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

Abstract

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 tRNA^His 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 tRNA^His, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNA^His transcripts independent of the presence of G-1; however, tRNA^His 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.

Keywords: CCA-addition; G-1 residue; tRNA maturation; tRNA nucleotidyltransferase; tRNAHis; tRNAHis guanylyltransferase.

Figure 1

Cytosolic tRNA^His processing in eukaryotes. After removal of 5′-leader and 3′-trailer sequences by the nucleus-localized RNase P and RNase Z (orange), CCA- (cyan) and G-1-addition (green) take place in the cytosol. However, it is not clear whether these events occur at random or follow a sequential order, due to different substrate preferences of the involved enzymes. Furthermore, these events can be affected by the nature of the discriminator base 73 that is located across the -1 position (as an example, A73 is indicated in red).

Figure 2

CCA-adding enzyme-catalyzed CCA incorporation on tRNA^His lacking G-1 (tRNA^HisΔG-1; A) and tRNA^His+G-1 (B) from S. cerevisiae. tRNA variants were incubated with increasing amounts of CCA-adding enzyme (0.5, 1.5, 3.0, 4.5, 6.0, 60 und 300 ng). The reaction products were separated on a denaturing polyacrylamide gel and visualized by ethidium bromide staining. Both tRNA variants were processed at comparable efficiencies, resulting in a complete substrate turnover. NC, negative control without enzyme. (C) Kinetic analysis of CCA-addition for both tRNA variants. Increasing amounts of tRNA^HisΔG-1 (black curve) and tRNA^His+G-1 (green curve) were incubated with CCA-adding enzyme, NTPs and α-³²P-ATP under steady-state conditions. Michaelis–Menten data were calculated from triplicates using GraphPadPrism software (Table 1).

Figure 3

The CCA triplet affects the fidelity of G-1-addition by Thg1. 5′-labeled tRNA^His lacking the CCA-end (tRNA^HisΔCCA); (A) or ending with CCA (tRNA^His+CCA); (B) were incubated with saturating amounts of Thg1 (15 µM) and assayed using a phosphatase protection assay, in which the 5′-³²P-label on unreacted tRNA substrate is accessible to phosphatase, and visualized as inorganic phosphate (*Pi). On tRNA^HisΔCCA, Thg1 exhibits a reduced fidelity and adds not only the correct G-1 (G-1*pGpC product) but to a certain amount also erroneously A-1 (A-1*pGpC product on panel A). However, when the tRNA substrate carries a 3′-terminal CCA sequence, Thg1 exclusively incorporates G-1, indicating that the CCA triplet contributes to the fidelity of the reaction. An additional non-enzymatic labeled species (♦) is visible in the no enzyme control (NC) and enzyme-containing reactions, as has been observed previously with these types of labeled tRNA assays [13,23]. (C) Single-turnover nucleotide incorporation was measured in triplicate and plotted as a function of time. 5′-labeled tRNA^HisΔCCA (blue) and tRNA^His+CCA (black) were incubated with 15 µM Thg1 in the presence of 0.1 mM ATP and 1 mM GTP.

Figure 4

Thg1 activity on cytosolic tRNA^His carrying a cytosine residue at the discriminator position 73 (red). (A) On tRNA^HisΔCCA A73C, the enzyme correctly adds a single G-1 residue (G-1*pGpC product), consistent with the absence of a 3′-end template sequence for further 3′-5′ polymerization. Other species observed in this assay include *Pi, which represents the remaining unreacted substrate tRNA, and ♦ which represents the non-enzymatic product that is visible in both no enzyme control (NC) and enzyme-containing reaction lanes. (B) If tRNA^His A73C additionally carries the 3′-CCA-end (cyan), Thg1 catalyzes multiple GTP incorporations in a 5′-template-dependent manner, resulting in two additional G-C base pairs with the 3′-CCA-end (G-2G-1*pGpC and G-3G-2G-1*pGpC products). In this case, the non-enzymatic side product is not visible because it is obscured by the strong signal from the multiple G-addition products. (C) Single-turnover nucleotide incorporation was measured in triplicate and plotted as a function of time. 5′-labeled tRNA^HisΔCCA A73C (red graph) or tRNA^His+CCA A73C (green graph) were incubated with saturating amounts of enzyme (15 µM), 0.1 mM ATP and 1 mM GTP.