CCA addition to tRNA: implications for tRNA quality control

Abstract

The CCA sequence is conserved at the 3' end of all mature tRNA molecules to function as the site of amino acid attachment. This sequence is acquired and maintained by stepwise nucleotide addition by the ubiquitous CCA enzyme, which is an unusual RNA polymerase that does not use a nucleic acid template for nucleotide addition. Crystal structural work has divided CCA enzymes into two structurally distinct classes, which differ in the mechanism of template-independent nucleotide selection. Recent kinetic work of the class II E. coli CCA enzyme has demonstrated a rapid and uniform rate constant for the chemistry of nucleotide addition at each step of CCA synthesis, although the enzyme uses different determinants to control the rate of each step. Importantly, the kinetic work reveals that, at each step of CCA synthesis, E. coli CCA enzyme has an innate ability to discriminate against tRNA backbone damage. This discrimination suggests the possibility of a previously unrecognized quality control mechanism that would prevent damaged tRNA from CCA maturation and from entering the ribosome machinery of protein synthesis. This quality control is relevant to cellular stress conditions that damage tRNA backbone and predicts a role of CCA addition in stress response.

Figure 1

Canonical tRNA structure. (a) Cloverleaf secondary structure of E. coli tRNA^Cys. (b) The L shaped tertiary structure of E. coli tRNA^Cys [72]. The minihelix domain encompassing the acceptor stem, T stem, and T loop is shown by brackets.

Figure 2

Crystal structures of two classes of CCA enzymes. (a) The structure of the class I Archaeoglobus fulgidus enzyme (Protein Data Bank (PDB) code 1R8A). (b) The structure of the class II Bacillus stearothermophilus enzyme (PDB code 1M1W). Both enzymes consist of the head, neck, body, and tail domains, with different orientation of α helices and β sheets.

Figure 3

The minihelix recognition model. (a) Crystal structure of the class I Archaeoglobus fulgidus CCA enzyme (PDB code 1SZ1), in which one enzyme monomer (blue) binds to the minihelix domain of one tRNA (green), while the other monomer (red) binds the minihelix domain of the other tRNA (yellow). Both monomers project the ASL domain of the tRNA anticodon stem-loop away from the enzyme. (b). Kinetic evaluation of the minihelix recognition model, using the sequence framework of E. coli tRNA^Val as the substrate. While E. coli CCA enzyme exhibits a high value of k_pol/K_d of 200 μM⁻¹s⁻¹ for addition of A76 to the full-length tRNA-C75 substrate, it discriminates against the minihelix domain with no measurable value of k_pol/K_d, and it exhibits only a k_pol/K_d value of 20 μM⁻¹s⁻¹ with a damaged tRNA harboring a backbone break in the ASL between positions 37 and 38 (indicated by an arrow). Data are from reference [7].

Figure 4

The two phases in the lifetime of a tRNA in E. coli. (a) In the maturation phase, the precursor tRNA is processed at the 3' end by 3' to 5' exonucleases, and by endonucleolytic cleavage at the 5' end. The matured tRNA is challenged by RNase T in E. coli for 3' end turnover and relies on the CCA enzyme for restoration of A76. It is during this restoration stage that the CCA quality control inspects the tRNA integrity and discriminates against stress-induced damage. The CCA sequence is rapidly repaired on intact tRNA and is aminoacylated by an aminoacyl-tRNA synthetase (aaRS), whereas the damaged tRNA is delayed for CCA addition and is degraded by 3' to 5' RNases as part of RNA surveillance machineries. (b) In the protein synthesis phase, the tRNA moiety of the intact aminoacyl-tRNA is shown in orange while the aminoacyl moiety is shown as a red dot. This intact aminoacyl-tRNA is escorted by elongation factor EF-Tu in complex with GTP to the ribosome A site, where it is adjacent to a peptidyl tRNA on the P site (in yellow) and a discharged tRNA on the E site (in green). After GTP hydrolysis and release of EF-Tu, the polypeptide chain of the P site tRNA is transferred to the aminoacyl moiety of the A-site tRNA. With the assistance of GTP hydrolysis, the elongation factor EF-G promotes translocation of the ribosome-tRNA-mRNA complex, moving the A site and P site tRNAs to the P site and E site, respectively, while releasing the E site tRNA from the ribosome. The ribosome with the vacant A site then awaits the entry of the next intact aminoacyl-tRNA.