The identity of the discriminator base has an impact on CCA addition
CCA-adding enzymes synthesize and maintain the C-C-A sequence at the tRNA 3′-end, generating the attachment site for amino acids. While tRNAs are the most prominent substrates for this polymerase, CCA additions on non-tRNA transcripts are described as well. To identify general features for substrate requirement, a pool of randomized transcripts was incubated with the human CCA-adding enzyme. Most of the RNAs accepted for CCA addition carry an acceptor stem-like terminal structure, consistent with tRNA as the main substrate group for this enzyme. While these RNAs show no sequence conservation, the position upstream of the CCA end was in most cases represented by an adenosine residue. In tRNA, this position is described as discriminator base, an important identity element for correct aminoacylation. Mutational analysis of the impact of the discriminator identity on CCA addition revealed that purine bases (with a preference for adenosine) are strongly favoured over pyrimidines. Furthermore, depending on the tRNA context, a cytosine discriminator can cause a dramatic number of misincorporations during CCA addition. The data correlate with a high frequency of adenosine residues at the discriminator position observed in vivo. Originally identified as a prominent identity element for aminoacylation, this position represents a likewise important element for efficient and accurate CCA addition.
[…] For steady-state Michaelis–Menten kinetics, 15–200 ng enzyme were incubated with RNA transcript titrated between 1 and 10 μM according to Wolf et al. (). Kinetic parameters of three to five independent experiments were analysed using curve-fitting by non-linear regression (GraphPadPrism). As the transcripts are not soluble at excessive saturating conditions, the obtained kinetic parameters represent apparent values (,). [...] RNA secondary structures were predicted using the RNA Vienna package (RNAfold) (). Structure presentations were done using VARNA (). For tRNA structures, dot bracket annotation of the tRNA database was used (). […]
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