Recombinant Xenopus laevis Cysteine--tRNA ligase, cytoplasmic (cars), partial

What is the genomic organization of cysteine tRNA genes in Xenopus laevis?

Xenopus laevis contains multiple cysteine tRNA genes organized in a distinctive genomic structure. Detailed characterization has revealed that a 1737 bp DNA fragment contains two cysteine tRNA genes with the anticodon 5'-GCA-3', oriented in the same direction . These sequences show 95.8% homology between their mature tRNA coding regions, differing by only 3 bp .

Several key features of these genes include:

• Independent transcription units that can be transcribed separately

• Absence of intervening sequences within the genes

• A 452 bp spacer DNA separating the two genes

This genomic arrangement appears to be part of a tandemly repeated DNA structure. While earlier reports suggested a 3.18 kb tandemly repeated fragment containing eight tRNA genes, research indicates that the characterized 1.74 kb fragment represents approximately 96% of a 1.85 kb tandem repeat, suggesting some repeats may contain as few as two tRNA genes .

What methodologies are most effective for cloning and expressing recombinant Xenopus laevis CARS?

For cloning tRNA-related genes from Xenopus laevis, the following methodological approach has proven effective:

• DNA isolation and enrichment:

  • Separate tRNA gene-enriched DNA fractions from genomic DNA using CsCl density gradient centrifugation in the presence of actinomycin D

  • Detect tRNA genes through Southern blot analysis after restriction enzyme digestion (e.g., EcoRI)

  • Hybridize with kinase-labeled tRNA to identify gene-containing fragments

• Cloning strategy:

  • Separate DNA fragments by size on 1% agarose gel

  • Elute fragments of appropriate size (e.g., 1600-1800 bp for tRNA genes)

  • Clone into an appropriate plasmid vector (e.g., pBR325 or pUC8)

• Expression verification:

  • Identify clones that hybridize to labeled tRNA

  • Verify production of tRNA-sized products through in vitro transcription using Xenopus S-100 cell extract

For expressing functional aminoacyl-tRNA synthetases, protocols similar to those used for PylRS in Xenopus genetic code expansion systems could be adapted, involving transcription of capped mRNA using the mMessage mMachine in vitro Transcription Kit with a pCS2 vector template containing a 3' SV40 polyA signal .

How can researchers verify the aminoacylation activity of recombinant CARS?

A systematic approach to verify aminoacylation activity includes:

• In vitro aminoacylation assay:

  • Incubate purified recombinant CARS with ATP, [³⁵S]-cysteine, and isolated tRNA^Cys

  • Measure tRNA charging by acid precipitation and scintillation counting

  • Include controls with denatured enzyme and non-cognate tRNAs

• Functional complementation:

  • Test if recombinant CARS can rescue CARS-deficient cellular systems

  • Measure protein synthesis rates in the presence of the recombinant enzyme

• Reporter systems:

  • Similar to luciferase reporter systems used for UAA incorporation, develop specialized reporters with cysteine-dependent luminescence

What are the key features of tRNA^Cys gene transcription in Xenopus laevis?

Transcription of tRNA^Cys genes in Xenopus laevis follows the general eukaryotic tRNA gene transcription pattern:

• Transcription machinery:

  • RNA polymerase III (molecular weight ~700,000 with at least 10 subunits) is responsible for tRNA gene transcription

  • Initiation occurs within 20 bases upstream from the mature tRNA coding sequence

  • Transcription starts with a purine nucleoside triphosphate at a site preceded by a pyrimidine nucleotide on the non-coding strand

• Transcription control elements:

  • Two internal promoter elements (A and B blocks) containing conserved sequences

  • Interaction between sequences in the D and TψCG loops forms a tRNA-like structure in DNA that is recognized by transcription factors

  • Only a few nucleotides in the A and B blocks are necessary for transcription factor binding

  • Some B block sequences function as regions of dyad symmetry

• Experimental evidence:

  • Deletion of either 5' or 3' half of a X. laevis tRNA^Met gene prevents transcription

  • The 3' half can inhibit transcription of reference genes by competing for transcription factors

  • Substitution of nucleotide C56 (involved in tertiary interactions) severely reduces transcription

How does post-transcriptional processing of tRNA^Cys occur?

