAPII Elongation Complex
In order to understand RNAPII collision at the basic biochemical level, the characteristics of separate elongation complexes first needed to be understood in the context of the experimental system used here. Stepwise assembly of highly purified RNA and DNA oligonucleotides and yeast RNA polymerase II can reconstitute fully functional RNAPII elongation complexes (Kireeva et al., 2000) (Figure 1A, upper panel; see Figure S1 A available online). The transcribed strand of such an elongation complex was radioactively labeled prior to assembly, and the footprint of the ternary complex was mapped with DNase I. RNAPII protected an ∼43 nt region extending approximately from position −20 to +23 relative to the polymerase active site (+1) (Figure 1A, lower panel), in good agreement with previous experiments using mammalian, promoter-driven RNAPII (see, for example, Linn and Luse, 1991). It also matched more precise estimates made from crystal structure models of yeast RNAPII (Wang et al., 2006, and references therein), though the enzymatic footprint, not surprisingly, extended beyond the protein-DNA interactions predicted by crystallography (Figures 1C and 1D).
To map the precise position of the single-stranded RNAPII transcription bubble exposed upon the formation of the RNA:DNA hybrid, we took advantage of the ability to assemble an elongation complex at a defined position and of being able to transcribe it to another position. The transcribed strand was designed so that it lacked guanines for 12 nucleotides downstream from the 3′ end of the RNA, meaning that transcription to this position (the "G stop") could be achieved by addition of ATP, GTP, and UTP (Figure 1B, upper panel; Figure S1 B). The elongation factor TFIIS, which promotes transcript cleavage and thereby forward translocation of backtracked elongation complexes, was also added to ensure that the transcribed polymerase did not occupy backtracked positions. The 3′ end of the nontranscribed DNA strand was labeled prior to assembly and transcription. To map single-stranded DNA, the elongation complexes were treated with potassium permanganate (which modifies, but does not cleave at, thymines in single-stranded DNA). Moreover, to ensure that the single-stranded DNA region for a positionally defined elongation complex was mapped, elongation complexes were isolated based on their location marked by T7 exonuclease, which catalyzes the stepwise hydrolysis of nucleotides from the 5′ end of double-stranded DNA but is prohibited by the polymerase bulk from digesting beyond a point 8–9 nt upstream from the elongation complex delineated by DNase I footprinting (Figure 1B, lanes 1–4, 77 and 66 nt, respectively; Figure 1D). The isolated DNA was then treated with piperidine (which induces strand cleavage at the potassium permanganate-modified thymines), and the products were resolved by denaturing PAGE, uncovering the single-stranded bases in the respective elongation complexes (Figure 1B, lanes 5 and 6). The results are summarized in Figure 1D. Our data suggest that, all in all, the transcription bubbles extend for 15–20 nt, with only minor differences observed between the types of elongation complexes tested (see also Figure 5). This is in agreement with previous data using mammalian, promoter-driven RNAPII transcription (Fiedler and Timmers, 2001) and unpublished RNAPII structural information (R.D. Kornberg, personal communication).
Collision between RNAPII Elongation Complexes
We now established a system in which the consequence of RNAPII collision could be studied (Figure 2A; Figure S1 B). In this approach, two separate elongation complexes were assembled on 150-mer DNA oligonucleotides, with a labeled and unlabeled 9-mer RNA, respectively, where transcription up to a G stop was achieved by the addition of ATP/GTP/UTP ("NTPs"). The polymerase closest to the G stop (termed the "leading" polymerase) could transcribe 12 nt (producing a 21-mer product) before stopping due to the lack of CTP. The second polymerase (termed the "trailing" polymerase) could transcribe 86 nt in the absence of CTP (to produce a 95-mer product) if there was no leading polymerase on the DNA. The native agarose gel in Figure 2B shows that assembly of dielongation complexes was efficient, with only a small amount of contaminating monocomplex present (compare lanes 3 and 4 with lanes 1 and 2, respectively). Upon transcription, the integrity of dielongation complexes remained intact (Figure 2B, lanes 5–8), indicating that RNAPII elongation complexes can collide without substantial dissociation of either polymerase from the DNA.
Knowing that the integrity of the elongation complexes remained intact upon collision, we now resolved the RNA products by denaturing PAGE (Figure 2C, box). As expected, in monocomplexes, the trailing polymerase produced a 95-mer, while the leading polymerase produced a 21-mer RNA product (Figure 2C, lanes 1 and 2). With dielongation complexes, the length of the product produced by the leading polymerase did not change upon collision (Figure 2C, lane 4). More importantly, transcription by the trailing polymerase was significantly affected by the presence of a leading polymerase. Major transcripts with a length of 68–70 nt were produced (Figure 2C, lane 3, denoted by asterisk), although a weak 95 nt transcript was also visible. To investigate whether this 95 nt transcript originated from contaminating monocomplexes or from trailing polymerases transcribing past the leading polymerase, the reaction products from the transcription reaction were first resolved by native agarose gel electrophoresis (as in Figure 2B). The relevant elongation complexes were then excised and RNA was analyzed by denaturing PAGE. Using this procedure, the 9 nt RNA oligonucleotide was no longer visible, showing that all elongation complexes transcribed (Figure 2C, lanes 5–8). Moreover, the 95 nt product was also no longer observed (Figure 2C, lane 7), indicating that it originated from contaminating monocomplexes and that, as expected, the trailing polymerase cannot pass the leading polymerase.
With the knowledge of the anatomy of RNAPII elongation complexes obtained previously (see Wang et al., 2006, and references therein; Figure 1; R.D. Kornberg, personal communication), the consequences of collision could be assessed. The models predicted that the longest RNA product the trailing polymerase can produce before touching the leading polymerase should be approximately 95 minus 43 nt (the size of the footprint), i.e., 52 nt, or 95 minus 33 nt (the minimum DNA area protected by RNAPII in the elongation complex crystal structure), i.e., 62 nt, but instead 68–70 nt products were produced. The trailing polymerase thus continued transcription for at least 6–8 nt after first touching the leading polymerase (Figures 2C–2E). In contrast to nucleosomes, for example, where DNA is wrapped around a histone core, DNA is surrounded by protein mass in RNAPII (see Figures 1C and 8A), so "stripping of DNA" off the surface of RNAPII cannot account for the observations. We surmised that the conformation of elongation complexes might instead be flexible, buffering the effect of collision. If so, this might potentially result in retrograde movement ("recoil") of the trailing elongation complex as normal conformation is regained. Alternatively, the polymerases might remain entangled, with potentially dramatic consequences for elongation complex integrity. In the following experiments, we therefore mapped the position of the trailing elongation complex and the transcription bubbles after collision and assessed the activity of the collided enzymes.
Evidence for Backtracking of Trailing RNAPII upon Collision
To determine the position of the trailing polymerase after collision, T7 exonuclease mapping was performed, with and without TFIIS (Figure 3). As previously observed (Figure 1B), transcribing monocomplexes caused T7 exonuclease to make products 66 nt long (corresponding to position −29 relative to the RNAPII active site at +1) whether they started from the leading (Figure 3A, lanes 1–3), or the trailing position (lanes 7–9). This was confirmed by the addition of TFIIS (Figure 3A, lanes 4–6 and 10–12; Figure 3D, panel 1). Surprisingly, however, upon polymerase collision, the trailing polymerase was much further upstream than expected, with the products of exonuclease digestion 116 and 119 nt long (corresponding to position −79 and −82 relative to the active site of the leading polymerase) (Figure 3A, lanes 13–15, thin line; Figure 3D, panel 3). It is important to note that, in the previous experiment (Figure 2C, lane 3), no transcription products of a length (42–45 nts) that could explain these major exonuclease-mapped RNAPII positions were observed. The positions mapped by exonuclease digestion thus indicated that, upon collision, the active site of the trailing polymerase did not remain at the place of impact ∼26 nt upstream from the leading polymerase indicated by the length of its transcript (see Figures 2C and 2E; Figure 3D, panel 2). Rather, it moved ∼25 nt back from this position so that its active site ended up ∼50 nt from the active site of the leading polymerase.
The addition of TFIIS resulted in the products of exonuclease digestion being ∼20 nt shorter than this (position −63 to −61; Figure 3A, lane 16, short line; Figure 3D, panel 4). This suggests that the active sites of the polymerases were now 32–34 nt apart, but still not the ∼26 nt indicated by transcript length. It is important to note that although addition of TFIIS dramatically changed the position of the trailing polymerase after collision, the transcription products were similarly sized whether TFIIS was added or not (Figure 3B, compare lanes 3 and 4).
All in all, the only reasonable explanation for these results is that the trailing polymerase backtracks substantially after collision but can be reactivated by TFIIS (Figure 3C). This in turn indicates that it retains function after collision and backtracking. Moreover, even in the presence of TFIIS, the trailing elongation complex was located 6–8 nt upstream from the position expected from transcript length. This suggests that the trailing polymerase continues to transcribe forward against the opposing force of the leading polymerase, eventually quickly backtracking before rapid motion is repeated following TFIIS-mediated transcript cleavage (Figure 3C, and Movie S1 ). Interestingly, the exonuclease footprint appeared to move forward during the course of the experiment (Figure 3A, lanes 16–18, indicated by short and long lines, respectively). The ability of the T7 exonuclease to "catch" elongation complex positions further and further forward with time contrasted the lack of change over time in the case of monopolymerases (Figure 3A, lanes 10–12). It supports the above interpretation and indicates constant motion and dynamic interactions between colliding elongation complexes.
