LeuRS can leucylate type I and type II tRNALeus in Streptomyces coelicolor.

Transfer RNAs (tRNAs) are divided into two types, type I with a short variable loop and type II with a long variable loop. Aminoacylation of type I or type II tRNALeu is catalyzed by their cognate leucyl-tRNA synthetases (LeuRSs). However, in Streptomyces coelicolor, there are two types of tRNALeu and only one LeuRS (ScoLeuRS). We found that the enzyme could leucylate both types of ScotRNALeu, and had a higher catalytic efficiency for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA). The results from tRNA and enzyme mutagenesis showed that ScoLeuRS did not interact with the canonical discriminator A73. The number of nucleotides, rather than the type of base of the variable loop in the two types of ScotRNALeus, was determined as important for aminoacylation. In vitro and in vivo assays showed that the tertiary structure formed by the D-loop and TψC-loop is more important for ScotRNALeu(UAA). We showed that the leucine-specific domain (LSD) of ScoLeuRS could help LeuRS, which originally only leucylates type II tRNALeu, to aminoacylate type I ScotRNALeu(CAA) and identified the crucial amino acid residues at the C-terminus of the LSD to recognize type I ScotRNALeu(CAA). Overall, our findings identified a rare recognition mechanism of LeuRS to tRNALeu.

INTRODUCTION

Transfer RNAs (tRNAs), short non-coding RNAs of ∼70–100 bases, act as adaptors by linking nucleotide sequences and amino acids through codon-anticodon pairing (1,2). To function as a substrate for proteins synthesis, tRNA is first charged with the corresponding amino acid by its cognate aminoacyl-tRNA synthetase (AARS), and then the aminoacyl-tRNA is delivered into the ribosome to biosynthesize proteins (1). The fidelity of the specific attachment of the amino acid with its cognate tRNA is governed by AARS, leading to the need to decipher the recognition set of tRNA by its cognate AARS.

tRNAs have a characteristic secondary structure consisting of the accepting stem, D stem-loop, anticodon stem-loop, variable loop and TψC stem-loop (2). Mature tRNAs are categorized into type I and II, based on the length of variable loop located between the anticodon stem and the TψC-stem (3). Type I tRNAs have a short variable loop, and type II tRNAs are characterized by a long variable loop (more than 10 nt) in most prokaryotes and in the cytoplasm of eukaryotes. tRNALeu, which can be leucylated by the cognate leucyl-tRNA synthetase (LeuRS), along with tRNASer and tRNATyr, belongs to the type II tRNA. However, in eukaryotic mitochondria, tRNALeu has a short variable loop, belonging to the type I tRNA, and can be leucylated by mitochondrial LeuRS. Cross-recognition between tRNALeu and LeuRS from the cytoplasm and mitochondria is blocked (1). This specific structure of the tRNA plays an important role in the recognition of its cognate AARS.

The identity elements of type II tRNALeu have been widely studied. The most important elements within Escherichia coli tRNALeu isoacceptors, which contribute to recognition and aminoacylation by the cognate LeuRSs, are A73 (4–6), A14 (4) and several structural features from the core of the tRNALeu molecules that are involved in tertiary interactions (A15-U48 (5,7), G18/G19-U55/C56 (4,7–8), U54-A58 (8) and the position of residue 47n in the variable region (6)). In Aquifex aeolicus, the recognition set of tRNALeu is similar to that of EctRNALeus (9). In the human cytoplasmic leucylation system, the discriminator base A73; three base pairs C3-G70, A4-U69 and G5-C68 in the acceptor stem; nucleotide C20a in the D-loop; and the variable region are major contributing elements to aminoacylation (10–12). In addition to the common A73 discriminator, in the yeast cytoplasmic leucylation system, the tRNALeu anticodon loop is the major determinant (13). A previous study showed that human mitochondrial LeuRS (hmLeuRS) recognizes human mitochondrial tRNALeu (hmtRNALeu) by the size of its anticodon loop and the length of its anticodon stem, rather than the specific nucleotides at positions 34–36 (14). In addition, footprinting experiments showed that the acceptor stem binds with its cognate AARS (14). The type I hmtRNALeu shares the discriminator A73 with type II tRNALeu (15).

Based on sequence homology and the structures of the catalytic active sites, AARSs are divided into two classes. LeuRS is a class I AARS and catalyzes the aminoacylation of tRNALeu (16). The reactions catalyzed by LeuRSs comprise a two-step process: The amino acid leucine is first activated with adenosine triphosphate (ATP) to synthesize the Leu-AMP intermediate; the Leu moiety of the intermediate is subsequently transferred to the tRNALeu bearing the cognate nucleotide triplet at its synthetic domain (17). LeuRS consists of a typical Rossmann dinucleotide-binding fold in the catalytic domain, an editing domain (CP1), ZN1 domain, leucine specific domain (LSD), anticodon binding domain (ABD) and a C-terminal domain (CTD) (16,18–20). The LSD, which is connected to the signature sequence KMSKS motif of class I AARS via a β-ribbon, modulates their aminoacylation activities (21–23). Crystal structure studies have also revealed that the LSD, together with the adjacent catalytically crucial KMSKS loop, plays a critical role in stabilizing A76 of tRNALeu and Leu-AMP during aminoacylation reactions (22,24). Our previous study showed that the LSD of E. coli LeuRS (EcLeuRS) participates in tRNALeu recognition, favors the binding of tRNAs harboring a large loop in the variable region, and modulates the aminoacylation and proofreading functional cycle (23).

Genome mining of Streptomyces coelicolor revealed that in this bacterium, there are five tRNALeu isoacceptors, among them four, (ScotRNALeu(UAA), ScotRNALeu(GAG), ScotRNALeu (CAG) and ScotRNALeu(UAG)), have a long variable loop like type II tRNALeus from bacteria, and one, ScotRNALeu(CAA), has a short variable loop likes type I tRNA. It is interesting that in one actinomyces species, two types of tRNALeu coexist in the cytoplasm without membrane separation (25,26). However, in S. coelicolor, there is only one leuS gene encoding LeuRS. Can this one ScoLeuRS recognize and leucylate both type I and type II ScotRNALeu? What is the difference in their recognition and catalysis by ScoLeuRS? Determining the recognition mechanism of ScoLeuRS of two types ScotRNALeu is interesting and important.

In the present study, we choose ScotRNALeu(UAA) among the four type II ScotRNALeu isoacceptors as the representative to study its aminoacylation. ScotRNALeu(CAA), ScotRNALeu(UAA) and their mutants were successfully transcribed and purified in vitro. In additional, the genes encoding ScotRNALeu(CAA) and ScotRNALeu(UAA) were cloned into E. coli, and the two ScotRNALeus were isolated and purified from the transformants. Similarly, ScoLeuRS was obtained from the E. coli transformants containing leuS. The affinity and kinetics of ScoLeuRS for the two types of ScotRNALeus were measured and compared. An unprecedented role of ACCA at the 3′ end of the accepting stem for leucylation of ScotRNALeus was identified. Moreover, we showed that the LSD of ScoLeuRS helps another LeuRS that only leucylates type II tRNALeu, leucylate type I ScotRNALeu(CAA). Our results highlighted the distinct determinants resulting from the scaffold structure of type I and II tRNAs for aminoacylation, and the structural flexibility of LeuRS to adapt to this difference between the two types of tRNAs, further indicating the co-evolution of this tRNA and its cognate synthetase.

