Recombinant Saccharomyces cerevisiae DNA-directed RNA polymerase I subunit RPA34 (RPA34)

What is the evolutionary conservation pattern of RPA34?

RPA34 is a nonessential subunit of RNA polymerase I that is conserved across species from yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) to humans . In human cells, the orthologous protein is known as PAF49, forming part of the Pol I complex. Despite sequence divergence, functional conservation appears strong, as demonstrated by complementation studies between different yeast species. The evolutionary retention of this subunit across diverse eukaryotes suggests its fundamental importance in optimizing ribosomal DNA transcription, despite being nonessential in laboratory growth conditions .

What are the key protein-protein interactions of RPA34?

RPA34 forms a heterodimer with another Pol I-specific subunit called RPA49. This interaction has been confirmed through multiple experimental approaches, including two-hybrid assays that revealed RPA34 binds specifically to an N-terminal region of RPA49 (between positions 63 and 119) . The interaction is functionally significant, as RPA34 is lost from RNA polymerase I in rpa49 mutants lacking this binding domain. Conversely, rpa34Δ weakens the binding of RPA49 to the RNA polymerase complex . The interdependence of these two subunits suggests they function as a structural and functional unit within Pol I, with RPA34 primarily acting to stabilize RPA49 in the polymerase structure .

What phenotypes are observed in RPA34 deletion mutants?

RPA34 deletion mutants (rpa34Δ) in S. cerevisiae show several distinctive phenotypes:

• They are viable with only a slight growth defect under standard conditions

• They display sensitivity to caffeine (trimethylxanthine)

• The rpa34Δ mutation becomes lethal when combined with certain other mutations, including:

  • top1Δ (topoisomerase I deletion)

  • rpa14Δ (deletion of another Pol I subunit)

  • rpa135(L656P) and rpa135(D395N) (specific mutations in the second-largest Pol I subunit)

Notably, overexpression of RPA49 suppresses all known phenotypic defects of rpa34Δ, suggesting that the primary function of RPA34 is to stabilize RPA49 within the Pol I complex .

How can researchers create and validate functional RPA34 mutants?

Creating functional RPA34 mutants requires precise molecular biology techniques. Based on established protocols, researchers should:

• Design deletion constructs: Target specific domains like the N-terminal region or the lysine-rich C-terminal domain. Studies have shown that truncated versions of RPA34 lacking the C-terminal domain remain functional, suggesting this domain is dispensable for core activities .

• Utilize homologous recombination: Replace the endogenous RPA34 gene with a selection marker (HIS3 has been successfully used) in haploid yeast strains .

• Complementation testing: Express mutant constructs from plasmids in rpa34Δ strains and assess rescue of phenotypes. When working with cross-species complementation, co-expression of the partner subunit (RPA49) may be necessary, as demonstrated with S. pombe RPA34 which could not complement S. cerevisiae rpa34Δ alone but could when co-expressed with S. pombe RPA49 .

• Validation assays:

  • Growth tests under various conditions (standard media, caffeine-containing media)

  • Test synthetic lethality with known interacting mutations (top1Δ, rpa14Δ, etc.)

  • Chromatin immunoprecipitation (ChIP) to assess incorporation into Pol I and recruitment to rDNA

What techniques are effective for studying RPA34's contribution to nucleolar structure?

Studying RPA34's role in nucleolar structure requires a multi-faceted approach:

How should researchers design experiments to study the RPA34-RPA49 heterodimer?

Effective experimental design for studying the RPA34-RPA49 heterodimer should include:

• Yeast two-hybrid assays: These have successfully mapped interaction domains, showing that RPA34 binds to an N-terminal region of RPA49 (between positions 63-119) .

• Co-immunoprecipitation: To confirm physical association in native conditions and test the impact of mutations.

• Domain deletion analysis: Creating truncated versions of both proteins to map functional domains. For example:

  • N-terminal deletions of RPA49 (rpa49-63,416, rpa49-89,416, rpa49-119,416)

  • C-terminal deletions of RPA49 (rpa49-1,260, rpa49-1,366)

  • Deletion of the lysine-rich C-terminal domain of RPA34

• Heterologous expression systems: Expressing the heterodimer in E. coli for biochemical and structural studies.