Substantial Backtracking of the Trailing Polymerase Is a Consequence of Collision
In theory, the backtracking of trailing polymerase observed above might simply be due to the underlying DNA sequence; i.e., the polymerase might rapidly backtrack from this position even if there were no collision. To investigate this point, we introduced a second G stop at position −27 relative to the active site of the leading polymerase, i.e., at the main position reached by the trailing polymerase during collision (Figure 4A, double G stop template; Figure S1 B). Because the leading polymerase initiates transcription downstream from this point, we could now examine if the presence of the leading polymerase were the cause of the substantial backtracking observed. Figure 4B shows that, both in the absence and presence of a leading polymerase, the trailing polymerase stopped transcription at the same position on this template (lanes 3 and 4), producing transcripts of a length similar to that observed with a single G stop with a leading polymerase stalled at it (lane 2). Importantly, exonuclease footprinting showed that, in the absence of TFIIS and a leading polymerase, polymerase transcribing from the trailing position remained in the area immediately upstream of the newly introduced G stop (Figure 4C, lanes 5 and 6), rather than moving far back, as it did when there was a leading polymerase in front of it on the single G stop template (Figure 4C, lanes 1 and 2). In contrast, when the leading polymerase was present on the double G stop template, the trailing polymerase backtracked for more than 20 nt to the same positions it moved to on a single G stop template (Figure 4C, compare lanes 9 and 10 and lanes 1 and 2).
As observed earlier (Figure 3), TFIIS allowed the polymerase to move forward again after collision-induced backtracking (Figure 4C, lanes 3 and 4 and lanes 11 and 12). However, while the trailing polymerase was detected around position −56 in the absence of a leading polymerase in front of it (Figure 4C, lane 7, indicated by black sphere), it was primarily found around positions −61 to −63 (5–7 nt further back) in the presence of one (lane 11). This highlights the elastic nature of the elongation complex as well as its TFIIS-mediated oscillation and again indicates that the residence time of the trailing elongation complex in the furthest position it transcribes to (and where it is completely blocked by the leading polymerase) is so short that T7 exonuclease cannot catch it and mark the position. In contrast, a monoelongation complex facing the same G stop can be caught at the position indicated by the length of its transcript (Figure 4C, lane 8; Figure 4D).
The results are summarized in Figure 4E. Together, they indicate that substantial backtracking of the trailing polymerase is not a consequence of DNA sequence but is indeed the result of collision with the leading polymerase.
Transcription Bubbles Remain Intact upon Collision
The exonuclease mapping presented above only provides information about the trailing polymerase. To obtain a view of both polymerases after collision and, moreover, to test if collision results in changes to the integrity of the transcription bubbles of the collided polymerases, potassium permanganate was used to detect thymines in single-stranded DNA regions (Figure 5A).
It was previously observed that upon polymerase collision, backtracking, and TFIIS-mediated forward motion, trailing polymerase stopped the exonuclease at positions ranging from approximately −55 to −62 relative to the G stop (see Figure 3, lane 17). If the overall organization of the colliding elongation complex were restored immediately after reaching the furthest position (−55), resulting in retrograde movement, the positions of the transcription bubbles of elongation complexes corresponding to the distinct exonuclease-generated bands should all be the same as that of the elongation complex at position −62. This was indeed the case: analysis of permanganate-treated DNA from these collided polymerases showed two well-separated transcription bubbles, which in all compared cases were similarly positioned (Figure 5B, lanes 4–6, 7–9, 10–12, and 13–15, respectively; position of the two bubbles is indicated by lines between lanes 4 and 5). Importantly, the transcription bubble also gave information about the position of the active site of the trailing polymerase, which was found to be ∼32 nt away from the active site of the leading one (with polymerases positioned in touching distance; see Figure 8C), rather than the ∼26 nt suggested by transcript length. Together, these data again point to a transient clash of stable, elastic elongation complexes and also further support the TFIIS-mediated oscillation indicated by the experiments in Figure 3 and Figure 4.
We also note that the transcription bubble of the leading polymerase was almost indistinguishable from that in the (uncollided) monocomplex (Figure 5B, compare lanes 1–3 with lanes 4–6, for example) and that there was no evidence for single-stranded DNA between the transcription bubbles, arguing that little or no permanent loss of transcription bubble integrity occurred on collision.
Transcription bubbles were also mapped in the absence of TFIIS, using the same approach (Figure 5C, lanes 1–6; refer also to Figure 3A, lanes 13–15, which shows the main backtracked positions). In this particular experiment, somewhat more background cleavage at all thymines was evident, but comparison to Figure 5B (with lanes 4–9 of Figure 5B corresponding to lanes 7–12 of Figure 5C) and within the experiment made it straightforward to make conclusions. First, the position of the leading polymerase remained largely the same as in the presence of TFIIS, though evidence for some backtracking of leading elongation complexes could be seen (Figure 5C, compare lines at bottom of lanes 1 and 2 and lanes 7 and 8, respectively). More importantly, the transcription bubble of the substantially backtracked, trailing polymerase was observed at the position expected from exonuclease footprinting, approximately 51–70 nt upstream from the active site of the leading polymerase (Figure 5C, indicated by line near top between lanes 1 and 2). The distance between the RNAPII active sites was thus about 53 nt, showing that the elongation complexes were completely separated after collision.
These data, summarized in Figure 5D, complement and extend the exonuclease footprinting experiments. They indicate that transcription bubbles for both the leading and trailing polymerases remain intact after collision: the bubbles have the expected size and are completely separated.
Further Evidence for Dynamic Interactions between Colliding Elongation Complexes
Besides defining the consequences of RNAPII collision, the footprint experiments also indicated that TFIIS allowed the trailing polymerase to "oscillate." To more directly investigate this, experiments were performed in which the nascent transcripts were labeled by using radioactive GTP during transcription, enabling the RNA oligonucleotides cast off by transcript cleavage to be visualized (Figure 6). Interpretation of this experiment has to be done mindful of the fact that shorter RNA species have fewer radioactive labels incorporated and so are relatively underrepresented in the autoradiogram. As observed previously (Figure 2C), in the absence of TFIIS (Figure 6A, lanes 7–9), the trailing polymerase was blocked by the leading polymerase and predominantly produced 68–70 nt products (labeled T68) (and a 95 nt product [T95] originating from contaminating monocomplexes), while the leading polymerase concomitantly produced a 21 nt product (labeled L21), similar to what the monocomplex produced alone (lanes 1 and 2). No increase in the amount of these transcripts was observed over time, showing that, as expected, transcription to the end (or the obstacle) was completed already before TFIIS addition. TFIIS reactivates backtracked polymerase by cleaving the nascent RNA labeled by incorporation of radioactive GMP. Therefore, the length of the cleaved nascent RNA designates the distance of backtracking. T7 exonuclease mapping and permanganate probing previously showed that the majority of trailing complexes backtracked for 24–27 nt after collision in the absence of TFIIS (Figure 3 and Figure 5). Indeed, cleaved transcripts corresponding to those lengths were observed when TFIIS was added (Figure 6A, lanes 11–15, thin line; Figure 6B, upper panel). Moreover, large amounts of shorter bands accumulated with time as a result of transcript cleavage, the majority of which were 5–15 nt, indicating that TFIIS captured backtracking polymerases at the corresponding positions.
These results support the observation made by footprinting in Figure 3 and Figure 4 and Figure 5 that the predominant "steady-state position" of the active site of trailing polymerase in the presence of TFIIS is ∼32 nt away from the active site of the leading polymerase (see lower panel in Figure 5B), rather that the 26–27 nt suggested by transcript length. Once TFIIS acts, the cleaved RNA intermediate is short lived (i.e., undetectable because subsequent forward translocation [chain elongation] is immediate), so that only the longer transcripts (which indicate a distance of ∼26 nt from the active site of the leading polymerase) are detected. The differences observed in the previous footprint experiments (Figure 3 and Figure 4 and Figure 5), 32 minus 26–27, i.e., 5–6 nt, are in this experiment appearing as the shortest of the oscillation range (5–15 nt). We note that, according to molecular modeling based on the polymerase crystal structure, the position of the trailing polymerases observed in the presence of TFIIS places the colliding polymerases in "touching distance," or beyond (5–15 nt), rather than in a "compressed" state, as expected if the elastic/flexible elongation complexes are in effect driving each other back while regaining their conformation (see also Figures 8B–8D, Movie S1 , and Discussion ).