MATERIALS AND METHODS

Materials

L-leucine, dithiothreitol (DTT), ATP, cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP), 5′-GMP, Tris–HCl, MgCl2, NaCl, β-mercaptoethanol (β-Me) and 3 M sodium acetate (NaAc) solution (pH 5.2) were purchased from Sigma-Aldrich Co. LLC (St Louis, MO, USA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was obtained from AMRESCO (OH, USA). [3H] Leucine was obtained from PerkinElmer Inc. (Waltham, MA, USA). Q5 high-fidelity DNA polymerase was purchased from New England Biolabs (Ipswich, MA, USA). A DNA fragment rapid purification kit and a plasmid extraction kit were obtained from Yuanpinghao Biotech (Tianjin, China). Oligonucleotide primers were synthesized by Biosune (Shanghai, China). The KOD-neo-plus DNA polymerase and KOD-plus mutagenesis were obtained from TOYOBO (Osaka, Japan); and all genes and their variants were confirmed by DNA sequencing performed by Biosune (Shanghai, China). Protein standard markers, T4 ligase, pyrophosphatase, and restriction endonucleases were obtained from Thermo Scientific (Waltham, MA, USA). The nickel-nitrilotriacetic acid Superflow was purchased from Qiagen (Hilden, Germany). Amicon ultra-15 filters were obtained from Merck (Darmstadt, Germany). Competent E. coli Top10 and BL21 (DE3) cells were prepared in our laboratory. Escherichia coli ET12567, used to perform intergeneric conjugation from E. coli to S. coelicolor, was a kind gift from Prof. Wei-Hong Jiang's laboratory, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences. The T7 RNA polymerase was purified from an overproducing strain maintained in our laboratory (27).

Gene cloning, mutagenesis and expression; and protein purification

All primers used to amplify genes are shown in Supplementary Table S1. The ScoLeuRS gene was amplified from S. coelicolor A3(2) M145 genomic DNA, which was a kind gift from Prof. Wei-Hong Jiang, and inserted into pET28a vector via the restriction enzymes NdeI and HindIII. We purchased the Streptomyces azureus strain from the China Center of Industrial Culture Collection and cultured it in our laboratory. The S. azureus genomic DNA was extracted using an Ezup Column Bacteria Genomic DNA purification kit from Sangon Biotech (Shanghai, China). The S. azureus LeuRS gene (SazLeuRS) was amplified from genomic DNA and inserted into pET28a via the restriction enzymes NdeI and HindIII. The gene encoding SpurLeuRS, which was synthesized according to the sequence from EBI-EMBL ensemble genome database (28), gene annotation ADL19_14010, and inserted between NdeI and XhoI sites in pET30a, was optimized for expression in E. coli. All constructs were confirmed by DNA sequencing at Biosune. The recombinant plasmids containing genes encoding EcLeuRS and hmLeuRS, pET30a-EcleuS (29) and pET22b(+)-hmleuS-40 (30), respectively, were constructed in our laboratory.

The definition of the LSD domain of ScoLeuRS and SpurLeuRS was based on the crystal structure of EcLeuRS (Val569 – Ser618, PDB ID: 4AQ7) and sequence alignment. The LSD domain (Val594 – Glu638) of SpurLeuRS was substituted by the LSD domain (Ile696 – Gly733) from ScoLeuRS to obtain SpurLeuRS-ScoLSD. We further constructed multiple alanine replacement mutants, listed in Supplementary Figure S8. Five single site mutants at the terminus of the LSD residues, SpurLeuRS-ScoLSD-R730A, -L731A, -L732A, -G733A and -L732I, and one double-site mutant, SpurLeuRS-ScoLSD–L732A/G733A, were constructed by gene mutagenesis using pET30a-SpurLeuRS-ScoLSD as a template. The numbers in the names of the mutants indicated the position of residues in ScoLeuRS. The genes encoding all the mutants were confirmed by DNA sequencing.

All constructs were transformed into E. coli BL21 (DE3) cells to produce the proteins from the transformants. A single colony of each transformant was chosen and cultured in 500 ml of 2 × YT medium at 37°C. When the cells reached mid-log phase (A600 = 0.6), expression of the recombinant protein was induced by the addition of 0.2 mM IPTG for 12 h at 16°C. Protein purification was performed according to a previously described method (31), except that the buffers were: buffer A (50 mM Tris–HCl, pH 8.0, 400 mM NaCl, 10 mM imidazole, 10% glycerol and 5 mM β-Me), buffer B (50 mM Tris–HCl, pH 8.0, 400 mM NaCl, 20 mM imidazole, 10% glycerol and 5 mM β-Me), buffer C (50 mM Tris–HCl, pH 8.0, 400 mM NaCl, 250 mM imidazole, 10% glycerol and 5 mM β-Me), and buffer D (20 mM Tris–HCl, pH 8.0, 150 mM NaCl and 2 mM β-Me). The protein concentrations were determined using UV absorbance at 280 nm, and the molar absorption coefficient was calculated according to the sequence of each protein (32).

Preparation of tRNALeu

The DNA sequence of the T7 promoter and the ScotRNALeu(CAA) and ScotRNALeu(UAA) genes were obtained by ligating three chemically synthetized DNA fragments for each strand, which were then ligated into plasmid pTrc99b (pre-cleaved with EcoRI/BamHI) to construct pTrc99b-T7-ScotRNALeu(CAA), pTrc99b-T7-ScotRNALeu(UAA) and other ScotRNALeu isoacceptors using previously published methods (33). Amplification of the template and transcription of tRNA were performed according to a previously described method (31). The corresponding mutants were prepared using the same method as that for the wild-type. The accepting capacity of ScotRNALeu(CAA) and -(UAA) are 1507 and 1574 pmol/A260, respectively. The tRNA construct of EctRNALeu(UAA) and the tRNA construct of hmtRNAleu(UUR) were constructed, and in vitro transcription was performed as reported previously (34,35); their amino acid accepting activity was ∼1500 pmol/A260.

Aminoacylation assay

Leucylation of ScotRNALeu was performed at 37°C in a reaction mixture containing 50 mM Tris–HCl (pH 8.5), 6 mM MgCl2, 2 mM DTT, 4 mM ATP, 20 μM [3H] leucine, 1 nM ScoLeuRS and 5 μM ScotRNALeu or its variants. The procedure was performed as previously described (29). Leucylation of ScotRNALeu by SazLeuRS and SpurLeuRS was performed under the same conditions; by SpurLeuRS-ScoLSD and its variant was under the same conditions except for 10 nM enzyme in the mixture; and that by EcLeuRS and hmLeuRS was performed as described previously (19,36). The kinetic parameters of ScoLeuRS for ScotRNALeu and its variants during aminoacylation were determined in the presence of varying concentrations of tRNALeu (0.5–32 μM).

Actinomyces strain, growth conditions and complementation plasmid construction

The S. coelicolor J1501 strain was a kind gift from Dr Mei-Feng Tao, Shanghai Jiaotong University. J1501 was grown on an MS plate for spore collection. Other strains were grown on R2YE plates with the required growth supplements: Histidine at 50 μg ml−1 and uracil at 7.5 μg ml−1. In all cases, S. coelicolor was grown at 30°C. For knockout of bldA (encoding ScotRNALeu(UAA)), the cosmid SCE25AT::apra was also a gift of Dr Tao, and the intergeneric mating was conducted as described before (37), yielding J1501▵bldA::apra. The plasmid pMS82, which was a present from Dr Maggie Smith, York University, UK, was used to construct a complementation plasmid. The bldA gene, together with the putative promoter region (38), was amplified from the S. coelicolor genome yielding a 387 bp product. The polymerase chain reaction product was digested with HindIII and KpnI, and cloned into the respective sites of vector pMS82 to obtain pMS82bldA. Other mutants were constructed by gene mutagenesis using pMS82bldA as a template. Conjugation of the plasmids from the donor strain to J1501▵bldA was performed according to the Practical Streptomyces Genetics handbook. For the time-course experiment, development was assessed through visual inspection.

RESULTS

ScoLeuRS has the same affinity for the two types of ScotRNALeus, but has a different aminoacylation activity

To study the interaction between ScoLeuRS and ScotRNALeus in vitro, we first obtained ScoLeuRS from E. coli transformants expressing its gene and then purified the protein to ∼95% homogeneity using affinity chromatography on Ni-NTA superflow (Supplementary Figure S1A). The transcripts of ScotRNALeu(CAA) and -(UAA) were obtained in vitro (Supplementary Figure S1B). To compare the thermal stabilities of the two types of ScotRNALeus, UV melting curves were obtained. Notably, the melting temperature of ScotRNALeu(CAA) was 55.6 ± 0.8°C, which was slightly higher than that of ScotRNALeu(UAA) (52.5 ± 0.1°C) (Supplementary Figure S2).