• Cross-species complementation: Testing whether heterodimers from different species can functionally substitute for each other, which has revealed that co-expression of both partners is often necessary for functional complementation .

How does the RPA34-RPA49 heterodimer affect RNA Polymerase I transcription cycles?

The RPA34-RPA49 heterodimer influences the RNA Polymerase I transcription cycle at multiple levels:

• Initiation complex formation: The heterodimer affects the recruitment of the essential transcription factor Rrn3 (TIF-IA in mammals) to the rDNA promoter. Mutations in RPA49 partially impair Rrn3 recruitment .

• Transition to elongation: One of the most critical functions of the heterodimer is facilitating the release of Rrn3 from the elongating polymerase. In rpa49 mutants, this release is strongly reduced, potentially causing defects in the transition from initiation to productive elongation .

• Elongation efficiency: The heterodimer promotes efficient elongation, as evidenced by the sensitivity of rpa49 mutants to elongation inhibitors like 6-azauracil and mycophenolate. In rpa49 mutants exposed to mycophenolate, Pol I almost completely dissociates from its rDNA template .

• Polymerase clustering: Analysis using Miller spreads has shown that the heterodimer promotes the clustering of elongating Pol I complexes on rRNA genes, which may optimize transcription through cooperative effects .

What is the significance of RPA34's lysine-rich C-terminal domain?

The lysine-rich C-terminal domain of RPA34 has been the subject of specific investigation:

• Dispensability for core function: Truncated versions of RPA34 lacking this domain remain functional in complementation tests, suggesting it is not essential for the basic functions of RPA34 in stabilizing RPA49 within Pol I .

• Species-specific functions: In higher eukaryotes, the orthologues of RPA34 appear to have additional specific functions mediated through this domain that are not present in yeast .

• Nucleolar localization: In budding yeast (S. cerevisiae), this domain may contribute to the ability of RPA34 to localize to the nucleolus independently of RPA49, a property not shared by its fission yeast (S. pombe) counterpart or the human orthologue (CAST) .

• Potential protein-nucleic acid interactions: The lysine-rich nature of this domain suggests possible interactions with nucleic acids, though this has not been conclusively demonstrated in the available research.

What factors influence RPA34 nucleolar localization in different species?

RPA34 nucleolar localization mechanisms vary significantly between species:

• In Saccharomyces cerevisiae: RPA34 localizes to the nucleolus even in the absence of RPA49, suggesting it contains an independent nucleolar localization signal .

• In Schizosaccharomyces pombe: The RPA34 orthologue (Sp-RPA34) requires RPA49 for proper nucleolar localization, indicating a dependency on the heterodimer formation .

• In human cells: CAST (the human orthologue) also requires the RPA49 counterpart (PAF53) for nucleolar localization .

This species-specific difference suggests evolutionary divergence in the mechanisms controlling nucleolar targeting of this Pol I subunit, with budding yeast evolving a more independent localization mechanism.

How can synthetic lethality screens with RPA34 be implemented and interpreted?

Synthetic lethality screens with RPA34 provide valuable insights into its functional network:

• Methodological approaches:

  • Global Genetic Interaction Mapping (GIM) has successfully identified genetic interactions of rpa34Δ

  • UV mutagenesis of rpa34::HIS3 strains transformed with RPA34-containing plasmids, followed by selection for mutants unable to lose the plasmid

  • Systematic crosses with deletion libraries followed by tetrad dissection

• Known synthetic lethal interactions:

  • top1Δ (topoisomerase I deletion)

  • rpa14Δ (deletion of another Pol I subunit)

  • rpa135(L656P) and rpa135(D395N) (specific mutations in Pol I subunit)

  • gcr2Δ and stb5Δ (transcription factors)

• Interpretation framework:

  • Synthetic lethality with other Pol I components (rpa14Δ, rpa135 mutations) suggests interdependent functions within the polymerase complex

  • Interactions with top1Δ point to a role in managing DNA topology during transcription

  • The fact that RPA49 overexpression suppresses all known synthetic lethal interactions of rpa34Δ indicates these interactions are mediated through effects on RPA49 function or stability

What is the relationship between RPA34 and topoisomerase functions?

