Positional control of selenium insertion in polypeptides for X-ray crystallography

A method for determining the structure of a heterologous polypeptide that does not contain selenocysteine in its native form, said method comprising

• (1) transfecting a cell with (i) a first nucleic acid encoding said polypeptide, wherein at least one, specific codon of mRNA transcribed from said first nucleic acid is replaced by the codon UGA, and (ii) a second nucleic acid operably linked to said first nucleic acid, said second nucleic acid directing the translation of said UGA codon as selenocysteine only when said cell can obtain selenium from the medium in which said cell is grown, wherein said second nucleic acid is derived from the 3' untranslated region of a gene encoding mammalian glutathione peroxidase and comprises a continuous stretch of at least 79 nucleotides comprising three stem elements, each having a 5' half and a 3' half, and three loop elements, each having a 5' end and a 3' end,
• wherein the stem elements comprise
• a) a base stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides,
• b) a lower stem comprising at least 16 nucleotides that can form 8 complementary pairs of nucleotides, the first nucleotide of the 5' half of the lower stem being bound to the last nucleotide of the 5' half of the base stem, and the first nucleotide of the 3' half of the lower stem being bound to the last nucleotide of the 3' half of the base stem, and
• c) an upper stem comprising at least 22 nucleotides that can form 11 complementary pairs of nucleotides,
• wherein the loop elements comprise
• d) a first loop consisting of 5'-AUGRG-3' (SEQ ID NO:2), the 5'-A being bound to the last nucleotide of the 5' half of the lower stem and the 3'-G being bound to the first nucleotide of the 5' half of the upper stem,
• e) a second loop consisting of 5'-YRNNNNUAV-3' (SEQ ID NO:3), the 5'-Y being bound to the first nucleotide of the 3' half of the upper stem and the 3'-V being bound to the last nucleotide of the 3' half of the lower stem, and
• f) a third, apical loop consisting of 5'-ARANNNN-NNNN-3' (SEQ ID NO:4), the 5'-A being bound to the last nucleotide of the 5' half of the upper stem and the 3'-N being bound to the last nucleotide of the 3' half of the upper stem, and wherein each A is adenine, G is quanine, N is adenine, guanine, cytosine, or uracil, R is quanine or adenine, U is uracil, V is any nucleotide except thymidine or uracil, and Y is uracil or cytosine;
• (2) growing said cell in selenium-containing growth medium under conditions in which said cell incorporates at least one selenocysteine residue into said polypeptide at a specific location;
• (3) isolating said polypeptide from said cell or said growth medium;
• (4) forming a crystal of said polypeptide;
• and
• (5) performing X-ray crystallography on said crystal, wherein said selenocysteine residue is used to determine the structure of said heterologous polypeptide.