The reaction conditions for the leucylation of the two ScotRNALeus by ScoLeuRS were optimized. The optimal pH value was 8.5, and the optimal concentration of Mg2+ and the magnesium/ATP ratio were 6 mM and 1.5, respectively (Supplementary Figure S3). The kinetic parameters of ScoLeuRS for the transcripts of the two types ScotRNALeus were assayed and are shown in Table 1. The Km and kcat values of ScoLeuRS for ScotRNALeu(CAA) were lower than those for ScotRNALeu(UAA), indicating that ScoLeuRS has a slightly lower affinity for type II ScotRNALeu and a higher catalytic rate of its leucylation. Therefore, the catalytic efficiency (kcat/Km) of ScoLeuRS for ScotRNALeu(CAA) is less than half that for ScotRNALeu(UAA). The kinetic parameters of ScoLeuRS for the ScotRNALeu(CAA) and ScotRNALeu(UAA) overexpressed and isolated from E. coli showed comparable results (data not shown).

Table 1.

Aminoacylation kinetics constants of transcripts of ScoLeuRS for ScotRNALeu(CAA), (UAA) and their mutants

ScotRNALeu	K m (μM)	k cat (s−1)	k cat/Km (s−1μM−1)	k cat/Km (relative %)
(CAA) type I	0.16 ± 0.03	1.00 ± 0.01	6.25	100
-A73C	0.45 ± 0.01	0.98 ± 0.07	2.17	35
-A73G	0.24 ± 0.08	1.24 ± 0.05	5.17	83
-A73U	0.22 ± 0.08	1.93 ± 0.09	8.81	141
-C74A	nm	nm	nm	nm
-C74G	nm	nm	nm	nm
-C74U	nm	nm	nm	nm
-C75A	0.20 ± 0.02	0.25 ± 0.01	1.25	20
-C75G	0.13 ± 0.03	0.06 ± 0.01	0.46	7
-C75U	0.19 ± 0.01	0.10 ± 0.01	0.53	8
(UAA) type II	0.24 ± 0.03	3.40 ± 0.22	14.2	100
-A73C	0.21 ± 0.01	2.02 ± 0.04	9.62	68
-A73G	0.31 ± 0.01	2.90 ± 0.07	9.35	66
-A73U	0.22 ± 0.01	2.75 ± 0.60	12.5	88
-C74A	nm	nm	nm	nm
-C74G	nm	nm	nm	nm
-C74U	nm	nm	nm	nm
-C75A	0.45 ± 0.05	0.63 ± 0.05	1.39	10
-C75G	0.20 ± 0.08	0.01 ± 0.002	0.07	0.5
-C75U	0.68 ± 0.07	0.47 ± 0.06	0.69	5

The results are the averages of three independent repeats, with the standard deviations indicated.

ScoLeuRS aminoacylates prokaryotic tRNALeu, but not eukaryotic mitochondrial tRNALeu

The above data showed that ScoLeuRS can leucylate both type I ScotRNALeu(CAA) and type II ScotRNALeu(UAA). Considering that hmLeuRS is the only reported LeuRS that can leucylate type I tRNALeus and ScoLeuRS has higher homology with well-studied EcLeuRS, we asked whether ScoLeuRS, hmLeuRS and EcLeuRS could cross-recognize their tRNALeus?

The aminoacylation activity of hmLeuRS and EcLeuRS for the two ScotRNALeus showed that hmLeuRS had similar leucylation activity for both types of ScotRNALeus compared with ScoLeuRS; EcLeuRS could catalyze aminoacylation of ScotRNALeu(UAA) with 50% activity; however, it could not leucylate ScotRNALeu(CAA) completely (Figure 1A and B). The sequence alignment of these three LeuRSs showed that ScoLeuRS and hmLeuRS have the same domains as EcLeuRS; however, ScoLeuRS has the longest N-terminus among them (Supplementary Figure S4), suggesting that domains in the N-terminus may play important roles in the aminoacylation of type I tRNALeus. Moreover, EcLeuRS has a lower activity for type II ScotRNALeu(UAA) despite the high homology, which suggested that ScotRNALeus may have different recognition sets from EctRNALeus.

Figure 1.

Cross-recognition between LeuRSs and tRNALeus from various species. (A and B) Aminoacylation of 1 nM ScoLeuRS, 1 nM EcLeuRS, 500 nM hmLeuRS or no LeuRS for 5 μM ScotRNALeu(CAA) (A) or 5 μM ScotRNALeu(UAA) (B), respectively. (C) Aminoacylation of 1 nM ScoLeuRS for 5 μM ScotRNALeu(UAA), 5 μM ScotRNALeu(CAA), 5 μM hmtRNALeu(UUR), 5 μM EctRNA or no tRNA, respectively. The data shown represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols. (D) Schematic demonstration of cross-recognition. The arrows indicate the ability to leucylate the indicated tRNALeu.

We also examined the aminoacylation of ScoLeuRS for type II EctRNALeu(UAA) and type I hmtRNALeu(UUR). ScoLeuRS could aminoacylate type II EctRNALeu(UAA) with a similar reaction rate to that of ScotRNALeu(UAA), but not type I hmtRNALeu(UUR) (Figure 1C), implying that ScotRNALeus and hmtRNALeus have distinct identity elements.

C74 of ScotRNALeu(CAA) and ScotRNALeu(UAA) instead of A73 is the identity element

The aminoacylation assay implied that ScotRNALeus may choose different identity elements to those of hmtRNALeu(UUR) and EctRNALeu(UAA). A73 of hmtRNALeu(UUR) and EctRNALeu(UAA) is an canonical identity element (4–6,15). To determine whether it plays the same role in ScotRNALeu(CAA) and -(UAA), a series of tRNA mutants was constructed (Figure 2B and D).

Figure 2.

Summary of ScotRNALeu constructs used in this study. (A) The L-shaped tertiary structure of ScotRNALeu(CAA) based on the structure of Staphylococcus aureus tRNAIle (PDB ID: 1FFY), (C) that of ScotRNALeu(UAA) based on the structure of EctRNALeu(UAA) (with EcLeuRS) (PDB ID: 4AQ7) and TttRNALeu(CAG) (PDB ID: 2BYT); dotted lines indicate the tertiary interactions. (B) The variants derived from ScotRNALeu(CAA), (D) those derived from ScotRNALeu(UAA); the arrows indicate the mutation locations, asterisks indicate the double substitution mutant(A14U/U8A); triangles indicate the deletion mutation (in ScotRNALeu(CAA) ΔG18/ΔG19 and ΔU55/ΔC56, or in ScotRNALeu(UAA) ΔG18 and ΔU55); and the underlines indicate the double substitution mutant (U54C:A58G). SVL, variable loop of ScotRNALeu(CAA), colored as dark yellow. LVL, variable loop of ScotRNALeu(UAA), colored as light yellow.

First, we mutated A73 to G, U, and C in the two ScotRNALeus, respectively, and the leucine accepting activities of these mutants were assayed (Table 1). For ScotRNALeu (CAA), the Km values of ScoLeuRS for all the mutants at A73 increased, indicating a weaker interaction between the tRNA and the enzyme; the kcat values for A73G and A73U increased, although that for A73C only declined slightly. For ScotRNALeu(UAA), the Km values of ScoLeuRS for A73G increased, showing a weaker interaction between the tRNA and the enzyme, although that for A73C and A73U decreased slightly, indicating a stronger interaction; the kcat values declined for the three mutants to varying degrees. However, ScoLeuRS still could recognize all six mutants at A73 (three for each ScotRNALeu) and had relatively higher catalytic efficiency (Table 1). The data showed that A73 was not an identity element in ScotRNALeu(CAA) and -(UAA) for ScoLeuRS.

Next, we determined whether A73 is an identity element in all isoacceptors of ScotRNALeu. The replacement mutants at A73 of all other ScotRNALeu-(GAG), -(CAG) and -(UAG) were constructed and their accepting activities were assayed (Supplementary Figure S5A–C). For all mutants at A73, ScoLeuRS maintained obvious charging capacities. The above results showed that in all ScotRNALeu isoacceptors, A73 is not a discriminator, unlike other most tRNALeus.

