Two sets of deletions, entering the TRP3 gene of Saccharomyces cerevisiae from the 3'- and the 5'-end were constructed. Complementation analysis with chromosomal trp3A, trp3B and trp3C mutations was done by introducing the 3'- and 5'-truncated gene on a multicopy 2 μm-vector. The N-terminal glutamine amido transferase function is encoded by a DNA fragment of 600-700 bp, and the C-terminal indole-3-glycerol-phosphate synthase function by a DNA fragment of about 900 bp, whereas both functions together are encoded by a contiguous DNA fragment of about 1,500 bp. The bi functional TRP3-peptide thus could be dissected into two catalytically independent peptides in vivo.For the indole-3-glycerol-phosphate synthase activity, independent catalytic activity was also demonstrated in vitro: deletions entering the TRP3 gene from the 5'-end, and lacking large parts of the sequence coding for the glutamine amidotransferase function, still are able to ex press a peptide exhibiting functional indole-3-glycerol phosphate synthase activity in vitro. Deletion plasmids pME505·De1C102·2μm and DelC10·2μm exhibited shorter TRP3 transcripts according to the deleted DNA-fragments (150 and 426 by respectively) but yielded peptides of invariable Mr of 35,000 d. Transcription and translation of these peptides, which probably represent the independently folding indole-3-glycerol-phosphate synthase core are discussed.

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Journal Article

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Aebi M, Furter R, Prantl F, Niederberger P, Hütter R

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