Recombinant Human GTPase ERas (ERAS)

What is GTPase Era and what is its primary function in human cells?

GTPase Era is a deeply conserved protein critically required for the assembly of bacterial-type ribosomes from Escherichia coli to humans. It functions as a key assembly factor in ribosome biogenesis, particularly for the small subunit (SSU). Era intervenes relatively early in small subunit biogenesis and is essential for the proper shaping of the ribosomal platform, which is a prerequisite for efficient translation . In humans, the homolog ERAL1 performs similar functions in mitochondrial ribosome assembly, and deficiencies in this protein are associated with pathological conditions including Perrault syndrome .

What domains are present in GTPase Era and what are their functions?

GTPase Era contains two primary functional domains:

• GTPase domain: Contains the typical motifs (G1-G5) found in GTPases and is responsible for binding and hydrolyzing GTP. This activity is critical for Era's function, as mutations in this domain (particularly in the G1 motif) render the protein inactive .

• KH domain: An RNA-binding domain that interacts with the 3′-minor domain of the small subunit ribosomal RNA (SSU rRNA). This domain is required for cellular function of Era, as mutations in the helix-turn-helix motif or in the h45-interacting loops show severe loss-of-function phenotypes in various species .

Both domains are essential for Era's function, and they appear to coordinate their activities to time Era's intervention in the ribosome biogenesis pathway.

How is GTPase Era involved in ribosomal assembly?

GTPase Era plays a critical role in the assembly of the 30S ribosomal subunit. It functions as a hub protein on the ribosome to direct enzymes involved in rRNA processing/degradation and ribosome subunit assembly to their site of action . In its GTP-bound form, Era associates with the immature 30S ribosomal subunit . Following GTP hydrolysis, Era dissociates from the ribosome, presumably after facilitating a key maturation event . This dynamic association and dissociation are essential for proper ribosome assembly and maturation.

What experimental techniques are most effective for studying recombinant ERAS protein interactions?

Based on published research, several effective techniques for studying ERAS protein interactions include:

• Protein interaction studies: Using techniques such as co-immunoprecipitation and pull-down assays to identify interacting partners. These approaches have revealed that Era interacts with proteins such as the 16S rRNA endonuclease YbeY and the DEAD-box RNA helicase CshA .

• X-ray crystallography: This method has been crucial for determining the structural conformations of Era in different nucleotide-bound states (apo-, GDP-bound, GTP-bound), revealing how the GTPase domain influences the orientation of the KH domain .

• In vitro activity assays: Measuring the GTPase activity of purified recombinant Era proteins under various conditions and in the presence of potential regulatory factors. For example, assays have shown that the stringent response alarmone (p)ppGpp can inhibit Era's GTPase activity, while the protein Rel_Sau can enhance it .

• Cryo-EM studies: Recent advances have allowed visualization of Era in complex with ribosomal assembly intermediates, providing insights into its binding position and conformational states .

How can researchers effectively express and purify recombinant human ERAS for functional studies?

While the search results don't provide specific purification protocols for human ERAS, the following methodological approach can be derived from existing research on GTPase Era proteins:

• Expression system selection: Bacterial expression systems (particularly E. coli) have been successfully used for expressing recombinant Era proteins from various species.

• Affinity tagging: Using affinity tags (such as His-tags) to facilitate purification while ensuring minimal interference with protein function.

• Quality control: Assessing protein folding and activity through GTPase activity assays is essential, as proper folding is critical for the coordination between the GTPase and KH domains .

• Nucleotide considerations: Since Era's conformation and activity are influenced by nucleotide binding (GTP vs. GDP), researchers should carefully consider the nucleotide state of the purified protein for subsequent functional studies .

How do guanosine nucleotides regulate Era's function in ribosome assembly?

The relationship between guanosine nucleotides and Era's function appears to be complex:

• Conformational changes: X-ray crystallographic studies have shown that apo- and GDP-bound Era demonstrate a rotated conformation of the KH domain, where the negatively charged helix αD partially blocks access to the RNA-binding groove. In contrast, GTP-bound Era reorients the KH domain to allow unimpeded RNA access .

• Contradictory observations: Interestingly, some in vitro studies show that only apo-Era significantly binds to 16S rRNA and mature SSU, while GDP or GTP addition abolishes these interactions . Recent structural studies of mitochondrial SSU assembly intermediates found ERAL1 (human Era homolog) in a nucleotide-free form with a conformation resembling the activated state .

• Recruitment hypothesis: Evidence suggests Era may be naturally recruited to nascent SSU in the apo-state and acquires GTP later during assembly .

• Stringent response regulation: The stringent response alarmone (p)ppGpp binds to Era with higher affinity than GTP, inhibiting its GTPase activity . This represents an additional regulatory mechanism linking ribosome assembly to cellular stress responses.

This complex regulation by nucleotides suggests that Era functions as a molecular switch in ribosome assembly, with its activity carefully timed through interactions with different guanosine nucleotides.

What are the known protein interaction partners of ERAS and their functional significance?

Research has identified several important interaction partners for GTPase Era:

• YbeY: Era interacts with this 16S rRNA endonuclease, which plays a role in rRNA processing. This interaction likely facilitates YbeY's activity at the appropriate site on the pre-ribosomal particle .

• CshA: Era interacts with this DEAD-box RNA helicase involved in rRNA processing. Both Era and CshA are required for growth at suboptimal temperatures, suggesting a functional relationship in cold adaptation .