The C74C75A76 at the 3′ end of all tRNAs is common, and A is a common nucleotide for accepting amino acid. The A73 next to C74C75A76 is no longer identity element of ScotRNALeus; therefore, we determined whether other bases at the 3′ end play important roles. Mutagenesis of C74 and C75 of ScotRNALeus was performed and the accepting activities of the tRNA mutants were assayed (Table 1). The accepting capacities of both ScotRNALeu(CAA) and -(UAA) were lost completely when C74 changed to the other three nucleotides, suggesting that this position is essential for both type I and type II ScotRNALeus (Table 1). However, our previous study showed that EcLeuRS could still leucylate C74 mutants of EctRNALeu, although with lower activities, which suggested that C74 is not as significant in EctRNALeus as it is in ScotRNALeus (29). The mutants at C75 of both ScotRNALeu(CAA) and -(UAA) were also constructed. The kinetic parameters of ScoLeuRS for the tRNA C75 mutants showed that although both the binding and catalysis of ScoLeuRS for the two mutants was weakened, C75 in the two types ScotRNALeus was not crucial to their accepting capacities compared with those of C74 (Table 1).

Based on the tertiary structure of EcLeuRS, Arg416 and Arg418 in the highly conserved motif 416R/KLRDWGVSRQRYWG429 interact with A73 of EctRNALeu(UAA) (Figure 3A) (22). Our constructed mutants EcLeuRS-R416A and -R418A showed obviously decreased aminoacylation activity (Figure 3B), indicating that disrupting the interaction between the residues of EcLeuRS and A73 of tRNALeu abrogated the activity of EcLeuRS; thus confirming that A73 is crucial to recognition by EcLeuRS, which has been reported previously (4–6). The high sequence homology between ScoLeuRS and EcLeuRS identified the homologous residues of Arg416 and Arg418 in EcLeuRS as Arg517 and Arg519 in ScoLeuRS, which were changed to alanine. The leucylation activities of ScoLeuRS-R517A and -R519A for ScotRNALeu(CAA) and -(UAA) were even higher than that of the wild-type ScoLeuRS (Figure 3C and D), suggesting that the interaction of Arg517 and Arg519 with A73 of ScotRNALeu(CAA) and -(UAA) is not important for the activity of ScoLeuRS, and confirming that A73 in the two types of ScotRNALeus is not the identity element, unlike the EcLeuRS-tRNALeu system.

Figure 3.

The interaction between residues in ScoLeuRS and A73 in ScotRNALeus. (A) Crystal structure of EctRNALeu (orange in the cartoon mode) in complex with EcLeuRS (green in the cartoon mode) during the aminoacylation conformation (PDB ID: 4AQ7, Ref. 22). Residues R416 and R418 (R517 and R519) of a conserved motif (yellow) are numbered and shown in the stick model with their distances to A73 of EctRNALeu. (B) Aminoacylation of 1 nM EcLeuRS-WT, -R416A, -R418A or no enzyme, respectively, for 5 μM EctRNALeu(UAA). (C and D) Aminoacylation of 1 nM ScoLeuRS-WT, -R517A, -R519A or no enzyme, respectively, for 5 μM ScotRNALeu(CAA) (C) or 5 μM ScotRNALeu(UAA) (D). The data shown represent the averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

In the EcLeuRS and EctRNALeu(UAA) complex, C74 of the tRNA contacts with Arg424 of EcLeuRS, which is the homolog of Arg525 of ScoLeuRS (Figure 4A) (22). EcLeuRS-R424A resulted in the loss of half the aminoacylation activity of EcLeuRS (Figure 4B), indicating that this residue is not crucial to aminoacylation. However, the aminoacylation activity of ScoLeuRS-R525A for both types ScotRNALeus was completely lost (Figure 4C and D). These results suggested that this Arg residue of ScoLeuRS is crucial to recognize C74 in both types of ScotRNALeus and is important for the activity of the enzyme.

Figure 4.

The interaction between residues in ScoLeuRS and C74 in ScotRNALeus. (A) Crystal structure of EctRNALeu (orange in the cartoon mode) in complex with EcLeuRS (yellow in the cartoon mode) during the aminoacylation conformation (PDB ID: 4AQ7, Ref. 22). Residues R424 (R525) of a conserved motif (yellow) are numbered and shown in the stick model with their distances to C74 of EctRNALeu. (B) Aminoacylation of 1 nM EcLeuRS-WT, -R424A or no enzyme, respectively, for 5 μM EctRNALeu(UAA). (C and D) Aminoacylation of 1 nM ScoLeuRS-WT, -R525A or no enzyme, respectively, for 5 μM ScotRNALeu(CAA) (C) or 5 μM ScotRNALeu(UAA) (D). The data shown represent the averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

To check whether the shift in the identity element from A73 to C74 is universal for all ScotRNALeus, we mutated C74 and C75 of type II ScotRNALeu(GAG) into other nucleotides, and obtained the same results as those for ScotRNALeu(UAA) (Supplementary Figure S5D).

The above results indicated that the interaction of ScoLeuRS with both types of ScotRNALeus was similar, and that C74 of ScotRNALeus is a crucial base at 3′ ACCA end instead of A73 in ScotRNALeus.

The number of nucleotides of the variable loop in ScotRNALeus is important for aminoacylation

The variable loop of ScotRNALeu(CAA) contains only five nucleotides, A44C45A46G47C48 (SVL); however, ScotRNALeu(UAA), like other tRNALeus, has a long variable loop with seventeen nucleotides (LVL), which could form five base pairs, U44·G47l, G45-C47k, C46-G47j, C47-G47i and C47a-G47h (Figure 2). We replaced the SVL of ScotRNALeu(CAA) with the LVL of ScotRNALeu(UAA) to produce a chimeric mutant ScotRNALeu(CAA)-LVL. The catalytic efficiency of ScoLeuRS for this chimeric tRNA mutant decreased, with a larger Km value (Table 2). Then, the nucleotides in the SVL of ScotRNALeu(CAA) were gradually deleted to form four deletion mutants, named ScotRNALeu(CAA)-SVLΔ1nt, -SVLΔ2nt, -SVLΔ3nt and -SVLΔ4nt (their names are shown in Figure 5E). Except for mutant ScotRNALeu(CAA)-SVLΔ1nt (deletion of A44), which retained a slight accepting activity, the other three completely lost their accepting activities (Figure 5A). Then, five substitution mutants in the SVL of ScotRNALeu(CAA) (ScotRNALeu-A44C, -C45A, -A46C, -G47U and -C48A) were constructed. The Km values of ScoLeuRS for A44C and C48A changed only slightly, and the kcat values decreased. The Km values for C45A decreased slightly; however, the kcat value increased. The Km values for A46C and G47U increased markedly; however, the kcat values also increased. These data indicated that although the affinity of LeuRS for these tRNA mutants varied, the substitution tRNA mutants affected the catalytic efficiency only slightly (Table 2). The variable loop forms a compact core of seven base layers with the D-stem/loop (39) (Figure 2A); therefore, these results implied that the number of nucleotides on the variable loop, rather than the type of base, is more significant for this structure.

Table 2.

