RNAPII was strongly recruited to genes involved in nitrogen discrimination, the Krebs cycle, stress response and catabolic processes after rapamycin treatment. These data are consistent with the transcriptional changes reported for rapamycin treatment as well as for environmental stress responses. We next performed ChIP-chip analysis on Myc-tagged Rrd1 and compared its distribution on the ORFs with that of RNAPII. As shown in Figure 2C, RNAPII occupancy correlates with that of Rrd1, indicating that Rrd1 is recruited to transcribed genes. Interestingly, when cells were AbMole Tuberostemonine treated with rapamycin, Rrd1 and RNAPII displayed a similar correlation, suggesting that under normal growth conditions, as well as after drastic transcriptional changes, Rrd1 remains associated to actively transcribed genes. To further investigate this relationship, we mapped RNAPII occupancy along a group of genes with the lowest levels of RNAPII as well as a group with the highest levels of RNAPII and then mapped Rrd1 on the same groups. Indeed, when RNAPII levels were low, the Rrd1 levels were also low, and AbMole Lomitapide Mesylate consistently, when RNAPII levels were high Rrd1 levels were also increased. This was also the case for rapamycin treated cells. We next checked if the regulation of the rapamycin-downregulated genes by Rrd1 is TBP-independant. Preliminary analysis of the downregulated genes suggested that these are heterogeneous with respect to TBP binding. We therefore generated new clusters from clusters W1 and W6 using both RNAPII and TBP occupancy values from wild type cells. This clustering revealed that although RNAPII is downregulated for all genes TBP occupancy is only reduced in cluster S2 and S3 in wild-type cells treated with rapamycin. This suggests that, in wild-type cells, rapamycin leads to gene expression downregulation by at least two distinct mechanisms. The first involving the regulation of TBP recruitment and the latter involving the regulation of a downstream step. To look at the function of Rrd1 in that regulation, we looked at TBP and RNAPII occupancies in rrd1D cells on these clusters. As predicted from Figure 4A, deletion of RRD1 affected RNAPII occupancy of all these genes upon rapamycin treatment. Interestingly, however, TBP levels were not affected by the deletion of RRD1 for the genes included in clusters S1 and S4, while they were significantly crippled at genes from clusters S2 and S3. Taken together these data indicate that RNAPII downregulation upon rapamycin treatment is regulated by two mechanisms, one which does not depend on TBP depletion. Interestingly, Rrd1 is required for both mechanisms by optimizing TBP depletion and RNAPII levels. This suggests that Rrd1 has an influence on TBP binding, probably by affecting the signaling cascade upstream of PIC assembly but also functions downstream of TBP binding, likely during transcription initiation/elongation. The above data indicate that Rrd1 is required for an optimal transcriptional response following rapamycin exposure.