Rear-End Collision Can Drive RNAPII through a Pause Site
The situation characterized so far was one in which the leading polymerase was blocked from further forward translocation. But what happens if the leading polymerase is merely stalled at a pause site at collision? To address this question, a DNA template containing a 9 nt adenine tract upstream from the G stop to induce transcriptional pausing was used (Figure 7A). The native gel in Figure 7B shows that RNAPII elongation complexes also under these conditions can collide without substantial dissociation of either polymerase from the DNA, as observed previously (Figure 2B). Next, the RNA products isolated from elongation complexes were resolved by denaturing PAGE (Figure 7C, box). The vast majority of polymerases in monocomplexes paused in the A tract whether transcribing from the leading (Figure 7C, lanes 1 and 5) or the trailing position (lanes 3 and 7), both at high and low nucleotide concentration. The presence of a trailing polymerase dramatically increased transcription through the A tract by the leading polymerase (Figure 7C, compare lanes 1 and 2 and lanes 5 and 6, respectively), indicating that collision did not perturb the activity of the leading polymerase but rather drove it through the pause site to continue transcription. The transcript pattern produced by leading polymerases making it through the pause site to the G stop was not markedly altered by collision (Figure 7C, compare lanes 1 and 2 and lanes 5 and 6, respectively), suggesting that their escape from the stall upon collision was not due to base skipping.
We also characterized the transcripts produced by the trailing polymerase upon collision on this template. This suggested the existence of two distinct trailing polymerase populations. The first was positioned only 25–26 nt upstream from the G stop (Figure 7C, lanes 4 and 8, black spheres) and must therefore represent trailing polymerases that had driven leading polymerases through the pause site and were blocked by this leading RNAPII at the G stop (indicated by gray spheres in lanes 2 and 6; see also Figure 7D, right). The second was positioned at several locations 32–33 nt, or more, upstream from the position of leading polymerases paused at the A tract. The furthest the trailing polymerases of this population transcribed was to position −45/−46 relative to the G stop (Figure 7C, lanes 4 and 8, black arrowheads). These transcripts therefore represent trailing polymerases that failed to interact sufficiently with the paused leading polymerase to push it (they only transcribed to position −13 relative to the G stop; Figure 7C, lanes 2 and 6; gray arrowheads) and which were therefore prohibited from progressing further. We note that the difference between polymerase positions (−13 versus −45/−46, i.e., 32–33 nt) again corresponds to their being in "touching distance" (Figure 8C). By contrast, no transcripts were observed in the region between −38 and −45 relative to the G stop (Figure 7C, bold line on right), indicating that all trailing polymerases that transcribed this close to a leading, stalled polymerase (25–-26 nt from its active site) drove it through the pause and so did not stop at these positions themselves (Figure 7D).
These results indicate that, as observed for the trailing polymerase previously, collision does not perturb the activity of the leading polymerase. Rather, collision greatly stimulates pause-site readthrough by this polymerase.
Polymerase Collision and TFIIS Action Cooperate to Allow Efficient Transcription through Pause Sites
TFIIS enhances transcription through pause sites (reviewed by Arndt and Kane, 2003). As expected, we found that at a high NTP concentration, TFIIS was capable of stimulating efficient pause-site readthrough. However, polymerase collision showed a significant additive effect, further promoting transcription (Figure S2 ). This was particularly clear at a limiting UTP concentration. Here, TFIIS enhanced transcription through the pause site when added at up to equimolar concentration relative to RNAPII (Figure 7E, compare lane 1 with lanes 2 and 3) but had an inhibitory effect when added at a higher concentration (lane 4). The stimulatory amounts of TFIIS were used in collision experiments. Notably, rear-end collision by the trailing polymerase stimulated pause-site readthrough by leading RNAPII to a higher degree than addition of the optimal TFIIS concentration (Figure 7F, compare lanes 3 and 4). Moreover, whereas less than 10% of polymerases transcribed through the pause site in the absence of both TFIIS and collision (Figure 7F, lane 1), the combination of TFIIS and RNAPII collision resulted in more than 60% of the polymerases transcribing through to the end (lane 6).
These results demonstrate that RNAPII collision and TFIIS action can cooperate to allow transcription through pause sites.
Mechanism of RNAPII Collision: Complementing the Information Gained from RNAPII Crystallography
Considerable progress in our understanding of RNAPII transcription has been achieved through solving crystal structures of RNAPII at different stages of transcription (Kornberg, 2007). The enzymological evidence reported here complements and extends these data. Arguably, one of the most surprising findings of this study is the prediction of a temporary state during impact where the colliding elongation complexes are structurally "intermingled" (Figure 8). This state requires structural changes in the protein component or/and the DNA component of the RNAPII elongation complexes. In the former case, it seems likely that the elongation complex is "elastic," with flexible domains of either (or both) polymerase(s) changing conformation (swivelling/rotating) to accommodate the presence of the bulk of the other polymerase and then regaining their original shape upon backtracking (Figure 8D, model 1). Interestingly, substantial movement of polymerase domains, not least of modules such as the jaw lobe and clamp, is indeed structurally feasible (Cramer et al., 2000,Cramer et al., 2001,Darst et al., 2002,Gnatt et al., 2001). While the precise extent of freedom of jaw lobe movement remains unclear, the clamp might be free to move out by as much as 20 degrees (Darst et al., 2002).
In the case of structural changes in the DNA component of elongation complexes (Figure 8D, model 2), helix unwinding (twisting) between the transcription bubbles by polymerase rotation might gain space between the colliding polymerases, which enables the trailing polymerase to continue forward translocation on DNA while remaining in touching distance of the leading polymerase. Alternatively, or additionally, the DNA between the transcription bubbles may temporarily become stretched as the trailing polymerase keeps moving forward on DNA from the point where it has reached the point of contact with the leading polymerase. Reformation of a stable DNA double helix might thus also help drive backtracking. Finally, if some stripping of DNA contacts with RNAPII occurs, reformation of these might contribute as well.
In any case, the mechanism outlined here differs fundamentally from that proposed for transcription into a nucleosome. When RNAPII transcribes into the nucleosome, it appears to unwrap the DNA from the histone core. When it pauses and backtracks in doing so, the backtracked conformation is stabilized by restoration of DNA-histone contacts in front of it, causing transcriptional arrest (Kireeva et al., 2005). In agreement with this model, the prominent pause and arrest sites in the nucleosome are observed at intrinsic pause sites of DNA. In contrast, DNA is not "wrapped around" the elongation complex, and the transcripts generated by trailing polymerase in the furthest position it transcribes to and where it is completely blocked by the leading polymerase are not observed on naked DNA; their production is more consistent with polymerase hitting a flexible protein "wall." Likewise, the backtracking resulting from this clash is initiated by the elastic nature of elongation complexes themselves (Figure 3 and Figure 4 and Figure 5).
Translocation by RNAPII is probably best explained by a Brownian ratchet model, according to which fluctuation between pre- and posttranslocated states is driven toward forward motion primarily by NTP binding and hydrolysis: binding of the incoming nucleotide increases dwell time in the posttranslocation position, so that chain elongation is favored (reviewed by Herbert et al., 2008). However, if NTP-mediated stabilization does not occur, backtracking is almost as likely as forward translocation because of Brownian motion. It thus seems reasonable to suggest that when collision with an immovable leading polymerase occurs, the transcription ratchet (the trailing polymerase) moves against greater and greater force so that when the next translocation step can no longer be secured (a state of low entropy, because the elongation complexes have fewer conformations available), rapid retrograde motion occurs, probably driven at least partly by the increase in entropy reforming the stable equilibrium states (high entropy) of both elongation complexes entails.
Oscillation in the Presence of TFIIS
The idea of conformational elasticity of elongation complexes was supported by exonuclease mapping of the trailing polymerase during collision in the presence of TFIIS. This polymerase's active site (as inferred from both T7 exonuclease mapping and permanganate footprinting; Figure 3 and Figure 4 and Figure 5 and Figure 6) was positioned 5–8 nt upstream from the position predicted from the length of the transcript it was generating. The only reasonable explanation for these data is that the trailing polymerase constantly oscillates at impact, quickly transcribing forward after TFIIS-promoted transcript cleavage to the foremost position (to produce the transcripts we detect) and then immediately backtracking to upstream positions (the shortest of which were detected by footprinting techniques) before TFIIS acts again (Movie S1 ). Indeed, 5–15 nt RNA cleavage products accumulated with time, in a collision- and TFIIS-dependent manner (Figure 6). Moreover, the most forward position in which the polymerases spend the vast majority of time in the presence of TFIIS is also the position at which only minimal elongation complex-elongation complex incursion occurs (Figure 8C). RNAPII oscillation might help ensure that, facing obstacles, the polymerase can adapt dexterously to changes in circumstances by keeping itself in the active state.
Cooperation between RNAPII Molecules
Our results indicate that during collision at a pause site, structural elasticity and dynamic interactions between trailing and leading polymerase in effect generate bias in Brownian motion for the leading polymerase toward the forward direction, so that its active site is kept in register with the end of the RNA, even through the long A tract. Evidence for cooperation between bacterial RNA polymerase was previously obtained (Epshtein and Nudler, 2003,Epshtein et al., 2003). Our results on transcription by RNAPII confirm and extend the work on the bacterial enzyme by Epshtein and Nudler by pointing to the elasticity and dynamic interactions of elongation complexes as a driving factor and underlying mechanism, and by showing that polymerase collision and TFIIS-promoted transcript cleavage can be cooperative, helping the majority of leading polymerases through a potent pause site (Figure 7). It seems reasonable to suggest that TFIIS allows polymerases to oscillate against obstacles (Figure 3 and Figure 4 and Figure 5 and Figure 6), while the resulting repeated collisions increase the time spent by the leading polymerase in the posttranslocation state, so that NTP can bind, and further progress is stimulated.
