Figure 2.

Structure of the 5' leader of the SAG1 transcript regulates its translation. (a) Distribution of SAG1 mRNA across polysome gradients in growing cells (filled circles) or cells treated with α-factor for 30 min (open circles). Cell lysates [3] were loaded onto 7-47% sucrose gradients and spun for 1.5 hours in a SW40 rotor at 39,000 rpm at 4°C. Levels of SAG1 transcript in each gradient fraction were determined by real-time polymerase chain reaction (QPCR) and the signal in each fraction was divided by the sum of the signals in all fractions. The top of the gradient is to the left and the position of the 80S monosome is marked with the arrow. (b) Northern blot analysis of SAG1 RNA from growing cells (lanes 1 and 2) or from cells after 45 min of α-factor treatment (lanes 3 and 4). Equal cell equivalents of RNA from pooled sucrose gradient fractions 1-14 (lanes 1 and 3) or pooled fractions 15-25 (lanes 2 and 4) were analyzed. (c) Relative levels of SAG1 mRNA at different time points after treatment with α-factor. Total RNA was isolated from cells treated with α-factor for the indicated times and cDNA was produced with reverse transcription by priming with oligo(dT)₂₅. SAG1 transcript levels were determined by QPCRusing either primers recognizing both transcripts or primers specific for the long transcript. Closed circles show the values for the long form and the open circles represent calculated values for the short SAG1 transcripts, computed as the differences between the values for both transcripts and those for the long transcript. The curves are normalized to a value of 1.0 for the long transcript at zero time of treatment. (d) RNase protection assay showing two forms of SAG1 : lane 1, probe only; lane 2, no RNA; lane 3, tRNA control; lanes 4 and 5, 50 µg total RNA from growing cells; lanes 6 and 7, 50 µg total RNA from cells treated for 30 min with α-factor; and lane 8, RNA markers. The antisense RNA probe was prepared from cloned genomic sequence and contained 55 nucleotides of open reading frame, 484 nucleotides of 5' leader, and 92 nucleotides of noncomplementary sequence. Independent RNA preparations were used in lanes 4-7. Locations of the protected probes corresponding to SAG1 long 5' leader (539 nucleotides) and short 5' leader (95 nucleotides) are indicated. (e) Western blot analysis of protein extracts from growing cells was performed to determine the relative levels of His3-HA protein from yeast strains transformed with reporter constructs containing the ADH1 promoter and SAG1 short 5' leader (lane 1), SAG1 long 5' leader (lane 3), or the empty vector (lane 2). The arrow indicates location of the His3-HA protein. As indicated in the figure, lanes 2 and 3 had 10 times more protein loaded than did lane 1.