• Rel_Sau: Era forms direct interactions with this (p)ppGpp synthetase. Interestingly, Rel_Sau positively impacts the GTPase activity of Era but negatively affects the helicase activity of CshA . This suggests a mechanism by which the stringent response can regulate ribosome assembly at multiple points.

These interactions support the model that Era acts as a hub protein on the ribosome, recruiting and coordinating the activities of multiple factors involved in ribosome assembly and rRNA processing.

How does the stringent response affect ERAS function and ribosome assembly?

The stringent response, mediated by the alarmone (p)ppGpp, affects ERAS function and ribosome assembly through multiple mechanisms:

• Direct inhibition: (p)ppGpp directly binds to Era with higher affinity than GTP, inhibiting its GTPase activity . This inhibition may prevent proper functioning of Era in ribosome assembly.

• Protein interactions: The (p)ppGpp synthetase Rel_Sau forms direct interactions with both Era and CshA. Rel_Sau has a positive effect on the GTPase activity of Era, which could promote premature dissociation of Era from immature 30S ribosomal subunits .

• Inhibition of CshA: Rel_Sau negatively affects the helicase activity of CshA, which may delay 17S rRNA processing or degradation, especially at lower temperatures (25°C) .

• Processing defects: Activation of the stringent response results in increased rRNA processing defects, particularly at lower temperatures .

These findings suggest that the stringent response can impact RA-GTPase function through direct interactions of Rel with both Era and CshA, contributing to the slowed growth phenotype characteristic of this stress response pathway .

What phenotypes are associated with ERAS deficiency or mutation?

Research has revealed several significant phenotypes associated with Era deficiency or mutation:

What are the most common pitfalls when designing experiments to study ERAS function?

Based on research findings, several methodological considerations should be kept in mind when studying ERAS:

• Nucleotide state considerations: The nucleotide-bound state of Era (apo-, GDP-bound, or GTP-bound) significantly impacts its conformation and interactions. Experiments should carefully control and consider the nucleotide status of the protein .

• Domain interdependence: The GTPase and KH domains of Era function interdependently. Studies focusing on only one domain may yield misleading results, as evidenced by the toxicity of KH domain overexpression in the absence of the GTPase domain .

• Temperature sensitivity: Era function appears particularly important at suboptimal temperatures. Experiments conducted at standard laboratory temperatures may miss important phenotypes that would be evident at lower temperatures .

• Complex interaction networks: Era interacts with multiple partners in ribosome assembly. Studying Era in isolation may not capture the full complexity of its in vivo function .

• Stringent response interactions: The function of Era is intricately linked to the stringent response pathway. Experimental conditions that activate or suppress this pathway may indirectly affect Era function .

How can researchers differentiate between direct and indirect effects of ERAS in experimental systems?

To differentiate between direct and indirect effects of ERAS, researchers can employ the following methodological approaches:

• In vitro reconstitution experiments: Using purified components to test direct biochemical activities and interactions.

• Structure-based mutational analysis: Creating specific mutations in Era based on structural information to disrupt particular functions or interactions while preserving others. For example, mutations in the KH domain's helix-turn-helix motif or in the h45-interacting loops can specifically disrupt RNA binding .

• Temporal analysis: Tracking the timing of effects after Era depletion or mutation can help distinguish primary (direct) from secondary (indirect) effects.

• Complementation studies: Testing whether wild-type Era can rescue mutant phenotypes, and using domain-specific mutants to determine which functions are essential for complementation.

• Correlation with biochemical activities: Measuring whether the severity of in vivo phenotypes correlates with specific biochemical activities (GTPase activity, RNA binding) across a panel of mutants.

What are the emerging techniques for studying ERAS dynamics in living cells?

Though not explicitly covered in the search results, based on current research approaches with Era, emerging techniques likely include:

• Fluorescence microscopy techniques: Methods such as Fluorescence Recovery After Photobleaching (FRAP) or single-molecule tracking could be used to monitor the dynamics of fluorescently tagged Era in living cells.

• Proximity labeling approaches: Techniques like BioID or APEX could be employed to identify proteins that transiently interact with Era under different conditions.

• Cryo-electron tomography: This technique could potentially visualize Era in the context of assembling ribosomes within intact cells.

• Time-resolved structural studies: Capturing Era at different stages of ribosome assembly to understand the dynamic conformational changes that occur during this process.

How does ERAS function change under various cellular stress conditions?

Research has shown that ERAS function is significantly affected by stress conditions, particularly:

• Nutritional stress/stringent response: The stringent response alarmone (p)ppGpp binds to Era with higher affinity than GTP, inhibiting its GTPase activity . Additionally, the (p)ppGpp synthetase Rel_Sau directly interacts with Era, potentially affecting its association with the ribosome .

• Cold stress: Era is particularly important for growth at suboptimal temperatures, with Era-deficient cells showing more pronounced defects under cold conditions . The activation of the stringent response results in increased rRNA processing defects particularly at lower temperatures (25°C) .

• Regulatory adaptations: Era is subject to sophisticated regulatory mechanisms at the transcriptional, post-transcriptional, and post-translational levels in response to various cellular conditions .

These findings suggest that Era function is dynamically regulated in response to stress conditions, potentially as part of a broader cellular strategy to modulate protein synthesis in accordance with environmental challenges.