Aminoacylation kinetics constants of transcripts of ScoLeuRS for ScotRNALeu(CAA), (UAA) and their mutants

ScotRNALeu	K m (μM)	k cat (s−1)	k cat/Km (s−1μM−1)	k cat/Km (relative %)
(CAA) type I	0.16 ± 0.03	1.00 ± 0.01	6.25	100
-LVL	0.75 ± 0.30	0.83 ± 0.03	1.10	18
-A44C	0.16 ± 0.03	0.88 ± 0.12	5.34	85
-C45A	0.14 ± 0.03	1.14 ± 0.17	8.41	130
-A46C	0.40 ± 0.04	1.43 ± 0.03	3.58	58
-G47U	0.54 ± 0.02	2.70 ± 0.43	5.00	80
-C48A	0.10 ± 0.04	0.65 ± 0.05	6.50	104
-A14U	2.33 ± 0.37	2.02 ± 0.35	0.86	14
-A14U/U8A	4.41 ± 0.93	2.22 ± 0.09	0.50	7.9
-ΔG18/ΔG19	0.18 ± 0.01	1.27 ± 0.11	7.03	113
-ΔU55/ΔC56	0.50 ± 0.07	3.00 ± 0.32	6.00	96
-U54C:A58G	0.47 ± 0.15	2.56 ± 0.28	5.49	87
(UAA) type II	0.24 ± 0.03	3.40 ± 0.22	14.2	100
-SVL	3.80 ± 0.14	7.70 ± 0.01	2.03	14
-A14U	1.27 ± 0.09	0.38 ± 0.01	0.30	2
-A14U/U8A	3.83 ± 0.19	0.54 ± 0.06	0.14	1
-▽G19	0.09 ± 0.02	1.85 ± 0.14	21.5	151
-ΔG18	4.28 ± 0.30	12.95 ± 0.25	3.03	21
-ΔU55	1.24 ± 0.13	10.31 ± 0.53	8.31	65
-U54C:A58G	0.20 ± 0.01	0.72 ± 0.11	3.60	25

The results are the averages of three independent repeats, with the standard deviations indicated.

Figure 5.

Aminoacylation activity of ScoLeuRS and mutants of ScotRNALeu in variable arms. (A) Aminoacylation of 1 nM ScoLeuRS for 5 μM ScotRNALeu(CAA) -WT, -SVLΔ1nt, - SVLΔ2nt, - SVLΔ3nt, - SVLΔ4nt or no tRNA, respectively. (B) Aminoacylation of 1 nM ScoLeuRS for 5 μM ScotRNALeu(UAA) -WT, -LVLΔ1bp, - LVLΔ2bp, - LVLΔ3bp, - LVLΔ4bp, - LVLΔ5bp or no tRNA, respectively. (C) Aminoacylation of 1 nM ScoLeuRS for 5 μM ScotRNALeu(UAA) -WT, -LVLΔ2nt, -LVLΔ4nt or no tRNA, respectively. (D) Aminoacylation of 1 nM ScoLeuRS for 5 μM ScotRNALeu(UAA) -WT, -LVLΔ(5bp+2nt), -LVLΔ(5 bp + 4 nt) or no tRNA, respectively. The data shown represent the averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols. (E) A summary of the accepting activities of the mutants. The sequence of the variable loop is shown, and the red letters indicate the nucleotides that were deleted in the mutants.

To understand the function of the LVL of ScotRNALeu(UAA), the LVL was replaced with the SVL of ScotRNALeu(CAA) to obtain another chimeric mutant ScotRNALeu(UAA)-SVL. The Km values of ScoLeuRS for the chimera increased 16-fold compared with that for ScotRNALeu(UAA), indicating a weaker affinity of the enzyme for the tRNA mutant; however the kcat value doubled, the catalytic efficiency decreased to 14% of that for ScotRNALeu(UAA), and was one third of that recorded for ScotRNALeu(CAA) with the same variable loop (Table 2). The data showed that other structural elements of ScotRNALeu play important roles in the catalysis by ScoLeuRS. A series of deletion mutants in the LVL were constructed to investigate the role of the LVL in recognition by ScoLeuRS (Figure 5E). Successive deletions of five base pairs in the LVL produced five mutants, which all maintained their accepting capacity (Figure 5B). The catalytic rate of ScoLeuRS for the tRNA mutants, which were deleted for the first and second base pair in the LVL, was even higher than that for ScotRNALeu(UAA) (Figure 5B). The mutant of ScotRNALeu(UAA) deleted for C47b and A47g in the LVL (LVLΔ2nt) or deleted for two more, U47c and G47f (LVLΔ4nt), still retained a higher accepting activity (Figure 5C). When the five base pairs of LVLΔ2nt were deleted, the accepting activity of the mutant LVLΔ(5bp+2nt) with only four nucleotides, 47d, -e, -f, -g in the LVL, decreased markedly; LVLΔ(5 bp + 4 nt) deleted for two more nucleotides, with only two nucleotides, 47d and 47e, in the LVL, lost its accepting activity completely (Figure 5D).

Notably, the accepting capacity of ScotRNALeu(CAA) and -(UAA) were lost completely when nucleotide 48 (C48 in ScotRNALeu(CAA) and U48 in ScotRNALeu(UAA)) were deleted (data not shown). Consequently, all deletion mutants retained nucleotide 48. Taken together, these results suggested that a length of five nucleotides (including nucleotide 48) in the variable loop of either ScotRNALeu(CAA) or -(UAA) is the shortest length required for relatively high aminoacylation.

The elbow structure plays different roles in two types of ScotRNALeu

Most tRNAs have a similar L-shaped tertiary structure, which complicates the ability of AARSs to discriminate their cognate tRNAs from other tRNA species. Previous studies have shown that the U8:A14 tertiary structure base pair of EctRNALeu(CAG) and hmtRNALeu(UUR) is the identity element (4,15). The elbow structure base pairs between the D- and TψC-loops of EctRNALeu (G18:U55, G19:C56 and U54:A58) play important roles in aminoacylation (8). The possible tertiary base pairs of ScotRNALeus are shown in Figure 2 A and C. To determine whether the tertiary structure base pairs mentioned above play an important role in recognition by ScoLeuRS, several mutations at these sites were constructed.

In either ScotRNALeu(CAA) or -(UAA), substitution of A14 with U to break U8:A14 decreased their leucine accepting capacities drastically (Table 2). Although an additional U8A mutation on ScotRNALeu(CAA)-A14U or ScotRNALeu(UAA)-A14U maintained the A8:U14 base pair, the accepting activities of the mutants also decreased (Table 2), indicating that leucylation of ScotRNALeu(CAA) or -(UAA), like hmtRNALeu(UAA) and EctRNALeu, requires a precise U8:A14 pair. The Km values of ScoLeuRS for the above four ScotRNALeu mutants increased, indicating that the binding of ScoLeuRS with the four mutants was weakened compared with their cognate wild-type ScotRNALeus and that the tertiary structure base pair between U8 and A14 is important to maintain the interaction between the tRNA and the enzyme. The kcat values of the enzyme for ScotRNALeu(CAA)-A14U and -A14U/U8A increased, whereas those of ScotRNALeu(UAA)-A14U and -A14U/U8A decreased obviously, implying that the U8:A14 tertiary structure base pair has different roles in the catalysis of ScoLeuRS. The catalytic efficiency (Km/kcat) of ScoLeuRS for the two ScotRNALeu(UAA) mutants and two -(CAA) mutants decreased by varying degrees (12 and 7.9% for (CAA), 2 and 1% (UAA), respectively) compared with their cognate wild-type tRNA (Table 2). The results showed that the U8:A14 base pair in both ScotRNALeus is significant for their aminoacylation, and especially, it is more crucial in ScotRNALeu(UAA).

Our previous work indicated that G18:U55 in EctRNALeu is not as important as that of G19:C56, and the base pair U54:A58 in the TψC-loop plays a more important role in aminoacylation (8). To compare the different contributions of these elbow structure base pairs of ScotRNALeu(CAA) to aminoacylation, several deletion and substitution mutants were constructed and their kinetic parameters were assayed (Table 2). The Km and kcat values of ScoLeuRS for the deletion mutant ScotRNALeu(CAA)-ΔG18/ΔG19 were slightly higher than those of the wild-type, leading to higher catalytic efficiency, although this mutant was predicted to have an impaired tertiary interaction. The Km and kcat values for another double deletion mutant, ScotRNALeu(CAA)-ΔU55/ΔC56, and a double substitution mutant -U54C:A58G both increased, leading to only a slight change in catalytic efficiency. These results indicated that the elbow structure formed by these three base pairs does not play a crucial role in aminoacylation of ScoLeuRS for ScotRNALeu(CAA).

