A downstream regulatory element located within the coding sequence mediates autoregulated expression of the yeast fatty acid synthase gene FAS2 by the FAS1 gene product

Abstract

The fatty acid synthase genes FAS1 and FAS2 of the yeast Saccharomyces cerevisiae are transcriptionally co-regulated by general transcription factors (such as Reb1, Rap1 and Abf1) and by the phospholipid-specific heterodimeric activator Ino2/Ino4, acting via their corresponding upstream binding sites. Here we provide evidence for a positive autoregulatory influence of FAS1 on FAS2 expression. Even with a constant FAS2 copy number, a 10-fold increase of FAS2 transcript amount was observed in the presence of FAS1 in multi-copy, compared to a fas1 null mutant. Surprisingly, the first 66 nt of the FAS2 coding region turned out as necessary and sufficient for FAS1-dependent gene expression. FAS2-lacZ fusion constructs deleted for this region showed high reporter gene expression even in the absence of FAS1, arguing for a negatively-acting downstream repression site (DRS) responsible for FAS1-dependent expression of FAS2. Our data suggest that the FAS1 gene product, in addition to its catalytic function, is also required for the coordinate biosynthetic control of the yeast FAS complex. An excess of uncomplexed Fas1 may be responsible for the deactivation of an FAS2-specific repressor, acting via the DRS.

Figure 1

FAS1-dependent steady-state concentration of the FAS2 mRNA. For the northern blot hybridization shown, total RNA was isolated from strains with identical FAS2 copy number (single chromosomal copy, chr. copy) but varying FAS1 gene dosage. Strains IKY1 (ura3 pra1 FAS1 FAS2, lanes 3 and 4) and IKY3 (ura3 pra1 Δfas1 FAS2, lanes 1 and 2) were transformed with plasmids YEp352 (vector control, 2 µm URA3, lanes 1 and 3) or pJS229 (copy number n; 2 µm URA3 FAS1, lanes 2 and 4) and subsequently grown in SCD-Ura, supplemented with fatty acids. The filter was simultaneously hybridized against FAS2 and ACT1 (internal control) DNA probes. Quantification of signal intensities was done by phosphoimager analysis. After background subtraction, the ratio of PSL values (photo stimulated luminescence) for FAS2 and ACT1 signals was calculated for each lane (PSL_FAS2/PSL_ACT1; lane 1, 0.15; lane 2, 1.39; lane 3, 0.76; lane 4, 1.9).

Figure 2

Specific FAS activity in transformants with varying FAS1 and FAS2 gene dosage. Strain IKY1 (ura3 leu2 pra1 FAS1 FAS2) was transformed in pairs with combinations of empty vectors (YEp352, 2 µm URA3; YEp351, 2 µm LEU2; total FAS copy numbers 1), or multi-copy plasmids (pJS229, 2 µm URA3 FAS1; pJS225, 2 µm LEU2 FAS2; total FAS copy numbers n). Transformants were grown in selective medium (SCD-Ura-Leu). Specific FAS activity is given in nanomoles NADPH oxidized per minute per milligram of protein (mU/mg). Standard deviation of the mean value is indicated by bars.

Figure 3

Deletion analysis of the FAS2 coding region responsible for FAS1-dependent gene expression. Deletion constructs contain identical upstream sequences (–1008) but differ with respect to the length of the FAS2 reading frame, fused to lacZ. Reporter plasmids [ARS CEN TRP1 FAS2(1/x)–lacZ; data are given in nucleotide positions] were transformed into fas1 mutant strain IKY3, containing either the empty vector YEp352 (FAS1 copy number 0) or FAS1 multi-copy plasmid pJS229 (copy number n). Transformants were grown in selective medium (SCD-Ura-Trp), supplemented with fatty acids. Specific β-galactosidase activities are given in nanomoles ONPG hydrolyzed per minute per milligram of protein. Standard deviation was ≤25% of the mean value. Af, activation factor of specific enzyme activity in the presence of multiple FAS1 copies, compared with the null mutant.

Figure 4

FAS1-dependent expression of FAS2–lacZ reporter constructs under heterologous promoter control. Various sequences of the FAS2 coding region (indicated by nucleotide positions) were inserted between the MET25 promoter and the lacZ reporter gene. To ensure similar translational efficiency, reading frame fragments with an artificial start codon contain the natural –6/–1 sequence of FAS2. Reporter plasmids [ARS CEN TRP1 MET25–FAS2(x/y)–lacZ] were transformed into fas1 mutant strain IKY3, containing either the empty vector YEp352 (FAS1 copy number 0) or FAS1 multi-copy plasmid pJS229 (copy number n). For a maximal promoter strength, transformants were grown in selective medium without methionine (SCD-Ura-Trp-Met), supplemented with fatty acids. Specific β-galactosidase activities are given in nanomoles ONPG hydrolyzed per minute per milligram of protein. Standard deviation was ≤25% of the mean value. Af, activation factor of specific enzyme activity in the presence of multiple FAS1 copies, compared to the null mutant.

Figure 5

(A) Hypothesis on coordinate control of FAS genes by Fas1-dependent anti-repression of FAS2 gene expression. In the absence of non-complexed Fas1 (β-subunit), the FAS2 control region (including downstream sequences) is substantially weaker than the FAS1 promoter. An excess of free β-subunit may directly or indirectly deactivate the repressor (Rep. X; acts via the DRS), leading to maximal FAS2 expression and subsequent synthesis of a balanced amount of α-subunit (FAS2 gene product), which may then associate with β to form a functional FAS complex (α₆β₆). Thereby, withdrawal of β again reduces FAS2 expression. The hypothesis considers FAS1 expression as the leading and FAS2 expression as the lagging step of FAS complex formation. It is unknown whether anti-repression acts at the level of transcriptional initiation or elongation. (B) Hypothetical stem–loop structure within FAS2 mRNA. Nucleotide positions refer to the start of the FAS2 reading frame.