Functional analysis of RPB4 and RPB7 subunits of RNA polymerase II in saccharomyces cerevisiae

Abstract

Eukaryotic RNA Polymerases: Roles of RPB4 and RPB7 Subunits in Saccharomyces cerevisiae

Eukaryotic RNA polymerases are complex multi-subunit enzymes that play a key role in regulating gene expression in response to both internal and external stimuli. Over the past several years, extensive research has focused on identifying proteins that associate with these polymerases and the mechanisms by which they regulate gene expression. However, our understanding of the polymerase itself remains limited. For instance, the functions of many smaller subunits and their modes of interaction with general transcription factors (GTFs) or gene-specific activator proteins are still largely unknown.

We have chosen Saccharomyces cerevisiae as the model system and initiated studies to understand the functions of two subunits of RNA polymerase II. RNA polymerase II purified from S. cerevisiae consists of 12 subunits, namely RPB1 to RPB12, ranging in molecular weight from 220 kDa to 10 kDa. Five of these subunits—RPB5, RPB6, RPB8, RPB10, and RPB12—are shared by all three RNA polymerases. All subunits, except RPB4 and RPB9, are essential for yeast cell viability.

The two largest subunits, RPB1 and RPB2, are functional homologs of the Escherichia coli RNA polymerase and subunits, respectively. Two subunits, RPB3 and RPB11, are considered homologs of the subunit of the bacterial polymerase. Subunit RPB9 is required for accurate start site selection by the polymerase (Hull et al., 1995). Recently, RPB3 and RPB5 have been shown to play roles in transcriptional activation (Tan et al., 2000; Miyao et al., 1998).

RPB4 is required for normal growth at moderate temperatures and is indispensable for growth under extreme temperatures. Yeast cells lacking RPB4 grow slowly at permissive temperatures and cease growth at high or low temperatures (Woychik and Young, 1989). The RPB7 subunit is essential for cell survival (McKune et al., 1993). Biochemical and genetic evidence suggests that RPB4 and RPB7 form an easily dissociable sub-complex within the polymerase. Both subunits are present at sub-stoichiometric levels relative to the rest of the polymerase during exponential growth, and this stoichiometry increases to 1.0 during the stationary phase (Choder and Young, 1993). This sub-complex has been implicated in the stress response and in the initiation of transcription in vitro (Woychik and Young, 1989; Edwards et al., 1991; Choder and Young, 1993).

Objectives

The objectives of the current study were:

To test whether a major function of Rpb4p is to stabilize the interaction of Rpb7p with the rest of the polymerase.

To elucidate the functions of RPB4 and RPB7 in RNA polymerase II-mediated transcription by: (a) Analyzing the effect of RPB4 on transcription from constitutive and inducible promoters. (b) Studying the effect of overexpressing cognate transcriptional activators on their respective promoter activities in the absence of RPB4. (c) Determining the effect of varying levels of RPB7 and the RPB7 dominant mutant on the activity of inducible promoters. (d) Testing the interaction of different transcriptional activators with RPB7 using Yeast Two-Hybrid Analysis.

To examine the effect of RPB4 and RPB7 on global gene transcription by identifying transcripts that are differentially expressed in the presence of RPB4 or varying levels of RPB7 in an RPB4 deletion mutant.

Results 1. Effect of Overexpression of RPB7 in RPB4 Deletion Mutant

RPB4 and RPB7 form a heterodimer that can dissociate from the rest of the polymerase under mild denaturing conditions. RNA polymerase II purified from cells lacking RPB4 does not contain detectable levels of RPB7 associated with the polymerase (Edwards et al., 1991). This suggests that one function of Rpb4p is to stabilize the otherwise weak interaction of Rpb7p with the polymerase.

We examined the effect of varying levels of RPB7 on phenotypes characteristic of an RPB4 null mutation, such as slow growth and heat/cold sensitivity. Growth curves and temperature-sensitivity assays showed that both slow growth and temperature-sensitive phenotypes were rescued by RPB7 in a dose-dependent manner. A dominant mutant of RPB7 partially rescued the temperature-sensitive phenotype at 34°C but not at 37°C. However, neither RPB7 overexpression nor the dominant mutant could support growth at 12°C, indicating that RPB4 is indispensable for growth at low temperatures.

