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A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Home P, Mukherjee S, Adhya S - Nucleic Acids Res. (2008)

Bottom Line: RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III).Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo.These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

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tRNA-binding activity of RIC8A mutants. (A and B) Gel-shift assays using 32P-labeled tRNAIle (tRI) (10 fmol) and wild-type (RIC8A) or indicated mutant recombinant proteins (100 fmol). RNP, ribonucleoprotein. (C) Examples of Scatchard plots for mutants H1 and P2. b, concentration of bound tRI; f, concentration of free RNA. (D) Dissociation constants (Kd, nM) of complexes of tRI with indicated RIC8A derivatives. All variants are recombinant proteins expressed in E. coli. P1–P7 are derived from clone N (Figure 1) spanning residues 1–82.
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Figure 2: tRNA-binding activity of RIC8A mutants. (A and B) Gel-shift assays using 32P-labeled tRNAIle (tRI) (10 fmol) and wild-type (RIC8A) or indicated mutant recombinant proteins (100 fmol). RNP, ribonucleoprotein. (C) Examples of Scatchard plots for mutants H1 and P2. b, concentration of bound tRI; f, concentration of free RNA. (D) Dissociation constants (Kd, nM) of complexes of tRI with indicated RIC8A derivatives. All variants are recombinant proteins expressed in E. coli. P1–P7 are derived from clone N (Figure 1) spanning residues 1–82.

Mentions: RIC8A has intrinsic affinity for a subset of tRNAs—the type II tRNAs—that are imported into mitochondria (8). To localize the tRNA-binding site within the N-terminal domain, deletion and point mutations were generated (Figure 1A), the derivatives expressed in E. coli, and the recombinant proteins tested for binding to tRNAIle, a type II tRNA (14), by gel-shift assay. Mutant D1 in which the N-terminal 33 residues had been deleted was active for tRNA binding, whereas D2 and D3, with N-terminal deletions mapping to residues 46 and 66, respectively, were inactive (Figure 2). Mutants D4–D6 have internal deletions with the same right endpoint (residue 81) and different left endpoints (residues 71, 50 and 40, respectively). D4 and D5 had the normal affinity for tRNAIle, whereas D6 was inactive (Figure 2B). Taken together, these results indicate that the region between residues 33 and 50 is critical for tRNA binding, which is almost coincident with the position of helix α1 (residues 36–49). This helix contains three basic amino acid residues spaced 3 residues apart, and therefore oriented on the same side of the helix (Figure 1). Point mutants K42A, R45A and R48A, and deletion mutant H1 lacking helix α1, all had reduced or abolished tRNA-binding activity (Figure 2B); quantitative analysis showed an ∼4-fold increase in the apparent dissociation constant (Kd) of the complex in each case, compared to the wild-type protein (Figure 2C and D). In contrast, tRNA binding was not affected in the point mutant K51A, in the linker region, or in mutants K54A, K57A and R61A, or in deletion mutant H2 (Figure 2B and D), mapping to helix α2. These results confirm the role of the α1 helix in the interaction of RIC8A with tRNA. Moreover, each positive residue located on the hydrophilic surface contributes to the docking of the tRNA substrate. Thus, the divergence of the RIC8A domain to Lys42 has resulted in increase in the RNA-binding affinity (and perhaps specificity) of the protein.Figure 2.


A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica.

Home P, Mukherjee S, Adhya S - Nucleic Acids Res. (2008)

tRNA-binding activity of RIC8A mutants. (A and B) Gel-shift assays using 32P-labeled tRNAIle (tRI) (10 fmol) and wild-type (RIC8A) or indicated mutant recombinant proteins (100 fmol). RNP, ribonucleoprotein. (C) Examples of Scatchard plots for mutants H1 and P2. b, concentration of bound tRI; f, concentration of free RNA. (D) Dissociation constants (Kd, nM) of complexes of tRI with indicated RIC8A derivatives. All variants are recombinant proteins expressed in E. coli. P1–P7 are derived from clone N (Figure 1) spanning residues 1–82.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: tRNA-binding activity of RIC8A mutants. (A and B) Gel-shift assays using 32P-labeled tRNAIle (tRI) (10 fmol) and wild-type (RIC8A) or indicated mutant recombinant proteins (100 fmol). RNP, ribonucleoprotein. (C) Examples of Scatchard plots for mutants H1 and P2. b, concentration of bound tRI; f, concentration of free RNA. (D) Dissociation constants (Kd, nM) of complexes of tRI with indicated RIC8A derivatives. All variants are recombinant proteins expressed in E. coli. P1–P7 are derived from clone N (Figure 1) spanning residues 1–82.
Mentions: RIC8A has intrinsic affinity for a subset of tRNAs—the type II tRNAs—that are imported into mitochondria (8). To localize the tRNA-binding site within the N-terminal domain, deletion and point mutations were generated (Figure 1A), the derivatives expressed in E. coli, and the recombinant proteins tested for binding to tRNAIle, a type II tRNA (14), by gel-shift assay. Mutant D1 in which the N-terminal 33 residues had been deleted was active for tRNA binding, whereas D2 and D3, with N-terminal deletions mapping to residues 46 and 66, respectively, were inactive (Figure 2). Mutants D4–D6 have internal deletions with the same right endpoint (residue 81) and different left endpoints (residues 71, 50 and 40, respectively). D4 and D5 had the normal affinity for tRNAIle, whereas D6 was inactive (Figure 2B). Taken together, these results indicate that the region between residues 33 and 50 is critical for tRNA binding, which is almost coincident with the position of helix α1 (residues 36–49). This helix contains three basic amino acid residues spaced 3 residues apart, and therefore oriented on the same side of the helix (Figure 1). Point mutants K42A, R45A and R48A, and deletion mutant H1 lacking helix α1, all had reduced or abolished tRNA-binding activity (Figure 2B); quantitative analysis showed an ∼4-fold increase in the apparent dissociation constant (Kd) of the complex in each case, compared to the wild-type protein (Figure 2C and D). In contrast, tRNA binding was not affected in the point mutant K51A, in the linker region, or in mutants K54A, K57A and R61A, or in deletion mutant H2 (Figure 2B and D), mapping to helix α2. These results confirm the role of the α1 helix in the interaction of RIC8A with tRNA. Moreover, each positive residue located on the hydrophilic surface contributes to the docking of the tRNA substrate. Thus, the divergence of the RIC8A domain to Lys42 has resulted in increase in the RNA-binding affinity (and perhaps specificity) of the protein.Figure 2.

Bottom Line: RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III).Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo.These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

View Article: PubMed Central - PubMed

Affiliation: Genetic Engineering Laboratory, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, India.

ABSTRACT
Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix-turn-helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix alpha1 contacts tRNA whereas helix alpha2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import.

Show MeSH
Related in: MedlinePlus