Compared with the D-loop sequence of ScotRNALeu(CAA), G19 is missing in that of ScotRNALeu(UAA). The tertiary structure base pairs between the TψC-loop and the D-loop in ScotRNALeu(UAA) only contain G18:U55 and U54:A58; however, it still retains a stable tertiary structure because of its higher accepting activity. We inserted G19 to form ScotRNALeu(UAA)-▿G19, which forms a new G19:C56 base pair and should stabilize the elbow structure of the L-shaped ScotRNALeu(UAA). The kcat value of ScoLeuRS for this mutant decreased by about 50%, the Km value decreased by about one third, and the catalytic efficiency of the enzyme for the mutant increased by 50%, indicating that the more stable the elbow structure, the stronger the affinity of ScoLeuRS for this mutant, leading to higher catalytic efficiency. As expected, deletion of G18 or U55 to break the hydrogen bond between them decreased the accepting capacity of these mutants. The Km values of the two deletion mutants increased, indicating weakening of the binding of ScoLeuRS to these mutants. However, the kcat values increased, suggesting a higher catalytic rate. The aminoacylation of the substitution mutant U54C:A58G declined, with a loss in its kcat value, indicating that this tertiary structure base pair at the elbow of the L-shape in ScotRNALeu(UAA) is more important than that in ScotRNALeu(CAA).

In vivo complementation assay of ScotRNALeu(UAA) matched the results in vitro

Earlier studies showed that in S. coelicolor, mutation in the gene bldA encoding ScotRNALeu(UAA) led to a ‘bald’ phenotype, in which the bacteria lack aerial hyphae and spores, as well as antibiotic production (37,40–43). Complementation of S. coelicolor with a functional bldA rescued this deficiency, which could be observed by production of the dark-blue pigment actinorhodin (38). To investigate whether the mutations that did not affect aminoacylation could restore the phenotype, a bald strain, J1501▵bldA::apra, was constructed as previously described (37) and introduced with different mutant genes of ScotRNALeu(UAA) which were recombined into the integrative vector pMS82, as described in the ‘Materials and Methods’ section (44). Wild-type bldA was used as a positive control and an empty vector as the negative control. The bldA gene contains 5′-flank and 3′-flank regions of pre-mature tRNA and a full sequence of ScotRNALeu(UAA). We mutated the given site of ScotRNALeu(UAA) in bldA to generate pMS82bldA mutants. If the tRNA transcribed by the pMS82bldA mutants could be successfully leucylated, then the mRNA containing UUA could be translated to Leu from Leu-tRNALeu during protein synthesis, otherwise the Leu-tRNALeu and correct translation could not be performed (Figure 6A). Successful restoration of the bald phenotype of J1501▵bldA::apra was inferred not only from the production of aerial mycelia and spores (data not shown), but also by the appearance of antibiotics and production of dark-blue pigment actinorhodin, as shown in Figure 6B.

Figure 6.

(A) Schematic representation of the complementation assay. (B) Complementation assay of the Streptomyces coelicolor knockout strain J1501ΔbldA::apra by different bldA genes and mutant constructs. The various constructs harbored on the integrative pMS82 plasmid were introduced into S. coelicolor, which were grown on R5 plates at 30°C for 5 days. The plates were photographed at the indicated times. The pMS82 empty vector was introduced as a negative control.

As expected, the wild-type bldA construct could restore the phenotype and the empty vector could not. Consistent with the results of the aminoacylation assay, no detectable difference was observed when comparing the negative control with the A14U and A14U/U8A mutants. Full restoration was observed for the three A73 mutants. Deletion of G18 and the crucial pair G18-U55, as well as substitution of U54-A58 to C54-G58, also failed to recover the bald phenotype. Inserting G19 induced a higher level of acitnorhodin production, which was consistent with the higher catalytic efficiency of ScoLeuRS for ScotRNALeu(UAA)-▿G19. These results matched the in vitro results for ScotRNALeu (UAA). However, we observed no restoration using ▵U55, although the in vitro assay of this mutant detected 65% of the accepting activity of the wild-type, which was probably caused by the lack of a ψ modification on this nucleotide (45).

Taken together, our results showed that the A14-U8 pair of the two types of ScotRNALeus is a mutual identity element, similar to other tRNALeus. The structural stability of the elbow formed by the D-loop and the TψC-loop is more important for type II ScotRNALeu(UAA) than for type I ScotRNALeu(CAA).

The LSD of ScoLeuRS is responsible for aminoacylation of type I tRNALeu

ScoLeuRS could leucylate the two types of ScotRNALeu with different lengths of variable loops; therefore, to reveal which domain of ScoLeuRS contributes to the charging ability to the two types of ScotRNALeu, we replaced various domains from ScoLeuRS in EcLeuRS, which only charges type II tRNALeu. However, none of the chimeras of LeuRSs from EcLeuRS and ScoLeuRS, including EcLeuRS-ScoCTD (C-terminal domain), -ScoLSD (leucine specific domain), and -ScoCP1 (CP1 domain) could charge both type I and II ScotRNALeu (data not shown), indicating the effect of the large conformational change in these chimeric enzymes from S. coelicolor (phylum Actinobacteria) and E. coli (phylum Proteobacteria) in the active site. We then turned to LeuRSs from other Streptomyces species to make the chimeric enzymes.

We screened the genomes of several Streptomyces strains and found that in the genomes of S. azureus and Streptomyces purpurogeneiscleroticus, there is no gene for type I tRNALeu, meaning that the LeuRS from these two Streptomyces strains probably cannot aminoacylate type I tRNALeu. Streptomyces azureus LeuRS (SazLeuRS) and S. purpurogeneiscleroticus LeuRS (SpurLeuRS) were obtained from E. coli transformants harboring their genes (Supplementary Figure S6A). We found that SazLeuRS could leucylate ScotRNALeu(CAA); however SpurLeuRS could not, like EcLeuRS (Supplementary Figures S6C and D). Phylogenetic analysis showed that SpurLeuRS is located on the same branch with EcLeuRS (Supplementary Figure S6B). Therefore, chimeric enzymes substituted with various domains of SpurLeuRS with that of ScoLeuRS, separately, might help to identify the domain responsible for ScoLeuRS’s ability to charge two types of ScotRNALeu.

We replaced the CTD (Leu805–Val870) of SpurLeuRS with its ScoLeuRS counterpart (Arg901–Ala967), and CP1 of SpurLeuRS (Ser234–Arg439) with its ScoLeuRS counterpart (Ser306–Arg517) (Supplementary Figure S7A). Neither of the chimeric enzymes could leucylate ScotRNALeu(CAA) (Supplementary Figure S7B and C). However, SpurLeuRS-ScoLSD, in which the LSD of SpurLeuRS (45 amino acid residues from Val594 to Glu638) was replaced with its ScoLeuRS counterpart (38 amino acid residues from Ile696 to Gly733) (Figure 7C), gained aminoacylation activity for type I tRNALeu (Figure 7B).

Figure 7.

The effects of ScoLeuRS-LSD in recognizing type I ScotRNALeus. (A) Three-dimensional view of the ScoLeuRS model showing the LSD (red) and KMSKS motifs (yellow) in the aminoacylation state. (B) Aminoacylation of 1 nM ScoLeuRS, 1 nM SpurLeuRS, 10 nM SpurLeuRS-ScoLSD or no enzyme, respectively, for 5 μM ScotRNALeu(CAA). (C) Schematic demonstration of the detailed fusion sites of SpurLeuRS-ScoLSD. The definition of LSD domain was based on the crystal structure of EcLeuRS and sequence alignment. Numbers represent the beginning and end of each LSD domain in the context of the full-length enzyme. (D and E) Aminoacylation of 10 nM SpurLRS-ScoLSD-WT, -(730–733)A, -L732A/G733A, -L732A, -L732I or no enzyme, respectively, for 5 μM ScotRNALeu(CAA) (D) or 5 μM ScotRNALeu(UAA) (E). The data shown represent averages of three independent experiments and the corresponding standard errors. Some error bars are hidden by the symbols.

