Constitutive and Nitrogen Catabolite Repression-sensitive Production of Gat1 Isoforms

Premature termination ofGAT1transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production

“…S5). Together with the observation that altering ATG M1 did not lead to a detectable phenotype, 44 the lack of overall conservation of the full-length open reading frame among S. cerevisiae S288C's closest relatives suggested that the 1st in-phase methionine in the S288C sequence of Gat1 did not function in vivo under the conditions we used in our experiments.…”

“…Interestingly, neither of these isoforms required the first in-frame methionine for their production. 44 Indeed, using amino acid substitutions, we showed that translation of Gat1 is initiated at M40 and M95 relative to the beginning of the open reading frame. 44 Phylogenetic analysis was carried out by comparing the amino acid sequences deduced from the open reading frames of Gat1 orthologs from 25 S. cerevisiae strains available on Saccharomyces Genome Database ( Fig.…”

“…38,43 In light of this background, our objective in the present work was to investigate 2 paradoxical observations: (i) full length Gat1 protein levels are unaffected by the cell's environmental nitrogen status, i.e., glutamine vs. proline, rather than being produced in the NCR-sensitive manner observed for steady state GAT1 mRNA levels, 33 and (ii) NCR-sensitive Gat1 protein production is observed when translation is artificially prematurely terminated, about midway through the protein. 44 To this end, we have characterized the GAT1 mRNA levels across the locus and identified an unexpected decrease in those levels using 3 0 vs. 5 0 GAT1 probes, suggesting the existence of a premature transcription termination that could account for the lack of correlation between GAT1 mRNA and Gat1 protein levels. Remarkably, synthesis of all GAT1 mRNA species, both constitutive and NCR-sensitive, was Gln3-dependent.…”

“…Remarkably, synthesis of all GAT1 mRNA species, both constitutive and NCR-sensitive, was Gln3-dependent. RACE PCR analyses identified different termini for the GAT1 transcripts: (i) 3 major 5 0 GAT1 mRNA termini, correlating with the detection of 2 fulllength, constitutively produced protein species beginning at 2 different translation start sites, 44 and (ii) 2 major 3 0 GAT1 mRNA termini, correlating with one small, NCR-sensitive GAT1 and one full length, constitutive GAT1 mRNA species. The site for premature transcription termination at the GAT1 locus has been defined, and the possible physiological significance investigated.…”

“…Western blotting was carried out as described previously 43,44 using anti-myc (Santa Cruz Biotechnology, 9E10), anti-ha (Santa Cruz Biotechnology, F-7) and anti-pgk1 (Invitrogen) antibodies.…”