The mutant with mutation in the gene (CEG1) coding for the guanylyltransferase subunit of the capping enzyme shows an elongation-defective phenotype. (A) The ceg1-63 mutant, one of the temperature-sensitive alleles of the gaunylyltransferase subunit, displays an allele-specific sensitivity to 6AU and caffeine. The 10-fold serial dilutions of the cells (A₆₀₀ = 1.0) from the wild-type CEG1 allele (YSB242) or the indicated strains carrying the ceg1 temperature-sensitive alleles (YSB228, ceg1-12; YSB229, ceg1-34; YSB230, ceg1-63; YSB231, ceg1-237; YSB232, ceg1-250) were applied as spots to SC−uracil medium containing 50-μg/ml 6AU or 10 mM caffeine. As a control, the same dilution was applied as spots to the YPD medium. Photographs were taken after growth at 30°C for 3 days (6AU and no drug) or 4 days (caffeine). The yeasts in the 6AU test are URA3 as provided by pRS316. (B) The ceg1-63 mutant strain was defective in the PUR5 induction. The yeast cells, as described in panel A, were grown at 30°C in the SC−uracil medium containing 2% glucose. When growth reached 0.5 to 0.8 U at A₆₀₀, the culture was divided into two parts and grown in the presence or absence of 6AU (50 μg/ml). The RNA was purified from each sample, and the induction of PUR5 was analyzed by RT-PCR, as described in Materials and Methods. The level of the SED1 transcript was included as an internal control. Each band was quantified, normalized by SED1 level, and compared to the value of the wild type obtained at an hour of treatment of 6AU (lane 3 for ceg1 mutants or lane 21 for rpb2/ppr2, assigned as 1). The experiments were repeated three times, and a representative experiment is shown. +, samples treated with 6AU. RT-PCR with RNA from DY108 (rpb2-10/ppr2Δ) and its isogenic parent DY103 (RPB2/PPR2) was included as a control.

The in vitro Ceg1-GMP formation assay shows that the ceg1-63 allele has reduced catalytic activity. Immunoprecipitation (IP) was performed with Ceg1 polyclonal antibody (top panel) or Cet1 antibody (middle panel) from the total extract (200 μg) of each strain grown at 30°C. The beads were pelleted and washed extensively. Capping enzyme in the pellet was detected by both an autoradiogram of enzyme-GMP formation (Ceg1-GMP*) and immunoblotting with anti-Cet1 antibody (α-Cet1). −, immunoprecipitation without antibody. Whole-cell extracts (60 μg) were probed with Ceg1 antibody (bottom panel).

ceg1-63 produces the GAL1 transcript with a higher steady-state level following the glucose shutoff. Each of the wild type CEG1 and mutant ceg1-63 and ceg1-250 strains was grown in SC medium supplemented with raffinose at 30°C. The culture was induced with galactose for an hour prior to the glucose treatment. The steady-state level of the GAL1 transcript at the indicated time points after glucose addition was analyzed by RT-PCR. −, GAL1 level from the cells grown in raffinose prior to galactose induction. The level of the SED1 transcript was included as described for Fig. 1B. The signal was quantified as in the legend to Fig. 1, normalized with SED1, and compared to that of the wild type obtained by an hour of treatment of galactose (lane 2 was considered as 100%).

The ceg1-63 allele is synthetically lethal with rpo21 mutations that are specifically compromised in transcription elongation. (A) The ceg1-250 and ceg1-63 mutants were analyzed in combination with the rpo21 elongation-defective mutant upon shuffling pYF1561 (empty vector), pYF1866 (RPO21), pYF1868 (rpo21-17), pYF1867 (rpo21-7), pYF1863 (rpo21-23), and pYF1864 (rpo21-24) into YC9 and YC50, respectively. For the genetic interaction assay with rpb1-18 and rpb1-15, the YSB516 shuffling strain was used for the ceg1-250 strain (31). Growth of the transformants carrying a double mutation were assayed by applying spots of a 10- or 100-fold dilution of an A₆₀₀ of 0.5 culture onto plates containing SC plus fluoroorotic acid (FOA) and incubating for 3 days at 30°C. (B) The 6AU- and caffeine-sensitive phenotype was not suppressed by overexpression of PPR2. CEG1, pRS316-CEG1; PPR2, YEpPPR2; empty, pRS426.

The ceg1-63 cell is unable to transcribe passing through the pause sites. (A) The reporter constructs containing a tandem array of the pause Ia sequence under the galactose-inducible promoter (GAL1) is shown. The arrowhead represents the orientation of the cloned Ia pause. The region flanked by two flags was amplified by RT-PCR. (B) The reporter assay was performed with the CEG1 wild type (YSB242) and the ceg1-250 (YSB232) and ceg1-63 (YSB230) mutants. Each strain was grown in SC−uracil medium containing raffinose. The reporter was induced by 2% galactose for an hour. RNA from each cell culture was purified and analyzed by RT-PCR with the primers CYC1TERf and CYC1₄₅₄, which were annealed immediately downstream of the pause. The RNA level was quantitated as in Fig. 1, normalized with SED1, and compared to that of wild type obtained in lane 4 (considered as 100%).

Schematic model of how capping enzyme plays a role in an early transcription.