We then identified the residues on ScoLSD that affected the aminoacylation activity of SpurLeuRS-ScoLSD for type I ScotRNALeu. The homology of LSDs is relatively low among various species (Supplementary Figure S9A); therefore, to narrow down our search, eight multi-alanine peptides were substituted for eight sequences from the N- to the C-terminus of ScoLSD, as shown in Figure 8. The leucylation activity of these substitution mutants for the two types of ScotRNALeus was assayed. Four substitution mutants, SpurLeuRS-ScoLSD-(696–699)A, -(700–703)A, -(709–714)A and-(715–720)A, could not charge either ScotRNALeu(UAA) or ScotRNALeu(CAA), indicating that the structure of these mutant enzymes was disrupted. The other four mutants retained their ability to charge both type I and II ScotRNALeu. Among the eight mutants, three, SpurLeuRS-ScoLSD-(704–708)A, -(721–725)A and -(726–729)A, showed high aminoacylation activity toward ScotRNALeu(UAA); however, their aminoacylation of ScotRNALeu(CAA) was reduced by about 50% (Figure 8; Supplementary Figure S8B and C). At last, the last mutant, SpurLeuRS-ScoLSD-(730–733)A at the C-terminus of ScoLSD, retained its activity for ScotRNALeu(UAA), but drastically lost its activity for ScotRNALeu(CAA) (Figure 7D and E). Thus, these sequences of ScoLSD are probably important for the recognition of ScotRNALeu(CAA).

Figure 8.

The aminoacylation of ScotRNALeu(CAA) by multiple alanine mutants of SpurLeuRS-ScoLSD. Schematic demonstration of detailed replacement sites of SpurLeuRS-ScoLSD multiple alanine mutants. The italic fonts indicate the original sequence of ScoLSD. Aminoacylation of each mutant for ScotRNALeu (CAA) and (UAA) is listed on the right compared with that of the wild-type.

A number of single-point mutants at these sites of ScoLSD were constructed. The aminoacylation assays revealed that residues from SpurLeuRS-ScoLSD-(704–708)A, -(721–725)A and -(726–729)A and three residues from SpurLeuRS-ScoLSD-(730–733)A showed no loss of aminoacylation activity of ScotRNALeu(CAA) (Supplementary Figure S9). However, SpurLeuRS-ScoLSD-L732A retained 75% aminoacylation of ScotRNALeu(UAA), but showed less than half the activity toward ScotRNALeu(CAA) (Figure 7D and E). The aminoacylation activity of the L732I mutants was not influenced, suggesting the size and hydrophobicity of side chain of L732 is important for charging type I ScotRNALeu. Notably, mutation of L732 was not sufficient to cause a reduction in activity to the same extent as SpurLeuRS-ScoLSD-(730–733)A. To test whether another amino acid residue among the four residues would strengthen the negative effect on the recognition of the enzyme to ScotRNALeu(CAA), we mutated both L732 and G733 to A. This double mutation indeed triggered a loss of leucylation of ScotRNALeu(CAA), however the mutant enzyme still leucylated ScotRNALeu(UAA).

In summary, the LSD is the key fragment that helps Streptomyces LeuRS to gain the ability to charge type I ScotRNALeu. The C-terminal residues of the LSD play an important role in distinguishing the two types of ScotRNALeu.

DISCUSSION

Coexistence of two types of tRNALeu in a single compartment

Type I tRNALeu does not exist in the same compartment with type II tRNALeu in most cases, especially in eukaryotes. The most conventional example is human cells: Type II tRNALeu exists in the cytoplasm, while type I tRNALeu exists in the mitochondria, and there is a cognate LeuRS that leucylates each tRNAleu. In prokaryotic bacteria, almost all tRNALeus belong to type II tRNA, having a long variable loop and are leucylated by their cognate LeuRSs. The present study is the first to show that one LeuRS can leucylate two types of tRNALeu in one actinomyces cell.

Notably, in eukaryotes, the two types of tRNALeu are separated by a membrane and have their own cognate LeuRS, and have optimal catalysis conditions, such as human cell cytoplasm for the hctRNAleu–hcLeuRS system and the human mitochondrial environment for hmtRNALeu–hmLeuRS system. However, S. coelicolor appears to overcome this micro-environment differences. The results showed that ScotRNALeu(CAA) and -(UAA) display similar basic features. ScotRNALeu(UAA) has a slightly lower Tm but twice the catalytic velocity of ScotRNALeu(CAA), yet their binding affinities for ScoLeuRS are equal. The higher Ttm of ScotRNALeu(CAA) might result from the additional G19-C56 pair. We also performed mis-charging and editing assays with non-cognate norvaline; the data showed that ScoLeuRS’s mischarging and editing of two types ScotRNALeu were comparable (data not shown). How ScoLeuRS binds two types of ScotRNALeu and why ScoLeuRS shows a distinct catalytic velocity prompted our interest.

However, the result of a cross-species recognition experiment indicated the specificity of LeuRS from Streptomyces, as it is the only prokaryotic LeuRS that could leucylate both type I tRNALeu and type II tRNALeu. The alignment analysis showed that ScoLeuRS has a longer sequence than other previously reported prokaryotic LeuRSs, suggesting that its more flexible structure may help to leucylate the two types of tRNALeu. Notably, ScoLeuRS cannot charge hmtRNALeu(UUR) even though the tRNALeu is a type I tRNA. We suspected that the micro-environment might affect this leucylation process. However, we cannot rule out the possibility that ScoLeuRS did not adapt to the special structure of hmtRNALeu(UUR).

The identity elements of two types of ScotRNALeu

Previous studies indicated that each type of tRNALeu has its own identity elements. ScotRNALeu(UAA) and ScotRNALeu(CAA) have some identity elements that are shared by earlier reported tRNALeus, such as the A14-U8 pair for both ScotRNALeus. However, this is the first report that the A73 residue of ScotRNALeus is not the identity element recognized by its cognate ScoLeuRS. Mutagenesis of ScoLeuRS also showed that the activity of ScoLeuRS was not affected by interaction with A73, as it is in EcLeuRS. The structure of the aminoacylation state of EcLeuRS-EctRNALeu(UAA) showed the A73 on EctRNALeu(UAA) was flipped and interacted with a conserved peptide, 416R/KLRDWGVSRQRYWG429 (22). In the ScoLeuRS-ScotRNALeu system, A73 may not be flipped to interact with this peptide. The crystal structure of EcLeuRS- EctRNALeu(UAA) also displayed the interaction between A73 or the G1-U72 pair of tRNA and peptide 290–298 of CP1 domain (22). However, our data suggested that mutants ScoLeuRS-V385A, -E386A and -R387A (in which the residues are homologs of A291, E292 and A293 of EcLeuRS, respectively. See Supplementary Figure S10A.) could leucylate ScotRNALeu similarly to the wild-type (data not shown). In addition, the residues of peptide 290–298 in EcLeuRS display low homology to those of ScoLeuRS (Supplementary Figure S10A). Hence, the structure formed by these residues of EcLeuRS may differ from those of ScoLeuRS. Our data suggested that C74 in the CCA tail remains tightly bound to LeuRS and is important for leucylation of ScotRNALeus. Thus, C74 seems to be the new identity element for recognition of ScoLeuRS.

The tertiary structure of tRNA associates the D/TψC-loop interaction with a compact core of seven base layers (39). Our results suggested that D/TψC-loop interaction plays different roles regarding different types of ScotRNALeu, even though the base pairs are similar. The core of seven base layers, which require the residues to form a variable loop, appeared distinct from ScotRNALeu(CAA) and -(UAA). Previous crystal structure studies indicated that the variable loop in type II tRNA would comprise a hairpin, while type I tRNA would not (22,24,46). The complex of EcLeuRS-EctRNALeu(UAA) implied an interaction between the CTD of EcLeuRS and the 47f-47i nucleotide of EctRNA (22); the complex of Thermus thermophilus LeuRS (TtLeuRS) and TttRNALeu(CAG) implied an interaction between R826 of TtLeuRS and C47e of TttRNA, although this tRNA was deleted for two base pairs of the variable loop (24). Our data further confirmed that deletion of all base pairs on the variable loop did not harm the aminoacylation of ScotRNA Leu(UAA). The residues that contacted with tRNA in EcLeuRS-EctRNALeu system had a relatively low homology with those of ScoLeuRS (Supplementary Figure S10B); therefore, we suspected that there is no contact between CTD of ScoLeuRS and the variable loop of ScotRNALeu. The variable loop participates in the core of seven base layers in type I ScotRNALeu(CAA) more than it does in ScotRNALeu(UAA). However, altering the bases of the variable loop of ScotRNALeu(CAA) showed no drastic loss of amino acid accepting activity. We concluded that the 5 nt length of the variable loop is important for aminoacylation.

