The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA
X. Tang et al., The tryptophan biosynthesis gene cluster trpCDEGFBA from Pyrococcus kodakaraensis KOD1 is regulated at the transcriptional level and expressed as a single mRNA, MOL G GENET, 262(4-5), 1999, pp. 815-821
The entire gene cluster encoding enzymes involved in biosynthesis of L-tryp
tophan in the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 has
been cloned and sequenced. Seven ORFs, which encode indole-3-glycerol phosp
hate synthase (trpC), anthranilate phosphoribosyltransferase (trpD), the tw
o subunits of anthranilate synthase (trpEG), phosphoribosyl :anthranilate i
somerase (trpF) and the two subunits of tryptophan synthase (trpAB), were i
dentified. The gene order is trpCDEGFBA, covering a region of 6045 bp. In o
rder to confirm the function of the gene products, we expressed the first g
ene, Pk-trpC, in Escherichia coli. The protein product was purified, and wa
s found to show the expected indole-3-glycerol phosphate:synthase activity,
with a temperature optimum of 85 degrees C. We could clearly identify a si
ngle mRNA transcript by Northern analysis using probes in the central and 3
'-regions of the gene cluster, indicating that the gene cluster is transcri
bed as an operon. A significant increase in try mRNA level was observed in
cells grown in medium depleted of L-tryptophan, compared to cells grown in
medium supplemented with L-tryptophan: indicating that expression of the ge
ne cluster is regulated at the transcriptional level.