Phosphorylated proteins play important roles in the regulation of many different cell networks. However, unlike the preparation of proteins containing unmodified proteinogenic amino acids, which can be altered readily by site directed mutagenesis and expressed in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro studies, we have explored the use of phosphorylated amino acids in which the phosphate moiety bears a chemical protecting group, thus eliminating the negative charges that have been shown to have a negative effect on protein translation. Bis-o-nitroberizyl protection of tyrosine phosphate enabled its incorporation into DHFR. and I kappa B-alpha using wild-type ribosomes, and the elaborated proteins could subsequently be deprotected by photolysis. Also investigated in patallel was the re-engineering of the 23S rRNA of Escherichia coli, guided by the use of a phosphorylated puromycin, to identify modified ribosomes capable of incorporating unprotected phosphotyrosine into proteins from a phosphotyrosyl-tRNA(CUA) by UAG codon suppression during in vitro translation. Selection of a library of modified ribosomal clones with phosphorylated puromycin identified six modified ribosome variants having mutations in nucleotides 2600-2605 of 23S rRNA; these had enhanced sensitivity to the phosphorylated puromycin. The six clones demonstrated some sequence homology in the region 2600-2605 and incorporated unprotected phosphotyrosine into I kappa B-alpha using a modified gene having a TAG codon in the position corresponding to amino acid 42 of the protein. The purified phosphorylated protein bound to a phosphotyrosine specific antibody and permitted NF-kappa B binding to a DNA duplex sequence corresponding to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated I kappa B-alpha also mediated the exchange of exogenous DNA into an NF-kappa B-cellular DNA complex isolated from the nucleus of activated Jurkat cells.