Recombinant Enterococcus faecalis CCA-adding enzyme (cca)

What is the function of the CCA-adding enzyme in bacterial systems?

The CCA-adding enzyme (CTP:(ATP) tRNA nucleotidyltransferase) is responsible for synthesizing and maintaining the 3'-CCA end of tRNAs, which is essential for aminoacylation and subsequent participation in protein synthesis. This enzyme operates without requiring a nucleic acid template, making it a unique RNA polymerase that can synthesize a specific sequence onto tRNA 3' ends . In bacterial systems such as E. coli, the CCA-adding enzyme is particularly important for repairing damaged tRNA 3' ends that result from spontaneous or enzymatic hydrolysis . While some bacterial tRNAs have encoded CCA ends, the CCA-adding enzyme remains crucial for quality control and repair functions.

How do I express recombinant CCA-adding enzyme from Enterococcus faecalis in E. coli?

To express recombinant CCA-adding enzyme, the following methodological approach is recommended:

• PCR-amplify the cca gene from Enterococcus faecalis genomic DNA using primers that incorporate appropriate restriction sites

• Clone the amplified gene into an expression vector (such as pET28a+ for His-tagged protein)

• Transform the construct into an E. coli expression strain (BL21(DE3) or similar)

• Induce protein expression with IPTG (typically 0.05-0.5 mM)

• Harvest cells and lyse using sonication or other mechanical disruption methods

• Purify the enzyme using metal-ion affinity chromatography followed by anion exchange chromatography

When designing your expression system, consider that basal expression levels may be sufficient for complementation studies, as demonstrated with other CCA-adding enzymes .

How can I assess the basic catalytic activity of purified recombinant CCA-adding enzyme?

The catalytic activity of purified CCA-adding enzyme can be assessed through the following experimental approach:

• Prepare synthetic minihelix RNA substrates that mimic the acceptor stem of tRNAs with various 3' end permutations

• Incubate the purified enzyme with these substrates in the presence of:

  • CTP alone

  • ATP alone

  • Both CTP and ATP

• Analyze the reaction products using denaturing gel electrophoresis or HPLC

• Monitor nucleotide incorporation using [α-32P]-labeled NTPs

Expected results: With both CTP and ATP present, the enzyme should correctly synthesize the CCA sequence. With CTP alone, some CCA-adding enzymes like the E. coli enzyme can add multiple Cs, generating poly(C) . This activity is regulated by ATP, which prevents poly(C) synthesis and switches specificity to CCA.

How can I develop an in vivo system to evaluate the efficiency of Enterococcus faecalis CCA-adding enzyme variants?

An effective in vivo system to evaluate CCA-adding enzyme variants can be established using the following approach:

• Generate an E. coli strain with a knockout of the endogenous cca gene (Δcca)

• Construct a dual-expression vector system containing:

  • RNase T under control of an inducible promoter (e.g., T7 promoter)

  • The Enterococcus faecalis cca gene or its variants under control of a constitutive promoter

• Transform the constructs into the E. coli Δcca strain

• Monitor bacterial growth as a direct readout of enzyme functionality

This system leverages the antagonistic activities of RNase T (which removes tRNA 3'-ends) and CCA-adding enzyme (which repairs them). In the absence of functional CCA-adding activity, RNase T expression will prevent cell growth . Enzyme variants with altered catalytic efficiency, temperature sensitivity, or substrate specificity will show different growth patterns, allowing for direct phenotypic evaluation in a natural cellular environment.

What structural elements determine nucleotide specificity in CCA-adding enzymes, and how can I investigate this in the Enterococcus faecalis enzyme?

Nucleotide specificity in CCA-adding enzymes is determined by collaborative interaction between the catalytic head domain and the nucleobase-interacting neck domain . To investigate this in Enterococcus faecalis CCA-adding enzyme:

• Perform structural analysis through:

  • X-ray crystallography of the enzyme in complex with CTP or ATP

  • Homology modeling based on related structures (e.g., T. maritima CCA-adding enzyme)

• Conduct domain-swap experiments:

  • Replace the head or neck domains with corresponding domains from related enzymes

  • Create chimeric enzymes to identify regions critical for nucleotide selection

  • Test the resulting chimeras both in vitro (nucleotide addition assays) and in vivo (complementation assays)

• Perform site-directed mutagenesis of:

  • Conserved residues in the neck domain that prevent non-specific nucleotide incorporation

  • Regions in the head domain that define terminal A addition after CC addition

Key residues identified from previous studies include two catalytic aspartates that coordinate metal ions essential for catalysis . Mutagenesis of these positions (e.g., D21 and D23 in E. coli CCA-adding enzyme) can generate variants with altered catalytic properties or nucleotide specificity.

How does the Enterococcus faecalis CCA-adding enzyme contribute to tRNA quality control, and how can this be experimentally assessed?

CCA-adding enzymes are involved in tRNA quality control through multiple mechanisms:

• Addition of CCACCA degradation tags to defective tRNAs

• Inefficient CCA-addition to tRNAs with nicks in the sugar-phosphate backbone

• Restoration of damaged CCA-ends

To experimentally assess these quality control functions:

• Generate synthetic tRNA substrates with various defects:

  • Structural instabilities in the tRNA core

  • Nicks in the sugar-phosphate backbone

  • Truncated 3' ends

• Incubate these substrates with purified Enterococcus faecalis CCA-adding enzyme under various conditions:

  • Different Mg²⁺ concentrations

  • Varying enzyme:substrate ratios

  • Various incubation times

• Analyze reaction products using:

  • High-resolution gel electrophoresis

  • Northern blotting

  • RNA sequencing

• In vivo assessment:

  • Construct a system where defective tRNAs are expressed in E. coli

  • Co-express the Enterococcus faecalis CCA-adding enzyme

  • Monitor tRNA stability and degradation patterns

Expected results: Defective tRNAs should receive CCACCA additions at higher rates than properly folded tRNAs, leading to their targeted degradation . tRNAs with nicks should show reduced CCA-addition efficiency compared to intact tRNAs.