The unique linkers in ScoLeuRS to help to aminoacylate two types of tRNALeu

LeuRS is a multi-domain class at AARS that comprises a main enzyme body (Rossmann-fold catalytic domain and class Ia anticodon-binding domain) and four flexibly linked additional domains, termed ZN1, CP1, LSD and CTD (17). We swapped the other three linked domains - CP1, CTD and LSD of ScoLeuRS, to another SpurLeuRS that could not leucylate type I ScotRNALeu to form three chimeric LeuRSs. Notably, aminoacylation assays of chimeric SpurLeuRS-ScoCP1 showed drastic loss of activity for both ScotRNALeu(CAA) and -(UAA), indicating that the CP1 domain plays a crucial role in aminoacylation. Previous studies of EcLeuRS and TtLeuRS hinted us that CP1 is a flexible structure (22,24,47), probably acting as an important regulating domain during aminoacylation and engaging in cross-talk with other domains (48). We suspected that a CP1 domain from other species would fail for this function, thus abrogating the aminoacylation activity of LeuRS. The CTD domain of EcLeuRS was also suggested to contact with C56 of TψC loop, A20a of D loop and 47f to 47i of the variable loop of tRNA by crystal structure analysis (22). However, the results implied that the chimeric enzyme SpurLeuRS-ScoCTD did not charge this type I ScotRNALeu. Moreover, ScoLeuRS showed high activity for mutant ScotRNALeu(CAA)-ΔU55/ΔC56 (Table 2). We concluded that the CTD does not specifically contribute to aminoacylation of ScotRNALeu. We then asked which domain of ScoLeuRS contributes to aminoacylation of type I ScotRNALeu? Our results showed that only the LSD help SpurLeuRS to gain the ability to charging type I ScotRNALeu. The crystal structure of EcLeuRS showed that the LSD is crucial for positioning the conserved catalytic signature sequence, the KMSKS loop, during aminoacylation reactions (20,24). However, the structure of the LSD varies from species to species. The three-dimensional structure of the TtLeuRS showed its LSD exhibits five β-strands and two short α-helices (24). In comparison, the LSD of EcLeuRS contains an additional extended β-hairpin (22). This diversity implies that the LSD might be related to LeuRS’s species specificity. Our results indicated that the LSD from ScoLeuRS contributes the capacity for charging type I tRNALeu. However, how the LSD works when an armless tRNALeu binds with LeuRS is not clear. The biochemical experiments showed that a gross deletion of LSD abolished the aminoacylation activity of EcLeuRS (21). A previous crystal structure study showed that coupling of the LSD with the KMSKS loop located the tRNA in the correct place (22), suggesting that the KMSKS loop goes through a conformational change from an open state to a semi-open state; therefore, one could hypothesize that the LSD acts as a regulatory peptide to adjust the position of KMSKS to bind with tRNALeu. For type I tRNALeu, the LSD in ScoLeuRS helps KMSKS to bind and then performs aminoacylation.

Our results further showed that mutation of both L732 and G733 at the C-terminus of the LSD in ScoLeuRS caused SpurLeuRS-ScoLSD to lose its charging capability of tRNALeu(CAA). Interestingly, L732 and G733 link the LSD and KMSKS motifs. Additionally, leucine and glycine are both flexible residues that can better adjust the location of adjacent peptides. The loss of the aminoacylation activity of LeuRS after substituting both L732 and G733 might have resulted from an inability to correctly locate the KMSKS motif. The hypothesis that LSD properly locates KMSKS to leucylate type I tRNALeu further supported our original assumption that ScoLeuRS contains a regulatory peptide to adjust each domain to the right position to bind type I tRNALeu.

The coevolution of LeuRS and tRNALeu

While analyzing the genomes of Streptomyces strains, we noticed that some strains have LeuRSs highly similar to ScoLeuRS, but lack a type I tRNALeu. A model, SazLeuRS, showed its capability of charging ScotRNALeu(CAA), suggesting one of them evolved before the other. However, no reasonable explanation has been proposed for this observation so far. We may have a provided clue as to why type I tRNALeu, such as ScotRNALeu(CAA) and hmtRNALeu(UUR), exist. The ancient tRNALeu probably belonged to type I tRNA and then evolved to type II tRNA, for whom LeuRS has a higher catalytic efficiency. The type I tRNALeu disappeared in response to some unknown pressure; however, some Streptomyces and human mitochondria did not encounter such pressure and thus retained the type I tRNALeu. Despite this unsolved question, we provided evidence showing that mitochondria originated from prokaryotes. The LeuRS from human mitochondria also recognized and charged two types of ScotRNALeu. The structural stability at the elbow region of the type I ScotRNALeu(CAA) is not so important to aminoacylation as it is for type II ScotRNALeu(UAA), which also illustrated the structural flexibility of type I tRNALeu, a feature of hmtRNALeu(UUR) showed in earlier studies (14,15).

Another subtle clue that these two types of ScotRNALeu probably function similarly came from previous in vivo studies related to secondary metabolism. ScotRNALeu(UAA) is encoded by a secondary metabolism regulatory gene in S. coelicolor, bldA (40). The bldA knockout strain of S. coelicolor displayed a phenotype that lacked spores and antibiotics production (49). bldA is widespread among Streptomyces species. In this study, we proved that a functional bldA gene is essential for secondary metabolism. A bldA gene with a G19 insertion, whose accepting activity was elevated in vitro, showed higher production of actinorhodin, suggesting a modified tRNALeu could help to refine the metabolic process.

A previous study showed that a single TTA codon is mistranslated efficiently in Streptomyces clavuligerus and the authors hypothesized that this codon might be translated by tRNALeu(CAA) (50), given that there is a former report showing that this isoacceptor also reads UUA codons in the absence of a UUA reader in Escherichia coli (51). Furthermore, previous data demonstrated that the modes of accumulation of ScotRNALeu(CAA) and ScotRNALeu(UAA) are similar (38). Most interestingly, ScotRNALeu(CAA) is the only isoacceptor that can replace ScotRNALeu(UAA) after altering its anticodon to UAA (38). Taken together, the results suggested that these two isoacceptors are related. However, ScotRNALeu(CAA), unlike ScotRNALeu(UAA), cannot be deleted from the S. coelicolor genome. Our effort to knockout the gene encoding ScotRNALeu(CAA) failed. This is probably related to the high codon usage of UUG (the codon deciphered by ScotRNALeu(CAA)). There are 6706 UUG codons in the S. coelicolor genome, but only 145 UUA codons (the codon deciphered by ScotRNALeu(UAA)). In addition, the higher G+C content in the S. coelicolor genome makes UUA a rare codon, unlike UUG. In present study, the biochemical evidence suggests how these two types of ScotRNALeu resemble each other. Thus, we hypothesized that evolutionarily, ScotRNALeu(CAA) and (UAA) were likely to have the same function until ScotRNALeu(UAA) evolved a more stable structure to completely replace ScotRNALeu(CAA).

In summary, our results provided several comparisons between these two isoacceptors. ScotRNALeu(CAA) has lower amino accepting capacity than ScotRNALeu(UAA). The major difference between ScotRNALeu and other reported tRNALeu is that they abrogate the canonical discriminator A73. However, C74 shows more contact with ScoLeuRS. They share the U8-A14 tertiary base pair as the identity elements. In addition, the more stable the tertiary structure at the elbow of L-shape, the higher the accepting activity of ScotRNALeu(UAA). The stem part in the long variable loop of ScotRNALeu(UAA) is not necessary for its leucylation; however, the number of nucleotides in the variable loop of either type I or type II ScotRNALeu is crucial to their charging activities. By swapping domains, we showed that the LSD is specific and crucial for ScoLeuRS charging of ScotRNALeu(CAA) and identified the key amino acid residues at C-terminus of the LSD for that function. Our study provided a deeper understanding of how two types of tRNAs work together in a prokaryotic single cell.

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