Use of nucleotide analogs by class I and class II CCA-adding enzymes (tRNA nucleotidyltransferase): deciphering the basis for nucleotide selection

Abstract

We explored the specificity and nature of the nucleotide-binding pocket of the CCA-adding enzyme (tRNA nucleotidyltransferase) by using CTP and ATP analogs as substrates for a panel of class I and class II enzymes. Overall, class I and class II enzymes displayed remarkably similar substrate requirements, implying that the mechanism of CCA addition is conserved between enzyme classes despite the absence of obvious sequence homology outside the active site signature sequence. CTP substrates are more tolerant of base modifications than ATP substrates, but sugar modifications prevent incorporation of both CTP and ATP analogs by class I and class II enzymes. Use of CTP analogs (zebularine, pseudoisocytidine, 6-azacytidine, but not 6-azauridine) suggests that base modifications generally do not interfere with recognition or incorporation of CTP analogs by either class I or class II enzymes, and that UTP is excluded because N-3 is a positive determinant and/or O-4 is an antideterminant. Use of ATP analogs (N6-methyladenosine, diaminopurine, purine, 2-aminopurine, and 7-deaza-adenosine, but not guanosine, deoxyadenosine, 2'-O-methyladenosine, 2'-deoxy-2'-fluoroadenosine, or inosine) suggests that base modifications generally do not interfere with recognition or incorporation of ATP analogs by either class I or class II enzymes, and that GTP is excluded because N-1 is a positive determinant and/or the 2-amino and 6-keto groups are antideterminants. We also found that the 3'-terminal sequence of the growing tRNA substrate can affect the efficiency or specificity of subsequent nucleotide addition. Our data set should allow rigorous evaluation of structural hypotheses for nucleotide selection based on existing and future crystal structures.

FIGURE 1.

Stereochemistry of CCA addition. Addition of the R_P and S_P isomers of CTP or ATP to tRNA lacking CA or A was assayed under standard conditions using 100 μM CTP (R_P and S_P) or 200 μM ATP (R_P and S_P). Although modest addition was observed when the concentration of the R_P isomer of ATP was increased to 600 μM (data not shown), this presumably reflects incorporation of S_P isomer slightly contaminating the R_P preparation; mixing experiments suggested that a 10% contamination of R_P with S_P could account for the data.

FIGURE 2.

Addition of CTP analogs to tRNA-DC. (A) CTP analogs tested, with modifications indicated in red. Analogs are 2′-deoxycytidine (lane 1), 5-fluorocytidine (lane 2), 2′-deoxy-2′-fluorocytidine (lane 3), zebularine (lane 4), pseudoisocytidine (lane 5), 6-azacytidine (lane 6), 5-methylcytidine (lane 7), and 6-azauridine (lane 8). (B) Incorporation of CTP analogs into uniformly labeled tRNA substrates lacking 3′-terminal CA at high analog concentration (200 μM). The CTP reaction goes to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. (C and D) CTP analogs that were efficiently incorporated in B were retested at lower analog concentration (25 μM) for shorter times using the class I enzyme of S. shibatae (C) and class II enyzme of B. stearothermophilus (D). (E) CTP analogs that were inefficiently incorporated in B were retested in the linear range; the class I enzyme could not incorporate 5-fluorocytidine or 5-methylcytidine (B). All products were resolved by 12% denaturing PAGE and visualized by phosphorimaging. Note that in this and subsequent figures, tRNA mobility can depend strongly on the identity of the 3′ terminal nucleotide (for example, cf. lanes 2,4,5,6,7 in the middle panel).

FIGURE 2.

Addition of CTP analogs to tRNA-DC. (A) CTP analogs tested, with modifications indicated in red. Analogs are 2′-deoxycytidine (lane 1), 5-fluorocytidine (lane 2), 2′-deoxy-2′-fluorocytidine (lane 3), zebularine (lane 4), pseudoisocytidine (lane 5), 6-azacytidine (lane 6), 5-methylcytidine (lane 7), and 6-azauridine (lane 8). (B) Incorporation of CTP analogs into uniformly labeled tRNA substrates lacking 3′-terminal CA at high analog concentration (200 μM). The CTP reaction goes to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. (C and D) CTP analogs that were efficiently incorporated in B were retested at lower analog concentration (25 μM) for shorter times using the class I enzyme of S. shibatae (C) and class II enyzme of B. stearothermophilus (D). (E) CTP analogs that were inefficiently incorporated in B were retested in the linear range; the class I enzyme could not incorporate 5-fluorocytidine or 5-methylcytidine (B). All products were resolved by 12% denaturing PAGE and visualized by phosphorimaging. Note that in this and subsequent figures, tRNA mobility can depend strongly on the identity of the 3′ terminal nucleotide (for example, cf. lanes 2,4,5,6,7 in the middle panel).

