Recombinant Picrophilus torridus tRNA pseudouridine synthase Pus10 (pus10)

What distinguishes Pus10 from other pseudouridine synthases?

Pus10 belongs to a unique class of pseudouridine synthases that does not align with any of the five commonly identified families of Ψ synthases . The primary distinction of archaeal Pus10 enzymes is their ability to catalyze pseudouridylation at both positions 54 and 55 in tRNA molecules, while bacterial TruB and yeast Pus4 modify only position 55 . Furthermore, archaeal Pus10 does not require specific structural features like the U54- A58 reverse Hoogstein base pair and pyrimidine at position 56 that are essential for bacterial and yeast enzymes to convert tRNA U55 to Ψ55 .

The distribution of Pus10 homologs correlates precisely with the presence of Ψ54 in tRNAs - both are found in nearly all sequenced archaeal genomes and some higher eukaryotes, but are absent in yeast and bacteria . This makes Pus10 the first identified tRNA Ψ54 synthase, filling a significant gap in our understanding of RNA modification enzymes.

How might the extreme acidophilic nature of P. torridus influence its Pus10 enzyme properties?

As an archaeon that thrives at extraordinarily low pH values (optimum pH ~0.7), P. torridus likely possesses a Pus10 enzyme with distinctive adaptations to acidic conditions. These adaptations could include:

• Altered amino acid composition with increased proportion of acidic residues on the protein surface

• Unique folding characteristics that maintain structural integrity under acidic conditions

• Modified catalytic mechanism optimized for function at low pH

• Potentially different salt concentration dependencies compared to Pus10 from neutralophilic archaea

The influence of pH on pseudouridylation activity would be a critical parameter to investigate, particularly in comparison with the salt-dependent activity variations observed in M. jannaschii and P. furiosus Pus10 proteins . Experimental protocols should be designed to accommodate testing at pH values ranging from 0.7 (the organism's optimum) to neutral pH for comparative analyses.

What are optimal protocols for expression and purification of recombinant P. torridus Pus10?

Successful expression and purification of P. torridus Pus10 would likely require adaptations to standard protocols used for other archaeal proteins:

• Expression system selection:

  • Codon-optimized gene synthesis for expression in E. coli

  • Consideration of archaeal expression systems for proper folding

  • Use of thermostable vectors with strong promoters (T7 or tac)

• Expression conditions:

  • Lower induction temperatures (16-25°C) to enhance proper folding

  • Extended expression time (16-24 hours)

  • Supplementation with rare tRNAs if using E. coli

• Purification strategy:

  • Initial heat treatment (60-65°C) to eliminate many E. coli proteins

  • Affinity chromatography using His-tag or other fusion tags

  • Ion exchange chromatography at acidic pH conditions

  • Size exclusion chromatography for final purification

• Buffer optimization:

  • Testing various pH ranges (3.0-7.5) for optimal stability

  • Inclusion of glycerol (10-20%) to enhance stability

  • Evaluation of salt concentration effects (300-500 mM)

Storage conditions should be carefully optimized, as protein from acidophiles may exhibit different stability profiles than those from neutrophilic organisms.

What assays are recommended for characterizing the pseudouridylation activity of P. torridus Pus10?

Several complementary approaches can be employed to comprehensively characterize P. torridus Pus10 activity:

• Radioisotope-based assays:

  • Preparation of [α-32P]UTP-labeled tRNA substrates via in vitro transcription

  • Incubation with purified P. torridus Pus10 under varied conditions

  • Analysis of pseudouridylation by nuclease P1 digestion followed by thin-layer chromatography (TLC)

  • Quantification via phosphorimaging

• Site-specific analysis:

  • Nearest-neighbor analysis using RNase T2 digestion of [α-32P]UTP-labeled tRNAs to specifically identify Ψ54 formation

  • CMCT-primer extension assays to map pseudouridylation sites precisely

• Activity parameter assessment:

• Substrate specificity testing:

  • Full-length tRNAs

  • Truncated tRNAs (3'-half)

  • Isolated TΨC-arm (stem-loop)

  • Variant substrates with mutations at key positions

These assays should be performed under varying conditions to establish the unique characteristics of P. torridus Pus10 compared to other archaeal homologs.

How do salt concentration effects on P. torridus Pus10 compare with other archaeal Pus10 enzymes?

• Total pseudouridylation activity (Ψ54 + Ψ55) should be measured across a salt gradient (150-900 mM NaCl)

• Site-specific activity for Ψ54 and Ψ55 formation should be determined separately

• Results should be compared with the documented patterns for other archaeal Pus10 proteins

Anticipated patterns based on homologous proteins:

P. torridus Pus10 might show a unique salt response profile reflecting its adaptation to extremely acidic environments, particularly if intracellular salt concentrations are involved in pH homeostasis mechanisms.