We note that the similarity between bacterial RNA polymerase and eukaryotic RNA polymerase II is confined to the protein core surrounding the active site (Cramer et al., 2000). In contrast, proteins and protein domains (such as Rpb9 and the Rpb1 jaw domain [on the jaw lobe side], as well as Rpb5 [on the clamp side]) that are likely to be touching the leading polymerase during collision are not conserved between prokaryotic and eukaryotic RNA polymerases (Figure 8A, upper panel, boxed). Therefore, the precise mechanism of RNA polymerase cooperation may have changed in the course of evolution. Indeed, the extension of the polymerase footprint downstream from the active site that has occurred during evolution (phage, prokaryote, eukaryote) might have served to improve the stability of elongation complexes during collision, enabling transcription of longer genes.
Biological Significance
During transcript elongation, RNAPII constantly encounters obstacles to its progression, such as DNA-binding proteins, nucleosomes, DNA damage, and other polymerases. Unfortunately, studies of RNAPII collision in vivo are hampered by the fact that conditions that lead to it occur randomly across the transcriptome, with recent evidence supporting the idea that longer genes are more likely to experience "traffic jams" than shorter ones (Swinburne et al., 2008).
Interestingly, results obtained in yeast using an artificial pause site (ARTAR) integrated into a GAL-driven lacZ gene (Kulish and Struhl, 2001) are potentially very informative. Expression of lacZ was dramatically reduced by insertion of the ARTAR sequence and almost undetectable if the gene encoding TFIIS was concomitantly deleted. However, if the gene was expressed at a very high level, the ARTAR had no, or little, negative effect, and TFIIS was no longer needed. Although other possibilities cannot be ruled out, it is tempting to speculate that during low-level expression, backtracking of not only ARTAR-paused RNAPII but also polymerases that collide with these would require TFIIS-stimulated transcript cleavage for reactivation. In contrast, in the highly expressed gene (where polymerases are transcribing "back to back"), the constant pushing by the polymerase behind might render such action unnecessary. We note that such polymerase "group action" could also underlie the transcriptional pulsing bursts with high polymerase density observed by others (Chubb et al., 2006).
Unfortunately, making firm conclusions about molecular events at pause sites (or even at complete blocks to transcription, such as DNA lesions) in vivo is greatly complicated by the fact that, in eukaryotes, numerous other elongation factors also affect transcription through obstacles (Arndt and Kane, 2003), and by the fact that elongating RNAPII can also be ubiquitylated and degraded in response to transcription problems (Somesh et al., 2005). Moreover, our recent results indicate that the (TFIIS-independent) intrinsic transcript cleavage activity of RNAPII itself, although typically virtually undetectable in vitro, may nevertheless be important for transcription and cell viability (S. Sigurdsson, A.B. Dirac-Svejstrup, and J.Q.S., unpublished data).
The data presented here suggest a mechanism by which the extraordinary robustness of transcription, with individual polymerases being required to traverse very long genes (in human cells up to a million nucleotides long) without dissociation, might be possible. In this model, the remarkable conformational flexibility of RNAPII elongation complexes buffers the effect of running into objects on DNA, so that when collisions occur, the polymerase in effect "bounces off" and retains full activity. The structural flexibility of RNAPII may be important to generally regulate the flow of polymerases on genes, and besides the help provided by numerous transcription elongation factors, actively transcribing polymerases can also assist each other.
Experimental Procedures
Oligonucleotides and Proteins
For the collision experiments, the sequence of the transcribed strand DNA (TS) was 5′-CCGCAACCACATTCCAACCACACATCACTTACCAGGATACACATCAAACATACACCCAACTAACCAACTACTAACACACAATCACACACTCACCTACCATAACCAACTCACAACAACAACACCTCTCCATACTCTACTCCTATACCACGC-3′. Full information about the sequences of the oligonucleotides can be found in Figure S1 . RP-HPLC purified oligos were purchased from DNA Technology A/S (Aarhus, Denmark) and purified further by 5.2% denaturing PAGE. Radioactive 5′ end labeling was by T4 polynucleotide kinase (NEB), or, alternatively, 3′ ends were labeled with dideoxy-ATP (Amersham), or 3′ deoxy-ATP (NEN), using terminal transferase (Roche). Prior to T7 exonuclease assays, end-labeled oligos were repurified by 5.2% denaturing PAGE.
Recombinant yeast TFIIS and wild-type, 12 subunit RNAPII were purified from E. coli and yeast (BJ926), respectively (Christie et al., 1994,Edwards et al., 1990), and dialyzed against 100 mM KOAc, 20 mM HEPES-NaOH (pH 7.6, 26°C), 0.1 mM EDTA, 10% glycerol, 2 mM DTT, 0.1 mM PMSF. Unless specified, TFIIS was used in equimolar concentration to RNAPII.
Reconstitution and Characterization of RNAPII Ternary Complexes
Elongation complexes (ECs) were reconstituted essentially as described by Kashlev and colleagues (e.g., Kireeva et al., 2000). All experiments were done using nonstick microtubes and tips (Axygen). Oligos were prepared at the following concentrations in reconstitution buffer (as transcription buffer described below except 0.2 mM MgCl2). Typically, 4 μl 1 pmol/μl TS and 2 μl each of 4 pmol/μl RNA oligos were mixed and incubated at 65°C for 5 min, followed by gradual cooling to 25°C (−0.1°C/6 s for 40 min in thermal cycler). After addition of 2.6–3 μl 4 pmol/μl RNAPII (a 2.5–3 molar excess over TS), the reaction was incubated at 25°C for 25–30 min and at 37°C for 1 min. Then, 8 μl 1–1.25 pmol/μl NTS (pretreated by heating to 65°C for 5 min, then on ice for 2 min, and finally at 37°C for 2 min) was added and incubated for 10–15 min at 37°C. The final concentration of TS was 0.10–0.14 pmol/μl after adding supplement buffer to obtain transcription conditions (40 mM KCl, 20 mM Tris-Cl [pH 7.9], 7 mM MgCl2, 20 μM ZnCl2, 5 mM DTT, ±0.75 μg/μl BSA). Assembled ECs were kept on ice until use.
After addition of an equal volume of loading buffer (0.75 μg/μl BSA, 5 mM 2-mercaptoethanol, and with glycerol added to a final concentration of 3%), samples were resolved by 0.7% agarose gel electrophoresis in 0.25 × TB buffer (22.3 mM Trizmabase/SIGMA, 22.3 mM boric acid, 0.1 mM MgCl2, 0.01 mM ZnCl2, 5 mM 2-mercaptoethanol) at 4°C.
Transcription was initiated by adding nucleotides, typically 0.5 mM of each (no CTP) at 26°C. Transcription of A tract DNA was at 30°C and was initiated by 0.025 or 0.1 mM UTP, 1 mM GTP, and 0.5 mM ATP. For analysis of transcription products by 6% denaturing PAGE, the reaction was terminated by addition of an equal volume of urea analysis buffer (10 M urea, 80 mM EDTA, 0.05% [w/v] xylene cyanol, 0.05% bromphenol blue). Gels were dried on DEAE paper (DE81/Whatman) and subjected to autoradiography, as well as exposure for phosphor imaging. Where RNA was isolated from purified ECs, the ECs were resolved by agarose gel electrophoresis after transcription as described above, stained with SYBR Green I at 4°C, and visualized by fluorescence laser scanner (FLA-5000 FUJI). EDTA (to give a final concentration of 7.5 mM) and tRNA (to give a final concentration of 8 μg/ml) were added to the excised bands corresponding to ECs. These were then crushed by using a micropestle in a microtube and filtrated by Nanosep MF/0.45 μm (Pall). RNA was phenol/chloroform extracted and ethanol precipitated.
Footprinting Analyses of Elongation Complexes
DNase I footprinting: radioactively end-labeled ECs were incubated with 0.0056, 0.011, or 0.022 units/μl DNase I (Roche) at 24°C for 1 min. As control, naked DNA was digested with 0.0028, 0.0056, or 0.011 units/μl DNase I. Reactions were stopped by the addition of EDTA, and samples were resolved by agarose gel electrophoresis as described above, without MgCl2 and ZnCl2, and visualized with SYBR Green I as described above. DNA was then extracted and purified by QIAEX II (QIAGEN) and finally analyzed by 7% denaturing PAGE.
