Our data indicate that Sub1 functions in the recruitment of polymerases to active RNAP II and III genes and also uncovers specific conditions in which the normally nonessential Sub1 becomes critical. We found that, in the absence of the HOG pathway, cells require Sub1 for survival under moderate osmolarity. Our analysis suggests that, without the HOG pathway, expression of necessary osmoresponse genes is possible only because the cell turns to an alternative pathway that makes use of Sub1 to target RNAP II to those genes. Supporting this, previous studies have postulated that Hog1-independent pathways participate in the stress-dependent expression of osmoresponse genes, such as GPD1
, which encodes a key enzyme involved in glycerol biosynthesis, is essential during osmotic stress (1
), and we found that, in hog1
Δ cells, both RNAP II recruitment to GPD1
expression depend on Sub1. The function of Sub1 in the osmotic stress response might therefore represent redundancy necessary to ensure survival if the HOG pathway fails.
Sub1 employs distinct, gene-specific mechanisms to facilitate transcription initiation. Whereas deletion of SUB1
did not affect TBP and TFIIB association with constitutively transcribed RNAP II gene promoters, TBP and TFIIB levels at the GPD1
promoters were both significantly reduced after induction by NaCl. This suggests that Sub1 uses a different strategy, involving preinitiation complex assembly or stability, for targeting RNAP II to these genes, which would then facilitate their rapid induction during osmotic stress. Physical interactions between Sub1 and GTFs (18
) might be required for targeting Sub1 to active promoters, whereas in the osmotic stress response, rapid targeting of Sub1 to promoters, perhaps by stress-responsive transcription factors, might activate transcription by recruiting GTFs through the same Sub1-GTF interactions. Recruitment of Sub1 to the inducible ARG1
promoter (our unpublished data) suggests that pathways besides the osmoregulation pathway might employ Sub1 for rapid induction. However, our large-scale genetic analysis did not reveal additional Sub1-dependent gene activation pathways (our unpublished data), suggesting specificity in its function in the osmoresponse pathway.
Our detailed analysis of Sub1 occupancy on the transcriptionally active STL1
, and PMA1
genes revealed that Sub1 is strongly promoter associated. In our previous studies, cells expressing a plasmid-encoded version of an N-terminally three-HA-tagged Sub1 were used in ChIP experiments to show that 3HA-Sub1 associates with the constitutively transcribed ACT1
, and PMA1
). In that study, approximately equal levels of 3HA-Sub1 at the promoter and at downstream positions of the coding regions of those genes were detected. We believe those results were due to elevated Sub1 levels in the cell, due to additional expression from a plasmid. When we compared Sub1 occupancy in a strain expressing C-terminally tagged Sub1 from the SUB1
locus with that in a strain expressing C-terminally tagged Sub1 from a plasmid, we found the plasmid-derived Sub1 cross-linked at coding regions, whereas the chromosomally expressed Sub1 did not (data not shown). Although this indicates the potential for Sub1 to associate with the elongating transcriptional machinery, consistent with a role in elongation (9
), our current results suggest that Sub1 is predominantly promoter localized when expressed from its natural promoter. In another study, cells expressing a tandem affinity purification (TAP)-tagged Sub1 (from the chromosomal SUB1
locus) were used to determine the occupancy of Sub1 along the PYK1
). High levels of Sub1-TAP were detected at the PYK1
promoter, low levels at a coding region, and an intermediate level near the polyadenylation site. The physical interaction of Sub1 with the cleavage/polyadenylation machinery (7
) supports a role for Sub1 at the 3′ ends of genes, although this may also reflect the presence of certain cleavage/polyadenylation factors at promoters (8
Intriguingly, we detected Sub1 at every type of RNAP III gene that we tested. Very few other factors are found at both RNAP II and RNAP III promoters; these are limited to TBP (reviewed in reference 16
) and five common subunits of the polymerases themselves (6
). Additionally, in a recent genome-wide analysis, the RNAP II initiation and elongation factor TFIIS was detected at RNAP III genes, where it may act as a general RNAP III transcription factor (13
). To our knowledge, however, besides TFIIS, a yeast transcription factor that regulates transcription of all types of RNAP II and RNAP III genes, but not RNAP I genes, has not yet been reported. A previous study detected PC4 in a complex containing the RNAP III general transcription factor TFIIIC and found it to be a potent stimulator of RNAP III transcription in vitro using a reconstituted transcription system (38
). Our observations therefore place Sub1 in an evolutionarily conserved and unusual position, as a putative transcriptional regulator for RNAP II and III.
Under normal growth conditions, we detected Sub1 at all constitutively transcribed RNAP II and RNAP III genes tested, correlating association of Sub1 with sites of active transcription. In general, promoters with high levels of Sub1 corresponded to genes that are highly transcribed and are associated with high levels of RNAP II (this study and our unpublished data), although in some cases there is a disproportionately high level of Sub1 (e.g., at the PYK1
promoter). This general correlation supports a role for Sub1 in facilitating the recruitment of polymerases to transcribed genes after the assembly of promoter complexes, including TBP (and TFIIB for RNAP II promoters). It is curious that Sub1, so abundantly and ubiquitously present at so many genes, would have only modest effects on transcription, as measured by steady-state transcript analysis. However, our data support the idea that an important function for Sub1 might involve facilitating the mobilization of transcription apparatuses during conditions of stress, such as exposure to NaCl. During osmotic shock, the expression of hundreds of genes is either up- or downregulated (31
). Our analysis indicates that the association of Sub1 with constitutively transcribed genes is reduced, while Sub1 accumulates at osmoresponse genes. As the transcriptional response to osmotic stress is transient (1
), this could allow for a temporary and modest reduction in the availability of RNAP II at constitutive genes, while facilitating recruitment of RNAP II to stress-induced genes and preinitiation complex assembly at critical genes. According to this model, Sub1 is involved in fine-tuning the level of polymerases at RNAP II and III genes, while poising the cell for changes in transcription patterns necessary when certain stresses are encountered. It will be key to determine what targets Sub1 to actively transcribed promoters in a general manner and during activation of specific genes in order to understand how Sub1 functions in both situations. In any event, our current study has uncovered novel promoter-associated functions for a protein with multiple roles at many stages of gene expression.