Structure and function of theTRP3gene ofSaccharomyces cerevisiae: Analysis of 3′- and 5′-deletions in vivo and in vitro

✍ Scribed by Markus Aebi; Rolf Furter; Franziska Prantl; Peter Niederberger; Ralf Hütter

• Publisher: Springer-Verlag
• Year: 1984
• Tongue: English
• Weight: 899 KB
• Volume: 8
• Category: Article
• ISSN: 0172-8083
• DOI: 10.1007/bf00417813

✦ Synopsis

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 2pro-vector. The N-terminal glutamine amidotransferase 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 bifunctional 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 express a peptide exhibiting functional indole-3-glycerolphosphate synthase activity in vitro. Deletion plasmids pME505"DelC102"2pm and DelC10.2pm exhibited shorter TRP3 transcripts according to the deleted DNAfragments (150 and 426 bp 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.