T7 exonuclease footprinting: radioactively end-labeled ECs were incubated with 0.4 units/μl T7 exonuclease (NEB) at 26°C. The reaction was terminated with an equal volume of urea analysis buffer as described above and analyzed by 5.2% denaturing PAGE. Where transcription bubbles were analyzed, samples were treated with freshly prepared KMnO4, and the reaction was stopped with an equal volume of stop buffer (2 M 2-mercaptoethanol, 40 mM EDTA, 100 μg/ml tRNA). This DNA was purified by phenol/chloroform extraction and ethanol precipitation, dissolved in TE (pH 8, 26°C) with an equal volume of urea isolation buffer (10 M urea, 20 mM EDTA, 5 mM Tris-Cl [pH 7.5, 26°C]) and resolved by 5.2% denaturing PAGE. After taking an autoradiograph of the wet gel, bands corresponding to the relevant T7 exonuclease (and permanganate-)- treated oligos were excised and crushed by micropestle. DNA was eluted in elution buffer (10 mM Tris-Cl [pH 7.5, 26°C], 1 mM EDTA, 0.1% SDS), filtered by Nanosep MF/0.2 μm (Pall) and purified by phenol/chloroform extraction and ethanol precipitation. Modified thymines were cleaved by piperidine, and the resulting DNA was analyzed by 6% denaturing PAGE.
Molecular Modeling
Modeling of elongation complex interaction was based on the crystal structure of the elongation complex (Wang et al., 2006) (PDB 2E2H) using PyMOL. TS (−9 to +18), NTS (+5 to +18), and RNA (−9 to −1) are shown, but TS (−10) and RNA (−10) were omitted. Numbers in brackets indicate positions relative to the active site (+1).
This work was supported by a grant from the European Community (Integrated Project DNA repair, grant number LSHG-CT-2005-512113) and by an in-house grant from Cancer Research UK (to J.Q.S.). We thank Stefan Sigurdsson for recombinant yeast TFIIS and helpful discussions and Caroline Kane for the TFIIS expression plasmid. Dave Bushnell and Roger Kornberg are thanked for sharing unpublished information and their insight into the RNAPII structure. Members of the Svejstrup lab, Dale Wigley, Craig Kaplan, Dave Bushnell, Dong Wang, Roger Kornberg, and Peter Verrijzer are thanked for comments on the manuscript.
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Christie et al., 1994 (1994). Purified yeast RNA polymerase II reads through intrinsic blocks to elongation in response to the yeast TFIIS analogue, P37. J. Biol. Chem. 269, 936–943. PubMed
Chubb et al., 2006 (2006). Transcriptional pulsing of a developmental gene. Curr. Biol. 16, 1018–1025. Abstract | Full Text | PDF (475 kb) | CrossRef | PubMed
Conaway et al., 2000 (2000). Control of elongation by RNA polymerase II. Trends Biochem. Sci. 25, 375–380. Abstract | Full Text | PDF (360 kb) | CrossRef | PubMed
Cramer et al., 2000 (2000). Architecture of RNA polymerase II and implications for the transcription mechanism. Science 288, 640–649. CrossRef | PubMed
Cramer et al., 2001 (2001). Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution. Science 292, 1863–1876. CrossRef | PubMed
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Darzacq et al., 2007 (2007). In vivo dynamics of RNA polymerase II transcription. Nat. Struct. Mol. Biol. 14, 796–806. CrossRef | PubMed
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Epshtein and Nudler, 2003 (2003). Cooperation between RNA polymerase molecules in transcription elongation. Science 300, 801–805. CrossRef | PubMed
Epshtein et al., 2003 (2003). Transcription through the roadblocks: the role of RNA polymerase cooperation. EMBO J. 22, 4719–4727. CrossRef | PubMed
Fiedler and Timmers, 2001 (2001). Analysis of the open region of RNA polymerase II transcription complexes in the early phase of elongation. Nucleic Acids Res. 29, 2706–2714. CrossRef | PubMed
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Kireeva et al., 2005 (2005). Nature of the nucleosomal barrier to RNA polymerase II. Mol. Cell 18, 97–108. Abstract | Full Text | PDF (809 kb) | CrossRef | PubMed
Kornberg, 2007 (2007). The molecular basis of eukaryotic transcription. Proc. Natl. Acad. Sci. USA 104, 12955–12961. CrossRef | PubMed
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Margaritis and Holstege, 2008 (2008). Poised RNA polymerase II gives pause for thought. Cell 133, 581–584. Abstract | Full Text | PDF (384 kb) | CrossRef | PubMed
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Publication Information
Received: December 16, 2008
Revised: April 8, 2009
Accepted: June 5, 2009
Published: July 30, 2009


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In addition to printing and viewing your local deals vouchers, you can now easily
manage your location, free subscriptions and credit card details.
Secure access to My Account requires a password. If you have not yet created a Travelzoo
password, we will ask you to go ahead and set one up.
Relax on Shelter Island this spring with this deal from The Candlelite Inn, and save more than 40% on current rates.
Arrive Sunday-Wednesday through May 23, and spend two nights at the charming 180-year-old Victorian home for $199.
The-bed-and-breakfast is situated in the center of the island, making it an ideal home base for casual bike rides or hiking through the Mashomack Nature Conservancy, within walking distance of the inn.
The hotel is a three-hour train ride to Greenport from New York City, or a 40-minute drive from the Orient Point ferry.
This deal is not available on: May 17-18, 20-22.

subset: commoncrawl


text:
Found December 25, 2012 on
DULUTH, Ga. — Former Atlanta Braves star center fielder Andruw Jones was free on bond after being arrested in suburban Atlanta early Tuesday on a battery charge, according to jail records.
Around 1:30 a.m. Tuesday, police responded to a call for a domestic dispute between Jones and his wife in Duluth.
Gwinnett County Detention Center records say Jones was booked into the jail around 3:45 a.m. and had been released on $2,400 bond by 11 a.m.
Once one of the premier players in the big leagues, Jones broke into the majors with the Atlanta Braves in 1996 and won 10 consecutive Gold Gloves from 1998-07 as their center fielder. He has 434 career home runs over the span of 17 seasons in the majors.
Jones earlier this month signed a $3.5 million, one-year contract with the Tohoku Rakuten Golden Eagles of Japan's Pacific League.
Original Story:
With his Christmas arrest, Andruw Jones re-establishes the rules of scandal: When they mess up, they're always "ex-Yankees."
Details emerge in Andruw Jones battery case
MLB Rumors: Will Andruw Jones Arrest Jeopardize Japanese Deal?
Ex-Brave Jones arrested on battery charge
Andruw Jones arrested in Atlanta
Andruw Jones Arrested For Allegedly Beating His Wife
Andruw Jones Accused of Dragging Wife Down Staircase, Threatening to Kill Her, According to Police
Andruw Jones free on bond after attest for battery charge
Former White Sox OF Andruw Jones arrested on Christmas for domestic violence
Rare photo of Andruw Jones not smiling even though Xmas battery was included
Andruw Jones Booked On Assault Charges
Former Brave Andruw Jones Jailed
Reports: Andruw Jones arrested
Andruw Jones arrested and Mugshotted after Abusing his Wife
Andruw Jones Arrested on Domestic Battery Charge
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Jacob Goldfield, former CIO at Soros Fund Management, might be the most high profile staff member to leave the $8.3 billion operation, but he is not the...
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Franklin has misplaced his blue blanket, and he can't sleep without it. He searches everywhere, but he still can't find it by bedtime. What will Franklin do?
Every young child will relate to this sweet tale in which Franklin loses his favorite blanket, the one he just can't sleep without. With a funny surprise ending, it's another charming story starring the turtle-hero.
- Ages
- 3, 6, 4, 7, 5
- Interest Level
- Grades PreK - 2
- Grade Level Equivalent
- 2.9
- Lexile Measure
- 24
- Guided Reading
- Publisher
- Tandem Library
- Book Type
- Lift the Flap Book
- Genre
- Bedtime Stories, Dictionaries, Reference, Series
About the Author and Illustrator
Paulette Bourgeois
When asked what she hopes children will learn from her books, Paulette Bourgeois explains: "It is most important to look for the wonder in ordinary things." Some of this wonder, and a curious anxiety, spill over into her character Franklin. The world's best-loved turtle overcomes the challenges of the everyday life of a five-year-old in a way that resonates with children.
Brenda Clark
Brenda Clark
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text:
For Trey's So Kodak Launch event in NYC, fans got tickets through twitter, Trey's Angels and radio stations, and arrived hours before the show was scheduled to start to stand in line. So Kodak took
Web Crew
My Activity
Type: Blog
Comments 23
Type: Blog
In case you missed the live stream of Trey's Rocawear in-store appearance, check out the exclusive footage above of TreyComments 24 -
Cake provided by Make My Cake (www.makemycake.com) | Photo Credit: Walik Goshorn
Trey hit up 106 & Park yesterday to help celebrate their 10 year anniversary with a performance of "Say Aah/Bottoms
Comments 28 -
Trey Songz says there are two sides to him. There's the club-going, "Bottoms Up" version and then there's the mature "Can't Be Friends" Trey.
