Rrd1 Regulates Elongation of RNA Polymerase II Figure 7. mRNA expression analyses in response to different stress conditions. GeXP multiplex expression analysis of 10 genes from untreated, rapamycin (100 ng/ml for 30 min), H2O2 (0.6 mM for 30 min), NaAs (1 mM for 30 min), heat shock (37uC for 30 min) treated conditions. The data is expressed in log 2 ratio and is calculated relative to an internal PCR control. (A) Five upregulated genes. (B) Three downregulated genes (C) Two control genes. doi:10.1371/journal.pone.0023159.g007 mapping studies revealed that Rrd1 association was restricted to the body of the ORFs, since it reached its highest levels downstream of the promoter as compared to RNAPII. In addition, at the 39 gene ends Rrd1 occupancy was reduced before RNAPII. Numerous other elongation factors such as Dst1 (TFIIS) were also found to interact with RNAPII and to be associated within the body of the gene [40,41]. These co-localisation events occurring only during elongation suggest a function of Rrd1 during this phase of transcription. This is further supported by the fact that rrd1D deletion affects RNAPII gene occupancy in response to rapamycin for up and down regulated genes. It is noteworthy that this effect is noticeable during elongation, as the alterations of RNAPII are visible within the body of the gene as opposed to accumulation at the promoter or 39 end region. This implies that Rrd1 regulates the amount of transcribing RNAPII, which could be through at least two different mechanisms; one by recruiting more RNAPII to these genes or two, by regulating the speed by which RNAPII is transcribing along the genes. Our analysis of TBP occupancy indicated that Rrd1 acts upstream as well as downstream of TBP association and thus might be involved in the recruitment as well as the regulation of RNAPII elongation (Figure 4). Upon rapamycin treatment, transcription factors translocate to the nucleus and are recruited to promoters to stimulate or repress transcription by regulating TBP and PIC assembly [4,5,8,42,43,44]. How Rrd1 might operate upstream of TBP is unclear, although it could be through the ability of Rrd1 to activate phosphatases [17,18,45]. For example, Rrd1 could regulate a specific phosphatase thereby altering the recruitment of transcription factors and PIC assembly. We note that TBP remained promoter-bound on a subset of down regulated genes from which RNAPII dissociated in response to rapamycin. This was partially dependent on Rrd1 since RNAPII was less drastically reduced in the rrd1D mutant (Fig. 4C cluster S1 and S4). This could be a mechanism to temporarily downregulate transcription, while allowing for rapid restart of gene expression once the stress is over. That Rrd1 influences a larger number of genes downstream of PIC assembly suggests that this is the major role of Rrd1 in the transcriptional response to rapamycin. This, and given that Rrd1 is associated within the ORF of most genes, indicates that it acts at the level of elongation. Also, although Rrd1 is constantly associated with RNAPII, deletion of RRD1 has only a tangible effect on RNAPII in response to stress, meaning that under normal growth conditions Rrd1 is not crucial for RNAPII recruitment to genes. There are several more evidences advocating that Rrd1 acts on RNAPII during initiation and/or elongation: (i) We have previously shown that Rrd1 is localized to the chromatin, PLoS ONE | www.plosone.org 10 August 2011 | Volume 6 | Issue 8 | e23159