The post-transcriptional processing pathway for tRNA^Cys in Xenopus includes:

• Initial transcript processing:

  • Primary transcripts contain 5' leader and 3' trailer sequences

  • The 5' and 3' trailer sequences are removed in stepwise fashion

• Modification and maturation:

  • Base modifications occur simultaneously with trailer removal

  • Addition of the CCA end occurs in the nucleus (tRNA genes do not encode this sequence)

  • For tRNAs with intervening sequences (not present in the characterized cysteine tRNA genes), excision occurs either in the nucleoplasm or at the nuclear membrane

• Cellular transport:

  • Mature tRNA is transported to the cytoplasm for protein synthesis

  • Unlike in some yeast species where dimeric precursors have been found, multimeric precursors have not been observed in higher eukaryotes including Xenopus

What molecular interactions govern CARS recognition of its cognate tRNA^Cys?

Recognition of tRNA^Cys by CARS involves specific structural features:

How can CARS be engineered for genetic code expansion applications?

Engineering CARS for genetic code expansion would follow methodological principles similar to those used with other aminoacyl-tRNA synthetases:

• Engineering strategy:

  • Modify the amino acid binding pocket to accommodate cysteine analogs

  • Maintain tRNA recognition capabilities

  • Screen synthetase libraries for variants that selectively charge tRNA with unnatural amino acids

• Implementation in Xenopus:

  • Generate mRNA for engineered CARS and target protein with amber stop codon

  • Transcribe cognate tRNA with CUA anticodon

  • Inject a mixture containing CARS mRNA (~250 pg), tRNA (~7.5 ng), target protein mRNA (~250 pg), and unnatural amino acid into one-cell stage embryos

• Delivery optimization:

  • For cysteine analogs with phenylalanine-like structures, incubation in media (1 mM) may be effective

  • For more polar analogs, direct injection (10-50 mM in injection solution) would likely be required

What unnatural cysteine analogs could be incorporated using engineered CARS?

Potential unnatural cysteine analogs for incorporation include:

Incorporation efficiency would likely vary based on structural similarity to native cysteine, with efficiency measurements possible using luciferase reporter systems similar to those used for other UAAs in Xenopus .

How does the efficiency of genetic code expansion in Xenopus compare to other model systems?

Xenopus laevis demonstrates exceptional efficiency for genetic code expansion:

• Comparative efficiency data:

  • Alloc-protected lysine showed 1714-fold increase in expression in Xenopus vs. 217-fold in zebrafish

  • Photocaged lysine showed 339-fold increase in Xenopus vs. 70-fold in zebrafish

• Contributing factors:

  • High protein production capacity of Xenopus embryos

  • Excellent orthogonality of the PylRS system in Xenopus (very low background activity)

  • Efficient transport mechanisms for certain amino acid analogs

• Optimization parameters:

  • UAA concentration: minimum 10 mM in injection solution (50 pmol total)

  • RNA amounts: 250 pg each of synthetase and target protein mRNA, 7.5 ng tRNA

  • Delivery method based on amino acid backbone structure (phenylalanine backbone facilitates transport from media)

What are common issues in CARS expression and purification, and how can they be resolved?

Common challenges and solutions include:

• Protein solubility issues:

  • Problem: CARS forming inclusion bodies during expression

  • Solution: Express at lower temperatures (16-18°C), use solubility tags (MBP, SUMO), or optimize buffer conditions with stabilizing additives

• Low enzymatic activity:

  • Problem: Purified CARS showing reduced aminoacylation activity

  • Solution: Include reducing agents (DTT, β-mercaptoethanol) to maintain cysteine residues, test activity immediately after purification, optimize storage conditions (glycerol, -80°C)

• tRNA co-purification:

  • Problem: Endogenous tRNA co-purifying with CARS

  • Solution: Include high-salt washes (500-800 mM NaCl), RNase treatment followed by size exclusion chromatography

• Expression level optimization:

  • Problem: Low yield of recombinant CARS

  • Solution: Optimize codon usage for expression system, test different promoters, scale up culture volume

How can researchers differentiate between native and recombinant CARS activity in Xenopus extracts?

