Mutational analysis of Rpb4 and Rpb7 : Insights into their role in transcription and stress response of saccharomyces cerevisiae

Abstract

The Yeast Saccharomyces cerevisiae as a Model System for Stress Response and Transcription

The yeast Saccharomyces cerevisiae is an excellent model system to study the molecular basis of stress response. Apart from its amenability to routine genetic and biochemical manipulations, it exhibits distinct and readily measurable responses to a wide variety of stresses. While the role of different RNA polymerase II–associated proteins, such as co-activators and general transcription factors (GTFs), in modulating changes in gene expression patterns in response to different stimuli is well documented, a similar role for some subunits of the core RNA polymerase II is also postulated (Hampsey and Reinberg, 1999; Miyao and Woychik, 1998; Sadhale et al., 1998).

We are interested in two subunits of the yeast RNA polymerase II-Rpb4 and Rpb7-that have been shown to play roles in stress response and transcription. Rpb4 and Rpb7 form a sub-complex within the polymerase that readily dissociates from the polymerase. The stoichiometry of this sub-complex in the polymerase increases from 0.2 to 1 as growth proceeds from log-phase to stationary phase (Choder and Young, 1993).

The rpb4 strain grows at moderate temperatures, albeit slowly, and exhibits a variety of stress response defects-temperature sensitivity above 32°C or below 15°C, poor recovery from stationary phase (Woychik and Young, 1989), predisposition to pseudohyphal morphology, and inability to sporulate (Pillai et al., 2003), the last two being hallmark responses to nutritional starvation. Overexpression of Rpb7, the essential interacting protein, can partially rescue some of these defects (Sharma and Sadhale, 1999; Sheffer et al., 1999).

In addition to these stress responses, rpb4 also exhibits defects in activated transcription from a certain subset of genes and in carbon and energy metabolism at moderate temperatures (Pillai et al., 2001). Overexpression of Rpb7 does not rescue the defects in activated transcription, suggesting that Rpb4 and Rpb7 play independent roles in stress response and transcription (Pillai et al., 2001). The multiple phenotypes affected by these subunits necessitate the identification and characterization of mutants that will help localize the domains of the proteins involved in independent phenotypes and also be useful in isolating phenotype-specific interacting partners.

Role of Conserved and Non-Conserved Regions of Rpb4 in Stress Response and Transcription

S. cerevisiae (Sc.) Rpb4 is considerably larger than its homologs. Comparison of Rpb4 sequences from different eukaryotic homologs shows that the N-terminal 45 amino acids and the C-terminal 80 amino acids are highly conserved. The Sc.-specific residues, having a high density of charged amino acids, map to the central region.

Deletion analysis was used to understand the roles played by these conserved and non-conserved regions in different phenotypes associated with Rpb4. Our results suggest that the Rpb7 interaction domain consists of both the conserved N- and C-terminal regions. Molecular modeling of the Rpb4/Rpb7 complex further corroborates this result.

The N-terminal region is not required for stress response and activated transcription from GAL10 and HSE-driven promoters but is required for efficient activated transcription from the INO1 promoter.

The C-terminal conserved region is important for certain stress response phenotypes and activated transcription from all promoters tested.

Neither of these regions is important for the role of Rpb4 in pseudohyphal growth.

Deletion analysis of the non-conserved regions shows that they influence phenotypes involving both the N- and C-terminus (interaction with Rpb7 and transcription from the INO1 promoter) but none of the stress-responsive phenotypes tested, suggesting that they might be involved in maintaining the conserved N- and C-terminal regions in an appropriate conformation for interaction with Rpb7 and other putative interacting partners.

Analysis of Rpb7 Mutants in Stress Response

Overexpression of Rpb7 in an rpb4 strain rescues its defects in temperature sensitivity and sporulation but results in exaggeration of pseudohyphal growth. As Rpb7 is an essential protein, a genetic screen was used to isolate temperature-sensitive mutants of Rpb7, and these mutants were characterized with respect to the phenotypes seen for wild-type Rpb7.