FIGURE 2.

Addition of CTP analogs to tRNA-DC. (A) CTP analogs tested, with modifications indicated in red. Analogs are 2′-deoxycytidine (lane 1), 5-fluorocytidine (lane 2), 2′-deoxy-2′-fluorocytidine (lane 3), zebularine (lane 4), pseudoisocytidine (lane 5), 6-azacytidine (lane 6), 5-methylcytidine (lane 7), and 6-azauridine (lane 8). (B) Incorporation of CTP analogs into uniformly labeled tRNA substrates lacking 3′-terminal CA at high analog concentration (200 μM). The CTP reaction goes to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. (C and D) CTP analogs that were efficiently incorporated in B were retested at lower analog concentration (25 μM) for shorter times using the class I enzyme of S. shibatae (C) and class II enyzme of B. stearothermophilus (D). (E) CTP analogs that were inefficiently incorporated in B were retested in the linear range; the class I enzyme could not incorporate 5-fluorocytidine or 5-methylcytidine (B). All products were resolved by 12% denaturing PAGE and visualized by phosphorimaging. Note that in this and subsequent figures, tRNA mobility can depend strongly on the identity of the 3′ terminal nucleotide (for example, cf. lanes 2,4,5,6,7 in the middle panel).

FIGURE 2.

Addition of CTP analogs to tRNA-DC. (A) CTP analogs tested, with modifications indicated in red. Analogs are 2′-deoxycytidine (lane 1), 5-fluorocytidine (lane 2), 2′-deoxy-2′-fluorocytidine (lane 3), zebularine (lane 4), pseudoisocytidine (lane 5), 6-azacytidine (lane 6), 5-methylcytidine (lane 7), and 6-azauridine (lane 8). (B) Incorporation of CTP analogs into uniformly labeled tRNA substrates lacking 3′-terminal CA at high analog concentration (200 μM). The CTP reaction goes to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. (C and D) CTP analogs that were efficiently incorporated in B were retested at lower analog concentration (25 μM) for shorter times using the class I enzyme of S. shibatae (C) and class II enyzme of B. stearothermophilus (D). (E) CTP analogs that were inefficiently incorporated in B were retested in the linear range; the class I enzyme could not incorporate 5-fluorocytidine or 5-methylcytidine (B). All products were resolved by 12% denaturing PAGE and visualized by phosphorimaging. Note that in this and subsequent figures, tRNA mobility can depend strongly on the identity of the 3′ terminal nucleotide (for example, cf. lanes 2,4,5,6,7 in the middle panel).

FIGURE 2.

Addition of CTP analogs to tRNA-DC. (A) CTP analogs tested, with modifications indicated in red. Analogs are 2′-deoxycytidine (lane 1), 5-fluorocytidine (lane 2), 2′-deoxy-2′-fluorocytidine (lane 3), zebularine (lane 4), pseudoisocytidine (lane 5), 6-azacytidine (lane 6), 5-methylcytidine (lane 7), and 6-azauridine (lane 8). (B) Incorporation of CTP analogs into uniformly labeled tRNA substrates lacking 3′-terminal CA at high analog concentration (200 μM). The CTP reaction goes to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. (C and D) CTP analogs that were efficiently incorporated in B were retested at lower analog concentration (25 μM) for shorter times using the class I enzyme of S. shibatae (C) and class II enyzme of B. stearothermophilus (D). (E) CTP analogs that were inefficiently incorporated in B were retested in the linear range; the class I enzyme could not incorporate 5-fluorocytidine or 5-methylcytidine (B). All products were resolved by 12% denaturing PAGE and visualized by phosphorimaging. Note that in this and subsequent figures, tRNA mobility can depend strongly on the identity of the 3′ terminal nucleotide (for example, cf. lanes 2,4,5,6,7 in the middle panel).

FIGURE 3.

