Compositions of tRNA and uses thereof
Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNA's, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNA's/synthetases are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins using these orthogonal pairs.
1. A composition comprising a tRNA having the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3, and the full length complementary polynucleotide sequence that encodes SEQ ID NO: 1, 2 and 3 in a buffer.
2. The composition of claim 1 , wherein said tRNA is aminoacylated.
3. The composition of claim 2 , wherein said tRNA is aminoacylated with a non-naturally encoded amino acid.
4. The composition of claim 3 , wherein said non-naturally encoded amino acid is para-acetylphenylalanine.
5. The composition of claim 2 , wherein said tRNA is chemically aminoacylated.
6. The composition of claim 2 , wherein said tRNA is enzymatically aminoacylated.
7. The composition of claim 6 , wherein said tRNA is enzymatically aminoacylated by an aminoacyl tRNA synthetase.
8. The composition of claim 6 , wherein said tRNA is enzymatically aminoacylated by a ribozyme.
9. The composition of claim 1 , further comprising an aminoacyl tRNA synthetase, wherein the tRNA is aminoacylated with an amino acid.
10. The composition of claim 9 , wherein said amino acid is a non-naturally encoded amino acid.
11. The composition of claim 10 , wherein said non-naturally encoded amino acid is para-acetylphenylalanine.
12. The composition of claim 1 , wherein the tRNA is derived from an archael tRNA.
13. The composition of claim 1 , wherein the tRNA is derived from M. janneschii.
14. The composition of claim 1 , further comprising a translation system.
15. The composition of claim 14 , wherein said translation system is a cell-free translation system.
16. The composition of claim 14 , wherein said translation system is a cell lysate.
17. The composition of claim 14 , wherein said translation system is a reconstituted system.
18. The composition of claim 14 , wherein said translation system is a cellular translation system.
19. A cell comprising a translation system, wherein the translation system comprises the tRNA of claim 1 .
20. The cell of claim 19 , wherein the cell is a eukaryotic cell.
21. The cell of claim 20 , wherein the eukaryotic cell is a yeast cell.
22. The cell of claim 20 , wherein the eukaryotic cell is a fungal cell.
23. The cell of claim 20 , wherein the eukaryotic cell is a mammalian cell.
24. The cell of claim 20 , wherein the eukaryotic cell is an insect cell.
25. The cell of claim 20 , wherein the eukaryotic cell is a plant cell.
26. The cell of claim 19 , wherein the cell is a non-eukaryotic cell.
27. The cell of claim 26 , wherein the non-eukaryotic cell is an E. coli cell.
28. The cell of claim 19 , further comprising a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon that is recognized by the tRNA.
29. The cell of claim 28 , wherein said polypeptide of interest is human growth hormone.
30. An E. coli cell comprising the tRNA of claim 1 .
31. A yeast cell comprising the tRNA of claim 1 .
32. A fungal cell comprising a tRNA of claim 1 .
33. A mammalian cell comprising a tRNA of claim 1 .
34. An insect cell comprising a tRNA of claim 1 .
35. A plant cell comprising a tRNA of claim 1 .
36. A vector comprising a polynucleotide encoding a tRNA having the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, 2, and 3, and the full-length complementary polynucleotide sequence that encodes SEQ ID NO: 1, 2 and 3.
37. The vector of claim 36 , wherein the vector comprises a plasmid, a cosmid, a phage, or a virus.
38. The vector of claim 36 , wherein the vector is an expression vector.
39. A cell comprising the vector of claim 36 .
40. A method of producing a polypeptide in a cell with a selected amino acid at a specified position, the method comprising: growing, in an appropriate medium, the cell, where the cell comprises a nucleic acid that comprises at least one selector codon and encodes a polypeptide; and, providing the selected amino acid; wherein the cell further comprises: an orthogonal tRNA (O-tRNA) that functions in the cell and recognizes the selector codon having the amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, and the full-length complementary polynucleotide sequence that encodes SEQ ID NO: 1, 2 and 3; and, an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein said O-RS aminoacylates the O-tRNA with the selected amino acid.
41. The method of claim 40 wherein said selected amino acid is para-acetyl phenylalanine.
42. The method of claim 40 wherein said polypeptide is human growth hormone.
43. A polynucleotide having a nucleic acid sequence selected from a group consisting of SEQ ID Nos. 1, 2, and 3, and the full-length complementary polynucleotide sequence thereof.