The synthetic lethality between rpa34Δ and top1Δ (topoisomerase I deletion) reveals an important functional relationship:

• Topological stress management: Topoisomerase I relieves torsional stress generated during transcription. In its absence, the RPA34-RPA49 heterodimer becomes essential, suggesting it may help Pol I cope with topological challenges during transcription .

• Mechanistic hypotheses:

  • The heterodimer might stabilize Pol I during transcription through regions of high torsional stress

  • It could recruit or facilitate the function of alternative topoisomerases when Top1 is absent

  • It may optimize Pol I conformational states to better handle topological challenges

• Experimental evidence: Both rpa34Δ and rpa49Δ are synthetically lethal with top1Δ, but RPA49 overexpression can suppress the lethality of the rpa34Δ top1Δ combination, suggesting the critical factor is maintaining adequate RPA49 function when topoisomerase I is absent .

What are the optimal expression systems for producing recombinant RPA34 protein?

For optimal recombinant RPA34 production, researchers should consider:

• Prokaryotic expression:

  • E. coli BL21(DE3) with pET-based vectors has been used for expression of Pol I subunits

  • Co-expression with RPA49 is recommended for proper folding and solubility

  • Fusion tags like 6xHis, GST, or MBP can improve solubility and facilitate purification

• Yeast expression systems:

  • S. cerevisiae expression using GAL1-10 promoters allows native-like post-translational modifications

  • TAP-tagging strategies have been successfully employed for purification of RPA34-containing complexes

• Insect cell expression:

  • Baculovirus expression systems may better accommodate folding of eukaryotic proteins

  • Particularly valuable for structural studies requiring higher protein yields

• Purification considerations:

  • RPA34 is often most stable when purified as part of the heterodimer with RPA49

  • Affinity chromatography followed by size exclusion has proven effective for isolating the heterodimer

How can researchers effectively study cross-species complementation with RPA34?

Cross-species complementation studies with RPA34 require careful experimental design:

• Vector selection:

  • Shuttle vectors that function in multiple yeast species

  • Plasmids with appropriate selectable markers and copy number

• Expression control:

  • Using native promoters may better recapitulate physiological expression levels

  • When testing heterologous proteins, consider co-expression of the partner subunit (RPA49)

• Complementation testing protocol:

  • Create clean deletion mutants in the recipient species

  • Transform with plasmids expressing the heterologous RPA34

  • Test growth under various conditions including standard media, low temperature, and caffeine-containing media

  • For borderline cases, test synthetic lethality rescue with known interacting mutations

• Demonstrated examples:

  • S. pombe RPA34 alone cannot complement S. cerevisiae rpa34Δ, but the co-expressed S. pombe RPA34/RPA49 heterodimer can restore growth

  • This suggests that species-specific co-evolution of the heterodimer components must be considered in complementation studies

What approaches are most effective for analyzing RPA34's contribution to nucleolar assembly?

To analyze RPA34's role in nucleolar assembly, researchers should employ:

• Live cell imaging:

  • Fluorescently tagged nucleolar markers (Nop1, fibrillarin) to visualize nucleolar morphology

  • Time-lapse microscopy to capture dynamic changes during nucleolar assembly/disassembly

  • Quantitative image analysis to measure nucleolar size, shape, and signal intensity

• Biochemical fractionation:

  • Nucleolar isolation protocols to assess composition changes in the absence of RPA34

  • Proteomic analysis of isolated nucleoli to identify proteins whose localization depends on RPA34

• Structured illumination microscopy (SIM) or STED:

  • Super-resolution approaches to visualize detailed nucleolar substructures

• Inducible depletion systems:

  • Auxin-inducible degron tagging of RPA34 to study immediate effects of its removal on nucleolar integrity

  • This approach can distinguish direct versus indirect effects on nucleolar structure

• Electron microscopy techniques:

  • Cryoimmobilization followed by low-temperature substitution for optimal preservation of nucleolar ultrastructure

  • These methods have already revealed that both rpa34Δ and rpa49Δ mutations alter nucleolar ultrastructure in distinct ways