Comments 2
Type: Blog
Presale tickets for the OMG Tour at the Verizon Center in Washington, DC go on sale TOMORROW Thursday, October, 7th at 10AM local venue time for Trey's Angels! Presale tickets will be available on a
Comments 0 -
Tune in to BET TONIGHT at 11pm ET to watch Trey perform "Can't Be Friends" and "Bottoms Up" on the Mo'Nique Show!
Comments 20 -
Tune in to BET TONIGHT at 6pm EST SHARP as Trey celebrates 10 years of 106 & Park with a special performance of "Bottoms Up" and "Say Aah"! Visit BET.com for more info on tonight's show, and makeComments 28 -
Trey and Drake performed at last night's So Kodak Campaign launch event in NYC. Check out Trey performing "Love Faces" off the new album PASSION, PAIN & PLEASURE at last night's event in the
Comments 9
hey web crew I would like to
hey web crew I would like to sign up for Treys Angels but can't access the page to do so. I 'm not sure if I'm going to the correct place to sign up? I clicked club under Angel network and it brought to the page I thought would give me the option to join, but there wasn't anything to click on to sign up? I tried going through the already a member link and got a error message. I really would like to sign up. Am I doing something wrong?
HI Web crew,
HI Web crew,
I have yet to receive my VIP package. I've sent emails and still no response from the membership team or you guys. I understand high capacity and etc but it would be nice for some sort of reassurance as to you guys are working on the situation. Please contact me on here or via email @ firstname.lastname@example.com
Thank you
Hi Web Crew,
Hi Web Crew,
First off, upon opening all packaging to my Ultimate Collector's Pack for Chapter V, I did not even receive a copy of the Chapter V CD. Secondly, I have had two customer service requests under the numbers #2098 and #2518. I am still waiting on a refund for my Trey's Angels membership because I received a coupon with my order(#13500002897) for my purchase of the Ultimate Collector's Pack. The coupon code was CEB5CD6545. The invoice number for the payment that was supposed to be refunded is 201481. I was told that I would see the refund in 5-7 business days on Sept. 6. After not receiving my refund, I reached out to the support team again on Sept. 24, and was once again told I would have a refund in 5-7 business days. I am still waiting for this refund, and I would like to know why it has taken over a month? I reached out once again on Oct. 9, resulting in request #2645, and have yet to hear from anyone and it is now Oct. 17. I would really just like my money back. Below is a copy of my payment confirmation email.
Thank you for your order. You may find order details below:
Your Invoice Number 201481 from 09/03/2012
Product/Subscription Title Price
Trey's Angels Premium Fan Club Membership $30.00
Subtotal $30.00
Shipping Amount $10.00
Total $40.00
Best regards,
Trey's Angels
Hi Web Crew....... I'm having
Hi Web Crew....... I'm having a serious issue with logging in for Angel store purchases. I can't even look at my account information to change my old info. to log into the Angel's store. I have things I want to order!
Hey. I was wondering is there
Hey. I was wondering is there gon be VIP tix for the Dublin venue on Feb 7 2013?
Thanks in advance
I notice that Trey is doing alot of stops on military bases. I was wondering whether or not Trey will be coming to Ft Dix/McGuire AFB anytime in the near future? Would like to hear back from you. Thanks!!
You can reach me at firstname.lastname@example.com or email@example.com
(912) 247-2382
SGT Tyreta Cespedes


text:
All / Free
Richard and Rosemary Wakelin Purchase Award
This event has finished. 4 November–9 December 2012,
The annual Richard and Rosemary Wakelin Purchase Award is given to a Welsh artist whose work is purchased for the Glynn Vivian Art Gallery's permanent collection.
As the Gallery is currently closed for a major refurbishment the exhibition of this year's recipient will be held in the Museum.


text:
Women's Day Theme Could Reflect in Female Religious Leadership
'Inspiring change' is this year's International Women's Day message; as time goes by even churches are seeing a stronger presence of females in higher positions.
'Inspiring change' is this year's International Women's Day message; as time goes by even churches are seeing a stronger presence of females in higher positions.
Cast & crew of Unscrewed Theater invite audience to join in the fun of improvisational comedy .
Known as Ash Wednesday, millions of Catholics, other Christians will have ashes placed on their foreheads today even if they can't make it to church.
Common bird is considered playful and intelligent, connected to mythology and symbolism.
Transgender Pima County lawyer says situation even more difficult for trans people; most states including AZ don't have laws protecting LGBTQ people in workplace.
Seniors majoring in Russian studies were selected for opportunity, recently returned to Tucson.
As Arizona celebrates its birthday, Margret F. Maxwell is honored with recognition for her cultural work in Arizona.
For those living without homes food, nourishment are constant needs; over past 7 years local volunteers have aided the issue.
Nonprofit organization making streets less-automobile oriented; city planning on Tucson's first parklet.
Thanks to parents who come from Germany, Mexico and school where 7-year-old learns English.
On Jan. 20 various local nonprofits will set up service projects to commemorate civil rights leader in national holiday.
Kristina Bishop of the School of Geography and Development discusses her travels to continent for research.
Festival of Lights through Dec. 28 allows residents to share seasonal spirit with community; food donations sought.
Use of artificial lights is increasing around the world; experts say tens of millions of people have never experiences truly dark skies.
While searching for friends at the All Souls Procession, a woman encounters a masked man who may be more than he seems...
Find out why successful film composer Brad Fiedel decided to leave Hollywood behind in a quest to rekindle his artistic inspiration.
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Welcome to this stunning stone Tudor located on a lush .94 acre lot in Westover Hills. Originally built in 1928 w/Avondale stone & Vermont slate roofing, this magnificent 6/7 BR, 6.1 BA home has been meticulously maintained & updated w/modern day conveniences while retaining every bit of its class & elegance. In 1998, an 1100 sq. ft. GR & full bath were added by Dewson's master craftsmen to overlook the pool retreat. The adjoining kitchen & butler's pantry have granite countertops & backsplash, gourmet-savvy appliances including Subzero refrigerator freezer, Scotsman ice-maker, a brand new GE Monogram gas cook-top & all the storage for which any chef could wish. The main BR is a spectacular suite w/dressing room/study/nursery. Other features worth noting are the formal LR & DR, paneled study, a cozy tiled sunroom, spacious closets, & inviting random-width oak flooring to connect each room. Additional amenities include beautiful beams & glass paneled doors, 2fp, & a finished wing w/2 add'l BRs.
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There are shavings of chocolate on the top. However, this does not save the chocolate, which is thinner than what is served at Angelina in Paris. The little square speaks volumes. Although the label says Sprungli, the chocolate is Felchlin. 65% Maracaibo to be exact. Inside the store, bars made with Felchlin chocolates are featured everywhere.
Albums: 100th anniversary of Max Felchlin AG Switzerland
Location: zurich switzerland (Show Map)
Follow Clay on:
Twitter :: @DiscoverChoc
F'Book :: TheChocolateLife
F'Book Group :: LaVidaCocoa
Paper.li :: @DiscoverChoc
© 2014 Created by Clay Gordon.


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Orange Gun Club, Inc.
357 West River St.
Orange, MA 01364
Contact: | Jim Rich 978-544-3525 or Mark Kovalsick 978-544-3525 |
Facilities: | Trap Range 9 am to dusk, Star Shoot Tower, pistol range, 200 yd rifle range, archery, 2 fishing ponds, clubhouse. |
Activities: | Trap Shoots Tuesdays and Sundays, Sporting Clay shoots once a month. |
Training: | Class A permit classes. Send two children to Jr. Conservation Camp per year. |
Membership: | Dues by December 31st $20. Sponsored by present member in good standing. Must be 21 years of age. |
Meetings: | 3rd Sunday of the Month Jan. Feb. Mar. Sept. Oct. Nov. Dec. - 3rd Monday Apr. May June July |
*This page is made possible by the membership of GOAL and their generous donations. If you found this page helpful please Join - Support - Donate to GOAL.
Follow Us: GOAL is the Official State Association of the National Rifle Association


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Matters of the Heart 2: How to be Happy in Christ
Mike Reeves
- Mike Reeves is UCCF's Head of Theology. Follow him on Twitter @mike_reeves View all resources by Mike Reeves
The second of four talks entitled 'Matters of the Heart' given by Mike Reeves at 2011's New Word Alive.
Mike Reeves - Matters of the Heart 2 - How to be Happy in Christ
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This is the second of four talks that were given at New Word Alive 2011. Come and join us at New Word Alive 2013!


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Just hours before an expected NHL lockout, the Toronto Maple Leafs made a number of roster moves involving some of their younger players.
Among the 22 players assigned to the AHL's Toronto Marlies were forward Nazem Kadri and defenceman Jake Gardiner.
The Leafs also sent four players back to their former junior teams, including their fifth overall selection in the 2012 Draft, Morgan Rielly, who returned to the WHL's Moose Jaw Warriors.
The 22-year-old Gardiner completed his first NHL season in 2011-12, finishing with seven goals and 30 points in 75 games.
A first round selection of the Anaheim Ducks in 2008, Gardiner also appeared in four regular season games and 17 playoff games for the Marlies last season, recording two goals and 11 points.
The 21-year-old Kardri has split his time the last two seasons between the Marlies and Maple Leafs.