Addition of CTP analogs to tRNA-D. (A) Incorporation of CTP analogs zebularine, pseudoisocytidine, and 6-azacytidine into tRNA substrates lacking 3′-terminal CCA. The CTP control was carried out for only 3 min using low CTP (25 μM) to show the mobility of tRNA-DC as well as tRNA-DCC. (B) Incorporation of CTP analogs 5-fluorocytidine and 5-methycytidine into tRNA substrates lacking 3′-terminal CCA. Assays as in Figure 2B ▶.

FIGURE 4.

Addition of ATP analogs to tRNA-DCC. (A) ATP analogs tested, with modifications indicated in red. (B) Incorporation of ATP analogs into tRNA substrates lacking 3′-terminal A was assayed for the class I S. shibatae enzyme at high analog concentration (1200 μM) and 70°C (upper panel). The class II B. stearothermophilus and E. coli enzymes were assayed at lower analog concentration (600 μM) and 55°C or 37°C, respectively (lower panel). The analogs are 2′-deoxyadenosine (lane 1), N6-methyladenosine (lane 2), diaminopurine riboside (lane 3), purine riboside (lane 4), 2-aminopurine riboside (lane 5), 7-deaza-adenosine (lane 6), 2′-O-methyladenosine (lane 7), 2′-deoxy-2′-fluoroadenosine (lane 8). ATP (rightmost lane) was 300 μM in all assays. Under these conditions, the ATP reactions go to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. Assays as in Figure 2B ▶.

FIGURE 4.

Addition of ATP analogs to tRNA-DCC. (A) ATP analogs tested, with modifications indicated in red. (B) Incorporation of ATP analogs into tRNA substrates lacking 3′-terminal A was assayed for the class I S. shibatae enzyme at high analog concentration (1200 μM) and 70°C (upper panel). The class II B. stearothermophilus and E. coli enzymes were assayed at lower analog concentration (600 μM) and 55°C or 37°C, respectively (lower panel). The analogs are 2′-deoxyadenosine (lane 1), N6-methyladenosine (lane 2), diaminopurine riboside (lane 3), purine riboside (lane 4), 2-aminopurine riboside (lane 5), 7-deaza-adenosine (lane 6), 2′-O-methyladenosine (lane 7), 2′-deoxy-2′-fluoroadenosine (lane 8). ATP (rightmost lane) was 300 μM in all assays. Under these conditions, the ATP reactions go to completion in 5 min (data not shown); these assays were carried out for 15 min to screen for analogs that are inefficiently incorporated. Assays as in Figure 2B ▶.

FIGURE 5.

The 3′-terminal C analogs block addition of ATP, but not CTP analogs. (A) Addition of CTP analogs zebularine, pseudoisocytidine, and 6-azacytidine (200 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class I S. shibatae was used in this assay. (B) Addition of CTP analogs 5-fluorocytidine and 5-methylcytidine (500 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class II B. stearothermophilus and E. coli enzymes were assayed at 55°C or 37°C, respectively. (C) Addition of the CTP analog 5-fluorocytidine (200 μM) in the presence of different concentrations of [α-³²P]ATP to unlabeled tRNA substrates lacking CCA. The class II B. Stearothermophilus enzyme was assayed for 15 min at 55°C and products were resolved by 12% denaturing PAGE. (F) 5-fluorocytidine. Assays as in Figures 2 ▶ and 3 ▶.

FIGURE 5.

The 3′-terminal C analogs block addition of ATP, but not CTP analogs. (A) Addition of CTP analogs zebularine, pseudoisocytidine, and 6-azacytidine (200 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class I S. shibatae was used in this assay. (B) Addition of CTP analogs 5-fluorocytidine and 5-methylcytidine (500 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class II B. stearothermophilus and E. coli enzymes were assayed at 55°C or 37°C, respectively. (C) Addition of the CTP analog 5-fluorocytidine (200 μM) in the presence of different concentrations of [α-³²P]ATP to unlabeled tRNA substrates lacking CCA. The class II B. Stearothermophilus enzyme was assayed for 15 min at 55°C and products were resolved by 12% denaturing PAGE. (F) 5-fluorocytidine. Assays as in Figures 2 ▶ and 3 ▶.

FIGURE 5.