Eight mutant alleles were classified based on their degree of temperature sensitivity in an rpb7 background: three “strong,” three “moderately” affected, and two “unaffected” alleles. Analysis of these mutants in phenotypes affected by rpb4 revealed the following:

The rpb7-7 allele is compromised for rescue of temperature sensitivity and sporulation.

The rpb7-61, rpb7-63, and rpb7-64 alleles are compromised only for rescue of sporulation.

None of these alleles are severely compromised for the ability of Rpb7 to exaggerate pseudohyphal growth, though minor differences exist.

Analysis of steady-state protein levels showed that only the rpb7-18 mutant is destabilized in both rpb4 and rpb7. Many mutant alleles were stabilized specifically in rpb47 but not in rpb7, suggesting a novel role for Rpb4 in stabilizing Rpb7 protein levels.

The rpb7-7 allele contains four mutations: F70L, V72M, A123E, S125T. The two N-terminal mutations potentially disrupt the RNP fold in Rpb7, whereas the C-terminal mutations fall in solvent-exposed loops. Swapping the N- and C-terminal domains of rpb7-7 independently with wild-type domains showed that the N-terminal mutations are responsible for the observed phenotypes. The rpb7-7 allele can rescue rpb7 lethality in the presence, but not in the absence, of Rpb4. These results suggest that the RNP fold is an important structural determinant for Rpb7’s role in stress response.

A gain-of-function mutant, rpb7-22, carries a T28A mutation that allows the mutant protein to rescue rpb47 temperature sensitivity better than wild type, even at low expression levels. This mutant also improves rescue of sporulation and pseudohyphal growth phenotypes but does not affect rescue of rpb7 lethality.

Role of the Conserved PY Motif of Rpb7 in Stress Response

All eukaryotic Rpb7 proteins contain a conserved PPXY (PY) motif in the L3a loop of the C-terminal OB fold. The PY motif in many proteins serves as the interaction site for WW domain–containing proteins (Sudol et al., 2001).

The rpb7 P127A P128A allele, generated by site-directed mutagenesis, shows that the PY motif is required for efficient sporulation and high-temperature growth but not for exaggeration of pseudohyphal growth in rpb4. Steady-state protein levels of this mutant are stabilized in rpb47 strains, suggesting that the mutant is defective for interactions with key proteins involved in stress response. Analysis of five WW domain proteins in yeast (Rsp5, Ess1, Prp40, Wwm1, and Ssm4) revealed that these are not direct Rpb7 partners, highlighting that the molecular basis of the PY motif’s role in stress response remains unclear.

Interactions of Rpb4 and Rpb7 with Other RNA Polymerase II Subunits

Two-hybrid analysis detected direct interactions between Rpb4/7 mutants and RNA polymerase II subunits to link mutant phenotypes with polymerase interactions. Wild-type Rpb4 and Rpb7 interact weakly but significantly with Rpb1. No interactions were detected with Rpb3 and Rpb6.

Rpb7’s interaction with Rpb1 is mediated through both the N-terminal “clamp” head-forming regions and the linker and CTD domains of Rpb1. These observations are consistent with the recently released ~4 Å crystal structure of the 12-subunit polymerase (Armache et al., 2003; Bushnell and Kornberg, 2003), which predicts that Rpb7 in the Rpb4/Rpb7 complex is positioned to interact with both the clamp and linker regions. Both rpb7-7 and rpb7 P127A P128A mutants fail to interact with Rpb1, suggesting that the observed phenotypes may result from impaired polymerase interaction.

Summary

These results predict the importance of different regions of Rpb4 and Rpb7 for effective stress response and transcription in S. cerevisiae. These mutants can now be used to identify interacting partners for Rpb4 and Rpb7 involved in each specific phenotype