Taken seventh overall in the 2009 Draft, Kadri totaled 23 goals and 47 points playing in both the AHL and NHL in 2011-12.
Besides Gardiner and Kadri, the Leafs assigned the following players to the Marlies: Spencer Abbott, Carter Ashton, Jesse Blacker, Tyler Brenner, Sam Carrick, Joe Colborne, Andrew Crescenzi, Jerry D'Amigo, Nicolas Deschamps, Jamie Devane, Simon Gysbers, Ryan Hamilton, Korbinian Holzer, Leo Komarov, Greg McKegg, Mark Owuya, Brad Ross, Kenny Ryan, Jussi Rynnas and Greg Scott. In addition to Rielly, the Leafs assigned the following players to their junior clubs: Tyler Biggs (Oshawa Generals, OHL), David Broll (Sault Ste. Marie Greyhounds, OHL) and Stuart Percy (Mississauga Steelheads, OHL).
Martin St. Louis is a Ranger, Matt Moulson is a member of the Minnesota Wild and the Montreal Canadiens have Thomas Vanek. Look back at all the big name deals that went down.
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Titles similar to Habibi
Thompson's follow-up to the landscape-altering Blankets (2003), one of the generous handful of "important" comics of the past decade, shows that he has done anything but rest on his laurels. At root, this is a love story about two have-nots in a desert society: Dodola, a young woman whose only currency is her body, and Zam, a slave boy she rescues and tries to shelter. Passages from the Qur'an provide reflection on Dodola's and Zam's lives as they connect, break apart, and find each other again. Arabic lettering and magic-square mysticism offer rich foundations of visual symbolism and theosophical inquiry. And, not least, the fictional state of Wanatolia, where you can travel in time thousands of years simply by stepping from the midden slums to the sultan's palaces to the rapid encroachment of high-rise development, provides a polarizing backdrop of social conflict. The character depth, plot complexity, and storytelling in this lyrical, sexual, and scholarly epic would make any novelist proud. But no graphic novel lives on narrative alone, and through it all, Thompson strings compositions that are often more tapestry than comics and that balance graphic design, illumination, calligraphy, and cartooning in steady alignment. It is unfair to expect two masterpieces in a row from anyone, but here Thompson sits securely in that rarefied air.


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Lebo, Raitter Finish First at XTERRA Nottingham XduroNottingham, PA – At the final race of the season, Scott Lebo, 41, and Sarah Raitter, 35, won their respective divisions at the XTERRA Nottingham Xduro 21km Trail Run this past Sunday. Pottstown local Lebo was the first to cross the finish line with his time of 1:29:55 beating runner-up Justin Harris in a close race by a mere 6 seconds. However, Harris, 24, took the title for being the furthest from home having traveled all the way from Eva Beach, Hawaii. Reno, NV resident Raitter topped the women's category finishing 10th overall in 1:43:33. The longtime XTERRA athlete ran a fantastic race coming in almost 22 minutes ahead of second place runner Corrine Banks of Ocean City, MD. In the 10km race, Stefan Pohl, 44, of Oxford, PA took first place with his time of 49:51 to beat Levittown resident Don Little coming in 23 seconds later. The women's race was won by Ulla Chwalisz, 49 and also from Oxford, in 1:01:30. Less than a minute behind her was Caroline Hamilton who took second in her hometown of Nottingham. Runners from 13 states came out to race in the final 2008 XTERRA Philly Series event. The hot and humid day gave runners an additional challenge, but race director Don Morrison had a great turnout for the debut of XTERRA's new half-marathon distance Xduro. XTERRA Trail Run Series Manager Kristin Francis was in attendance for the event saying "The race went very well. The XTERRA Philly Trail Run Series has been a big success and a lot of fun. We can't wait until next year." Jerilyn Kucharski, who ran in all five races this season, said "I thoroughly enjoyed the rocky, rolling, muddy, and sometimes soaking wet terrain of the Wednesday evening series, and the 21km race was also a pleasure. I am already looking forward to next summer and more XTERRA trail races." This was the fifth and final race in the 2008 XTERRA Philly Trail Run Series. Runners who finished with top series points will be invited to race for free at the XTERRA Trail Running National Championship on September 27, 2008. Here they will compete against the best-of-the-best trail runners from across the country. From there, athletes can go on to race in the XTERRA Trail Running World Championship on December 7 in beautiful Hawaii. For more information, visit www.xterratrailrun.com. Complete race results are attached and more information can be found online at www.xterratrailrun.com. Media interested in photos from this event should contact firstname.lastname@example.com. |
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Human rights groups: No in-game war crimes
Here's one that will undoubtedly send some gamers into a craze. A study from two Swiss human rights organizations, Trial and Pro Juventute, has found that some video games depict war and battle actions that in real life would violate international human rights laws.
The study attempted to determine if the acts gamers engage in while they play violent titles would "lead to violations of rules of international law, in particular International Humanitarian Law (IHL), basic norms of International Human Rights Law (IHRL), or International Criminal Law (ICL)."
To find out, Trial and Pro Juventute picked up 20 games, including Call of Duty 4, Metal Gear Solid 4, Far Cry 2, and others. It had "young gamers" play the games as three attorneys watched to find actions in games that in real life would violate rules and regulations that govern armed conflict.
The organizations said the study is not intended to "prohibit the games, to make them less violent or to turn them into IHL or IHRL training tools." Instead, the groups want to work with developers to ensure that in the future, their games observe real-life human-rights laws.
After evaluating the 20 games, the group found that in many cases, "shooter" games failed to take into consideration international humanitarian law.… Read more


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The language courses are designed to help students practice and interact in Chinese with their homestay families and during site visits on the program. Field trips and lectures are integrated into the academic curriculum to provide a deeper understanding of what is learned in the classroom. Complementing the classroom work is a series of visits to Chinese companies and factories, government agencies, museums, temples, and other sites.
CIEE encourages students to get immersed in Chinese culture and to enrich their experience in Shanghai by: • reimbursing students for their participation in local cultural activities, • offering peer language tutors • maintaining volunteer opportunities • organizing an optional language clinic for students who need additional help • planning monthly group meals with Chinese speakers
Students have a mid-semester break during which they may travel around China.


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Ethanol is a high octane, clean burning, American-made renewable fuel. Its production and use offer a myriad of benefits to the United States and its citizens.
The production of ethanol is an economic engine for the United States, adding value to U.S. agricultural products and bringing billions of dollars to the nation's economy each year. The use of ethanol reduces harmful auto emissions, offers consumers a cost-effective choice at the pump, and decreases the amount of expensive crude oil needed to satisfy the nation's thirst for transportation fuel.
Ethanol is produced at 200 facilities across the nation and blended in to unleaded gasoline in varying percentages. Ethanol is most commonly retailed as E10, the blend of 10 percent ethanol (90% gasoline) for use in all automobiles. Increasingly, ethanol is also available as E85, the 85 percent ethanol blend for use in Flexible Fuel Vehicles. The E15 blend has recently been approved by the U.S. EPA for use in 2001 and newer model year vehices (read more here).
This All About Ethanol section contains a wealth of information about the production and use of ethanol. Visit these subsections to learn more:
Ethanol 101 - ethanol basics, benefits
Ethanol Facilities - how and where ethanol is made
Ethanol & Your Vehicle - the types and use of ethanol-blended fuel
Ethanol Research - research studies on various ethanol topics
Ethanol Co-Products - the co-products of the ethanol production process
Alternative Uses - ethanol for industrial, beverage, aviation purposes
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Banjos | Banjos
Buddy and Buck Bunion, known as the dumbest boys in Texas and worthless rascals, have met their match with Granny Gert. After being chased out of Amarillo, the Bunion Brothers ask Granny Gert for a job. She agrees to give them a chance but threatens to sic Mad Dog on them if they don't behave....
