Substrate Affinity Versus Catalytic Efficiency: Ancestral Sequence Reconstruction of tRNA Nucleotidyltransferases Solves an Enzyme Puzzle.

In tRNA maturation, CCA-addition by tRNA nucleotidyltransferase is a unique and highly accurate reaction. While the mechanism of nucleotide selection and polymerization is well understood, it remains a mystery why bacterial and eukaryotic enzymes exhibit an unexpected and surprisingly low tRNA substrate affinity while they efficiently catalyze the CCA-addition. To get insights into the evolution of this high-fidelity RNA synthesis, the reconstruction and characterization of ancestral enzymes is a versatile tool. Here, we investigate a reconstructed candidate of a 2 billion years old CCA-adding enzyme from Gammaproteobacteria and compare it to the corresponding modern enzyme of Escherichia coli. We show that the ancestral candidate catalyzes an error-free CCA-addition, but has a much higher tRNA affinity compared with the extant enzyme. The consequence of this increased substrate binding is an enhanced reverse reaction, where the enzyme removes the CCA end from the mature tRNA. As a result, the ancestral candidate exhibits a lower catalytic efficiency in vitro as well as in vivo. Furthermore, the efficient tRNA interaction leads to a processive polymerization, while the extant enzyme catalyzes nucleotide addition in a distributive way. Thus, the modern enzymes increased their polymerization efficiency by lowering the binding affinity to tRNA, so that CCA synthesis is efficiently promoted due to a reduced reverse reaction. Hence, the puzzling and at a first glance contradicting and detrimental weak substrate interaction represents a distinct activity enhancement in the evolution of CCA-adding enzymes.

Dual expression of CCA-adding enzyme and RNase T in Escherichia coli generates a distinct cca growth phenotype with diverse applications.

Correct synthesis and maintenance of functional tRNA 3'-CCA-ends is a crucial prerequisite for aminoacylation and must be achieved by the phylogenetically diverse group of tRNA nucleotidyltransferases. While numerous reports on the in vitro characterization exist, robust analysis under in vivo conditions is lacking. Here, we utilize Escherichia coli RNase T, a tRNA-processing enzyme responsible for the tRNA-CCA-end turnover, to generate an in vivo system for the evaluation of A-adding activity. Expression of RNase T results in a prominent growth phenotype that renders the presence of a CCA- or A-adding enzyme essential for cell survival in an E. coli Δcca background. The distinct growth fitness allows for both complementation and selection of enzyme variants in a natural environment. We demonstrate the potential of our system via detection of altered catalytic efficiency and temperature sensitivity. Furthermore, we select functional enzyme variants out of a sequence pool carrying a randomized codon for a highly conserved position essential for catalysis. The presented E. coli-based approach opens up a wide field of future studies including the investigation of tRNA nucleotidyltransferases from all domains of life and the biological relevance of in vitro data concerning their functionality and mode of operation.

A Temporal Order in 5'- and 3'- Processing of Eukaryotic tRNAHis.

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3'-terminal CCA sequence that includes the site of aminoacylation, the additional 5'-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3'-CCA and 5'-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3'-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.