What is the relationship between CCA-adding enzymes and polyadenylation, and how might this be investigated in Enterococcus faecalis?

Recent research has revealed interesting connections between CCA-adding enzymes and RNA polyadenylation:

• CCA-adding enzymes and poly(A) polymerases are evolutionarily related within the nucleotidyltransferase family

• The E. coli CCA enzyme can function as a poly(C) polymerase when incubated with CTP alone

• Depletion of CCA-adding enzyme in mycobacteria results in increased RNA polyadenylation

To investigate this relationship in Enterococcus faecalis:

• Generate a conditional knockdown system for the cca gene in a suitable host

• Analyze the global RNA polyadenylation state:

  • Perform 3'-RACE to identify polyadenylated transcripts

  • Use RNA-Seq with poly(A) enrichment before and after cca depletion

  • Quantify polyadenylated tRNA precursors specifically

• In vitro analysis:

  • Test the ability of purified E. faecalis CCA-adding enzyme to add poly(C) or poly(A) tails

  • Examine the regulatory effect of ATP on potential poly(C) polymerase activity

  • Compare kinetic parameters for CCA addition versus potential poly(C) addition

Expected results: Based on findings in mycobacteria, depletion of CCA-adding enzyme might lead to increased RNA polyadenylation, particularly of tRNA precursors . The enzyme itself might exhibit poly(C) polymerase activity in vitro when incubated with CTP alone, similar to the E. coli enzyme .

How can I set up a selection system to identify functional variants of the Enterococcus faecalis CCA-adding enzyme?

A powerful selection system for functional CCA-adding enzyme variants can be established based on the following methodology:

• Create a randomized library of the E. faecalis cca gene focusing on conserved catalytic residues (e.g., the two aspartates that coordinate metal ions)

• Clone this library into a dual-expression vector containing RNase T

• Transform the library into E. coli Δcca cells

• Plate transformants on selective media

• Only cells harboring functional CCA-adding enzyme variants will form colonies

This approach leverages the growth phenotype observed in the absence of A-adding activity when RNase T is expressed . The selection stringency can be adjusted by modulating RNase T expression levels through different IPTG concentrations.

For semi-randomization of specific codons, use degenerate primers in a site-directed mutagenesis approach. For example, using 'GNN' degenerate codons at position D23 would generate variants with aspartate, glutamate, glycine, valine or alanine at this position .

What controls and experimental conditions should be included when characterizing CCA-adding enzyme temperature sensitivity?

When characterizing temperature sensitivity of CCA-adding enzymes, include the following controls and conditions:

• Control enzymes:

  • Wild-type E. faecalis CCA-adding enzyme

  • A known temperature-sensitive variant (if available)

  • CCA-adding enzymes from thermophilic (e.g., T. maritima) and mesophilic organisms

• Temperature range:

  • Test at minimum 5 temperatures (e.g., 25°C, 30°C, 37°C, 42°C, 45°C)

  • Include temperatures above and below the optimal growth temperature of E. faecalis

• Assay methods:

  • In vitro activity assays at different temperatures

  • Pre-incubation of enzyme at elevated temperatures before assaying at standard temperature (to test thermal stability)

  • In vivo complementation in E. coli Δcca at different growth temperatures

• Analysis parameters:

  • Initial reaction rates

  • Endpoint measurements

  • Thermal inactivation kinetics (half-life at elevated temperatures)

Temperature-sensitive variants may show normal activity at permissive temperatures but rapid decline at non-permissive temperatures.

How might Enterococcus faecalis CCA-adding enzyme be leveraged for synthetic biology applications?

The CCA-adding enzyme's unique ability to add a specific sequence without a template makes it valuable for synthetic biology applications. Consider these research approaches:

• Engineering altered specificity:

  • Generate variants that can add alternative 3' terminal sequences

  • Develop orthogonal tRNA-CCA enzyme pairs for specialized translation systems

• RNA tagging applications:

  • Modify the enzyme to add detectable tags to RNAs of interest

  • Create systems where specific RNAs are marked for degradation or protection

• Methodology development:

  • Clone the E. faecalis cca gene into modular expression systems

  • Test chimeric enzymes with domains from different organisms

  • Evaluate activity in cell-free expression systems

These applications would require detailed characterization of domain functions and nucleotide selection mechanisms, similar to studies performed with T. maritima and A. aeolicus enzymes .

What is the relationship between CCA-adding enzyme function and bacterial stress response, and how can this be studied in Enterococcus faecalis?

The connection between CCA-adding enzymes and stress response is an emerging area of research. To investigate this relationship in E. faecalis:

• Create a conditional expression system for the cca gene

• Subject bacteria to various stresses while modulating cca expression:

  • Oxidative stress (H₂O₂, paraquat)

  • Antibiotic exposure (sub-lethal concentrations)

  • Nutrient limitation

  • Temperature stress

• Analyze:

  • Changes in tRNA charging levels

  • Alterations in the tRNA pool composition

  • Global transcriptome and proteome responses

  • Stress survival rates

Based on research in other organisms, CCA-adding enzyme may play critical roles in adapting to environmental stresses by ensuring proper tRNA maturation and turnover. Depleting the enzyme during stress conditions may reveal its importance in stress response pathways, similar to findings in mycobacteria where CCA depletion affected bacterial growth .