Methodological approaches include:

• Epitope tagging:

  • Add small epitope tags (His, FLAG, or HA) to recombinant CARS

  • Use immunoprecipitation to isolate tag-specific activity

  • Compare specific activity of tagged vs. untagged enzyme

• Selective inhibition:

  • Develop antibodies against unique epitopes in recombinant CARS

  • Test activity in the presence of selective inhibitors

  • Measure activity after immunodepletion of native CARS

• Kinetic discrimination:

  • Engineer recombinant CARS with altered substrate specificity

  • Measure activity with non-natural cysteine analogs that are preferentially recognized by the recombinant enzyme

  • Compare temperature or pH optima that might differ between native and recombinant forms

What strategies can resolve data contradictions in CARS functional studies?

When facing contradictory data, consider:

• Methodological approach:

  • Systematically compare experimental conditions (buffer composition, pH, temperature)

  • Standardize enzyme preparations and activity assays

  • Implement internal controls within each experiment

• Technical validation:

  • Use multiple independent methods to measure the same parameter

  • Verify protein integrity by mass spectrometry

  • Evaluate potential interfering factors in assay systems

• Statistical analysis:

  • Implement appropriate statistical tests for significance

  • Increase sample size to improve statistical power

  • Consider Bayesian approaches to integrate prior knowledge with new data

How might CARS research contribute to understanding translational regulation during Xenopus development?

CARS research offers unique insights into developmental regulation:

• Developmental expression patterns:

  • Quantify CARS expression across developmental stages

  • Map spatial distribution using in situ hybridization

  • Correlate with periods of high protein synthesis demand

• Regulatory mechanisms:

  • Investigate post-translational modifications of CARS during development

  • Examine compartmentalization and localized translation

  • Study potential non-canonical functions beyond aminoacylation

• Experimental approaches:

  • Conditional knockout or knockdown of CARS at specific developmental stages

  • Rescue experiments with wild-type vs. mutant CARS

  • Proteomics to identify CARS-interacting proteins across development

What CRISPR-Cas9 strategies would be most effective for studying CARS function?

CRISPR-Cas9 approaches for CARS functional studies might include:

• Genome editing strategy:

  • Design guide RNAs targeting conserved CARS domains

  • Create conditional knockout systems using floxed alleles

  • Generate precise point mutations to study structure-function relationships

• Delivery methods:

  • Inject Cas9 protein with guide RNAs into fertilized eggs

  • Use tissue-specific promoters for spatially restricted editing

  • Implement inducible systems for temporal control

• Phenotypic analysis:

  • Assess developmental defects in CARS mutants

  • Measure global protein synthesis rates

  • Examine specificity of effects through rescue experiments with wild-type CARS mRNA

How might systems biology approaches enhance our understanding of CARS function in the broader context of tRNA synthetases?

Systems-level approaches offer comprehensive insights:

• Multi-omics integration:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Map interaction networks of all aminoacyl-tRNA synthetases

  • Identify emergent properties not apparent from studying CARS in isolation

• Mathematical modeling:

  • Develop kinetic models of the complete aminoacylation system

  • Simulate the effects of CARS perturbations on global translation

  • Predict compensatory mechanisms when CARS function is compromised

• Comparative genomics:

  • Analyze evolutionary relationships among CARS proteins across species

  • Identify conserved vs. species-specific features

  • Correlate structural differences with functional adaptations

These comprehensive approaches would position CARS research within the broader context of translational regulation and protein synthesis machinery.