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Hospital Reduces Climate Footprint in Health Care Food Services to Win National Award | |
Date: 05/02/2013 Contact: Rachel Champeau (email@example.com) Phone: 310-794-2270 |
Ronald Reagan UCLA Medical Center was honored with a "Food, Climate, Health Connection Award" from Health Care Without Harm , an international coalition whose Healthy Food in Health Care program works with hospitals across the country to help improve the environmental sustainability of their food services. Ronald Reagan UCLA Medical Center was one of only three hospitals to receive an award in the category that recognizes facilities that are taking significant steps toward reducing their climate footprint. The awards were presented at CleanMed 2013, which was held April 24-26 in Boston, MA. "We are honored to receive this prestigious award from Health Care without Harm that validates our efforts to promote optimal health, while also helping to protect the environment," said Dr. David Feinberg, president of the UCLA Health. "Our nutrition team has been at the forefront in developing initiatives that encourage healthier food choices to purchasing sustainable foods to recycling and composting in our food service areas. This is part of our vision to support a healthier community." The Ronald Reagan UCLA Medical Center's hospital food service team took the following steps in 2012 that the award acknowledges:
"It is increasingly important that the world recognize the link between our food system, climate change and human health, and take steps to reduce our environmental footprint," said Gary Cohen, president and founder, Health Care Without Harm. "Ronald Reagan UCLA Medical Center has demonstrated an overall excellence in understanding this connection, and is engaging their hospitals food service department as part of the solution." The award is one of four categories of sustainable food awards given this year by the Healthy Food in Health Care program to recognize significant achievement as well as leadership in health care food service. By serving foods that are produced and distributed in ways that reduce resource and energy consumption as well as taking other operational steps, health care food service operations can play an influential role in decreasing the food system impacts on the environment. Award applicants were given points based on levels of achievement in the following areas: reduction of the amount of meat and poultry purchased; prioritizing purchase of food and beverages that are produced without use of fossil-fuel based fertilizers and pesticides; waste prevention, donation and composting; and energy and water conservation. The UCLA Health, which comprises the UCLA Hospital System and the UCLA Medical Group and its affiliates, has provided a high quality of health care and the most advanced treatment options to the people of Los Angeles and the world for more than half a century. Ronald Reagan UCLA Medical Center, the Resnick Neuropsychiatric Hospital at UCLA, Mattel Children's Hospital UCLA, and UCLA Medical Center, Santa Monica (which includes the Los Angeles Orthopaedic Hospital) deliver hospital care that is unparalleled in California. Ronald Reagan UCLA Medical Center is consistently ranked one of the top five hospitals in the nation and the best in the western United States by U.S. News & World Report. UCLA physicians and hospitals continue to be world leaders in the full range of care, from maintaining the health of families to the diagnosis and treatment of complex illnesses. -UCLA- |
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Cedar Rapids Weather
WBB: No. 24 Hawkeyes Commit 26 Turnovers, Lose At Illinois
CHAMPAIGN, Ill. (AP) — Karisma Penn scored 18 points, Adrienne GodBold and Amber Moore added 17 apiece, and Illinois closed with a 14-2 run over the final 5:11 to knock off No. 24 Iowa 74-62 on Thursday night.
Penn pulled down eight rebounds for the Illini (12-8, 5-3 Big Ten), whose last victory over Iowa in Champaign was Feb. 9, 2003. Ivory Crawford chipped in 14 points to help Illinois surpass its win total from the 2011-12 season. Samantha Logic had 13 points, 10 rebounds and six assists for the Hawkeyes (16-6, 5-3) but also committed eight of Iowa's 27 turnovers. Illinois converted those turnovers into 30 points. What had been a back-and-forth game swung the Illini's way after Iowa's Jaime Printy tied it at 60 with a layup with 5:31 to go. The Hawkeyes wouldn't score again until Kayla Timmerman's jumper with 13 seconds left, after Illinois had reeled off 14 unanswered points. Conversation GuidelinesBe KindDon't use abusive, offensive, threatening, racist, vulgar or sexually-oriented language. Share KnowledgeBe truthful. Share what you know and what you are passionate about. Stay focusedPromote lively and healthy debate. Stay on topic. Ask questions and give feedback on the story's topic. Report TroubleHelp us maintain a quality comment section by reporting comments that are offensive. If you see a comment that is offensive, or you feel violates our guidelines, simply click on the "x" to the far right of the comment to report it. read the full guidelines here » Commenting will be disabled on stories dealing with the following subject matter: Crime, sexual abuse, property fires, automobile accidents, Amber Alerts, Operation Quickfinds and suicides. |
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Jessica Brown Lindsay is a British actress best known for her role as Lady Sybil Crawley on the hit TV series Downton Abbey.
Robert Cascoyne-Cecil, 1st Viscount Cecil of Chelwood, is best known as one of the chief architects behind the League of Nations.
Morten Harket is best known for being lead singer of the Norwegian pop band A-ha, which produced the 1980s hit "Take On Me" and its innovative music video.
Actor Walter Koenig rose to fame in the 1960s as Mr. Chekov on the television series Star Trek. He has reprised his role in several film adaptations of the show, including Star Trek: The Motion Picture.
Melissa Leo is an American actress best known for her Academy Award-winning performance in the 2010 film The Fighter.
Dmitry Medvedev served under Vladimir Putin and succeeded him as president of Russia in 2008.
Actor Clayton Moore appeared in approximately 70 feature films and played the Lone Ranger on a ABC network television version of the radio program.
Constance Baker Motley was a legal advocate in the Civil Rights Movement. She became the first female African-American federal judge in 1966.
East Coast rapper Nas made it big with albums Illmatic (1994), It Was Written (1996) and Stillmatic (2001).
Russian physiologist Ivan Pavlov developed his concept of the conditioned reflex through a famous study with dogs and won a Nobel Prize Award in 1904.
Margaret Sanger was an early feminist and women's rights activist who coined the term "birth control" and worked towards its legalization.
One of the most famous filmmakers of all time, Hal B. Wallis produced more than 400 movies starting from the 1920s.
James Wilson was a Supreme Court associate justice. He signed both the U.S. Constitution and the Declaration of Independence.
Amy Winehouse won five Grammy Awards for her 2006 album Back to Black, and is remembered for songs like "Rehab," "Back to Black" and "Valerie." She died in 2011, at age 27.


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Oscars 2013: Seth MacFarlane's opening skit gets mixed reviews
(AP) - Seth MacFarlane accepted an assist from William Shatner to smooth his nerves during his first time as Academy Awards host.
MacFarlane, best known for behind-the-scenes Hollywood work as creator of the television series "Family Guy" and movie "Ted," seemed nervous in the traditional role telling jokes, although he did get in one quick poke at the academy for snubbing Ben Affleck as a best director nominee for the movie "Argo."
"The story is so top secret that the film's director is unknown to the academy," MacFarlane said.
One joke about "Amour" flew by so quickly that it was difficult to understand what he said.
He joked about "Lincoln" best actor nominee Daniel Day Lewis' habit of staying in character during filming days even when the cameras were turned off.
"If you bumped into Don Cheadle in the studio lot, would you try and free him?" MacFarlane asked.
A giant video screen was lowered near where MacFarlane stood, revealing Shatner in "Star Trek"-like regalia. Shatner said he was there trying to save MacFarlane from himself, showing a supposed headline from the future that declared MacFarlane the worst Oscar host ever.
It sent MacFarlane into more high concept comedy, with mixed results. MacFarlane was shown in a song and dance routine, backed by the Los Angeles Gay Men's Chorus, naming famous actresses and the films where they displayed their breasts. During a quick cutaway, Charlize Theron - one of the actresses named - did not appear amused.
In another skit, scenes from the movie "Flight" were performed by sock puppets. An overly long skit - again designed to illustrate MacFarlane's "bad taste" - showed him wearing a Flying Nun costume in an attempt to pick up Sally Field backstage.
MacFarlane made self-deprecation a theme of his Oscars hosting turn even before taking the stage, saying in interviews that he expected to get negative reviews.
He seemed to relax when the spotlight moved away and he had the quick job of introducing presenters with a handful of jokes. He had a handful of solid jokes when talking about Quvenzhane Wallis, the 9-year-old best actress nominess for "Beasts of the Southern Wild."
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Joni Eareckson Tada at Belhaven UniversityBelhaven University hosted an uplifting night with author, speaker and artist Joni Eareckson Tada. This free event on Saturday, March 1 was in the Belhaven University Center for the Arts on Riverside Drive. read more... Track and Field Athletes Compete for First TimeThe Blazer Mens and Womens Track and Field had a historic day on Saturday, March 1 and competed in their inaugural track and field event at the James E. Parkman Track for the Mississippi College Season Opener read more... Belhaven University Applies For NCAA Division III MembershipThe Belhaven University Board of Trustees has voted unanimously to approve application for NCAA Division III membership as Belhavens future national athletics affiliation. read more... Student Composers ConcertThe Belhaven Composers Forum invites you to an interactive showcase of engaging new works by Belhaven's student composers on Thursday, March 6 at 7:30 p.m. in the Center for the Arts Recital Room. read more... Faculty Concert Features Dr. Shelt with StudentsPast and present Belhaven music students will join together with Dr. Christopher Shelt, Professor of Music, on March 4 for a special concert celebrating the teacher-student relationship. read more... Best of Belhaven Showcases Top Music PerformancesBest of Belhaven will feature a wide variety of musical performances from the gifted students in Belhavens Music Department. This free performance on Thursday, Febuary 27 will highlight the finest concerts read more... 2014 Belhaven University Career FairThe 2014 Belhaven Career Fair is this Thursday, February 27 from 10:00 a.m. to 2:00 p.m. in the Student Center. This career fair draws top employers in the Jackson area and statewide read more... Communication Majors Work with ESPN at Sugar BowlThere is nothing bigger in college football than the bowl season, and Belhaven University communication majors Kelsie Hughes and Drew Garnder were right in the thick of it this year. read more... Belhaven Named One of 33 Schools in the Nation Accredited in Four ArtsBelhaven University is one of only 33 institutions in the nation accredited in all four areas of the arts - visual art, music, theater, and dance. read more... Spring Discover Day at Belhaven UniversityBelhaven Universitys Discover Day on February 14 is our open invitation for any and all interested high school students to visit and experience the energy on campus and witness an academically charged read more... |
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