The 3′-terminal C analogs block addition of ATP, but not CTP analogs. (A) Addition of CTP analogs zebularine, pseudoisocytidine, and 6-azacytidine (200 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class I S. shibatae was used in this assay. (B) Addition of CTP analogs 5-fluorocytidine and 5-methylcytidine (500 μM) with or without ATP (1 mM) to tRNA substrates lacking CA (upper panel) or CCA (lower panel). The class II B. stearothermophilus and E. coli enzymes were assayed at 55°C or 37°C, respectively. (C) Addition of the CTP analog 5-fluorocytidine (200 μM) in the presence of different concentrations of [α-³²P]ATP to unlabeled tRNA substrates lacking CCA. The class II B. Stearothermophilus enzyme was assayed for 15 min at 55°C and products were resolved by 12% denaturing PAGE. (F) 5-fluorocytidine. Assays as in Figures 2 ▶ and 3 ▶.

FIGURE 6.

The 3′-terminal C analogs do not relax the specificity of nucleotide addition. (A) Addition of GTP or UTP by class I and class II enzymes tRNA-DCX where X is one of the CTP analogs zebularine, pseudoisocytidine, or 6-azacytidine. Assays as in Figure 5 ▶, using CTP analogs (200 μM) with or without GTP and UTP (1 mM each). Input tRNA-DC (leftmost lane) is converted to tRNA-DCX (left three lanes) but cannot accept GTP or UTP (right three lanes). (B) Addition of ATP analogs (1 mM) to the tRNA-DCX substrates where X is as in A. ATP analogs (1 mM) numbered as in Figure 4A ▶. The class I S. shibatae enzyme was used in this assay. (C) Addition of ATP analogs (1 mM) to tRNA-DC by class I and class II enzymes. Analogs numbered as in Figure 4A ▶ (numbered lanes); tRNA-DC substrate only (leftmost lane); positive control containing CTP as well as ATP (second lane from left).

FIGURE 6.

The 3′-terminal C analogs do not relax the specificity of nucleotide addition. (A) Addition of GTP or UTP by class I and class II enzymes tRNA-DCX where X is one of the CTP analogs zebularine, pseudoisocytidine, or 6-azacytidine. Assays as in Figure 5 ▶, using CTP analogs (200 μM) with or without GTP and UTP (1 mM each). Input tRNA-DC (leftmost lane) is converted to tRNA-DCX (left three lanes) but cannot accept GTP or UTP (right three lanes). (B) Addition of ATP analogs (1 mM) to the tRNA-DCX substrates where X is as in A. ATP analogs (1 mM) numbered as in Figure 4A ▶. The class I S. shibatae enzyme was used in this assay. (C) Addition of ATP analogs (1 mM) to tRNA-DC by class I and class II enzymes. Analogs numbered as in Figure 4A ▶ (numbered lanes); tRNA-DC substrate only (leftmost lane); positive control containing CTP as well as ATP (second lane from left).

FIGURE 6.

The 3′-terminal C analogs do not relax the specificity of nucleotide addition. (A) Addition of GTP or UTP by class I and class II enzymes tRNA-DCX where X is one of the CTP analogs zebularine, pseudoisocytidine, or 6-azacytidine. Assays as in Figure 5 ▶, using CTP analogs (200 μM) with or without GTP and UTP (1 mM each). Input tRNA-DC (leftmost lane) is converted to tRNA-DCX (left three lanes) but cannot accept GTP or UTP (right three lanes). (B) Addition of ATP analogs (1 mM) to the tRNA-DCX substrates where X is as in A. ATP analogs (1 mM) numbered as in Figure 4A ▶. The class I S. shibatae enzyme was used in this assay. (C) Addition of ATP analogs (1 mM) to tRNA-DC by class I and class II enzymes. Analogs numbered as in Figure 4A ▶ (numbered lanes); tRNA-DC substrate only (leftmost lane); positive control containing CTP as well as ATP (second lane from left).

FIGURE 7.

The enzyme may recognize C and A, and discriminate against U and G, by forming hydrogen bonds (“base pairing”) with the 3 and 4 positions of pyrimidines and the equivalent 1 and 6 positions of purines (indicated by brackets, where + indicates hydrogen bond donor and − hydrogen bond acceptor). The cartoon combines functional data (this study) with structural data for the B. stearothermophilus class I CCA-adding enzyme complexed with CTP and ATP (Li et al. 2002). In the crystal structure, the nucleotide-binding site also “base pairs” with N-3 of ATP.