Can P. torridus Pus10 modify minimal RNA substrates, and how does this compare to other archaeal Pus10 proteins?

Research on M. jannaschii Pus10 has demonstrated that it can modify not only full-length tRNAs but also smaller RNA fragments containing just the TΨC-arm (stem-loop) . This raises important questions about substrate recognition:

• Testing minimal substrates:

  • The 17-base RNA containing the TΨC-stem-loop should be evaluated as a substrate for P. torridus Pus10

  • Activity comparison between full-length tRNA and the minimal substrate

  • Assessment of relative efficiency for Ψ54 versus Ψ55 formation in the minimal substrate

• Comparative analysis with other archaeal Pus10:

  • M. jannaschii Pus10 shows differential efficiency for Ψ54 versus Ψ55 in minimal substrates, with Ψ55 formation being more efficient

  • Salt concentration effects are more pronounced for Ψ54 formation in minimal substrates

  • Whether P. torridus Pus10 follows similar patterns would provide insights into evolutionary conservation of recognition mechanisms

What structural features of P. torridus Pus10 might account for its dual specificity for U54 and U55?

The dual specificity of archaeal Pus10 for both U54 and U55 represents a fascinating case of enzyme evolution. Based on comparative studies, several structural elements likely contribute to this capability:

• The catalytic domain likely contains a conserved aspartate residue essential for pseudouridylation activity

• The N-terminal domain may contain specific residues that influence substrate recognition and positioning

• The "forefinger loop" structure is likely involved in RNA binding and specificity determination

Sequence comparison with other archaeal Pus10 proteins would reveal whether P. torridus Pus10 contains the full complement of residues found in M. jannaschii Pus10 or if it lacks certain elements like P. furiosus Pus10, which could explain potential differences in activity .

Experimental approaches to investigate structure-function relationships would include:

• Site-directed mutagenesis of predicted key residues

• Truncation analysis to determine the contribution of different domains

• Homology modeling based on known structures of related proteins

• Crystallographic studies if possible

How might the independent nature of Ψ54 and Ψ55 formation be explained mechanistically?

Studies of archaeal Pus10 proteins indicate that formation of Ψ54 and Ψ55 appear to occur independently . This raises mechanistic questions about how a single enzyme can catalyze modifications at adjacent positions:

• Possible mechanisms include:

  • Sequential binding events with conformational changes between modifications

  • Different binding modes for the two target uridines

  • Distinct catalytic residues or binding pockets for each position

• Experimental approaches to investigate:

  • Kinetic analysis to determine if one modification precedes the other

  • Substrate variants with either U54 or U55 mutated to assess independence

  • Protein variants with mutations affecting activity at one position but not the other

Understanding this mechanism in P. torridus Pus10 would provide insights into how RNA-modifying enzymes achieve site-specificity and could reveal unique adaptations related to the extremophilic nature of the source organism.

What can comparative analysis of P. torridus Pus10 with other archaeal homologs reveal about adaptation to extreme environments?

P. torridus represents one of the most extreme acidophiles known, with an optimal growth pH of approximately 0.7. Comparative analysis of its Pus10 with homologs from archaea adapted to different extreme conditions can reveal evolutionary strategies for enzyme adaptation:

A comprehensive comparative study would involve:

• Sequence alignment and phylogenetic analysis

• Comparison of activity profiles under various conditions

• Identification of clade-specific insertions or deletions

• Correlation of enzymatic properties with environmental adaptations

What is the in vivo significance of Pus10-catalyzed pseudouridylation in P. torridus?

The biological significance of Ψ54 and Ψ55 in the tRNAs of an extreme acidophile like P. torridus raises intriguing questions:

• Potential functional roles:

  • Enhancement of tRNA structural stability under acidic conditions

  • Modification of tRNA-ribosome interactions in acidic cytoplasm

  • Influence on codon-anticodon interactions in extreme conditions

• Possible experimental approaches:

  • Gene knockout or depletion studies if genetic systems are available

  • Structural studies of modified versus unmodified tRNAs under acidic conditions

  • Translational efficiency assays comparing native and in vitro transcribed tRNAs

In archaeal systems, there appears to be some redundancy in Ψ55 formation mechanisms, with both Pus10 and Cbf5 (in complex with accessory proteins) capable of this modification . This raises questions about whether P. torridus employs both systems and whether their relative importance differs from other archaea.