Interestingly, overexpression of RPB7 in the absence of RPB4 caused elongation of haploid yeast cells and a shift in the budding pattern from axial to predominantly unipolar. The presence of RPB4 restored normal morphology and budding, suggesting that a specific RPB4:RPB7 ratio governs the transcription of different gene classes.

2. Functions of RPB4 and RPB7 in RNA Polymerase II-Mediated Transcription

(a) Effect of RPB4 on constitutive and inducible promoters

The transcriptional activity of four constitutive promoters (ACT1, ADH1, GPD1, TEF2) and six inducible promoters (INO1, GAL1, GAL10, HSE, PHO5, CUP1) was measured using lacZ fusions. RPB4 had minimal effect on constitutive promoters but was necessary for transcription from most inducible promoters.

(b) Effect of overexpressing transcriptional activators

Overexpression of transcriptional activators in RPB4 deletion mutants rescued transcriptional deficiencies at their respective inducible promoters, suggesting that these activators may interact directly or indirectly with the Rpb4p/Rpb7p sub-complex to recruit polymerase.

(c) Effect of varying levels of RPB7 and its dominant mutant

Increasing RPB7 dosage or expressing the RPB7 dominant mutant could not suppress the activation defect at inducible promoters in the RPB4 deletion mutant, despite rescuing temperature-sensitive and slow-growth phenotypes.

(d) Interaction of Rpb7p with transcriptional activators

Yeast Two-Hybrid analysis indicated that Rpb7p did not directly interact with the activators Ino2p, Ino4p, or Gal4p.

3. Effect on Global Gene Transcription

RNA Arbitrary Primed PCR (RAP-PCR) was used to assess transcriptional changes in RPB4 deletion mutants with varying RPB7 levels. Some genes showed similar expression across strains, while others were differentially expressed depending on RPB4 presence or RPB7 levels. Nine RAP-PCR products were cloned and partially sequenced. Northern and dot-blot analyses confirmed the expression patterns. Comparison with the Saccharomyces cerevisiae genome database revealed homology to a nucleolin-like protein, CAP2, an actin-bundling protein, and GPD2.

Conclusions

Rpb4p functions:

Stabilizes the interaction of Rpb7p with the polymerase.

May interact (directly or indirectly) with transcriptional activators, explaining its essential role in inducible transcription.

Rpb7p functions:

Plays an independent role in regulating transcription of specific genes, including those involved in agar-invasive growth of haploids.

Sub-complex regulation:

RPB4 and RPB7 regulate polymerase activity as a sub-complex under both permissive and stress conditions.

They may also independently regulate transcription of different gene classes.

References

Choder, M., & Young, R. A. (1993). A portion of RNA polymerase II molecules has a component essential for stress response and stress survival. Molecular and Cellular Biology, 13, 6984–6991.

Edwards, A. M., Kane, C. M., Young, R. A., & Kornberg, R. D. (1991). Two dissociable subunits of yeast RNA polymerase II stimulate initiation of transcription at a promoter in vitro. J. Biol. Chem., 266, 71–75.

Hull, M. W., McKune, K., & Woychik, N. A. (1995). RNA polymerase II subunit RPB9 is required for accurate start site selection. Genes & Development, 9, 481–490.

McKune, K., Richards, K. L., Edwards, A. M., Young, R. A., & Woychik, N. A. (1993). RPB7, one of the two dissociable subunits of yeast RNA polymerase II, is essential for cell viability. Yeast, 9, 295–299.

Miyao, T., & Woychik, N. A. (1998). RNA polymerase subunit RPB5 plays a role in transcriptional activation. PNAS USA, 95, 15281–15286.

Tan, Q., Linask, K. L., Ebright, R. H., & Woychik, N. A. (2000). Activation mutants in yeast RNA polymerase II subunit RPB3 provide evidence for a structurally conserved surface required for activation in eukaryotes and bacteria. Genes & Development, 14, 339–348.

Woychik, N. A., & Young, R. A. (1989). RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth. Molecular and Cellular Biology, 9, 2854–2859.