A stb1Δ mutant is defective in MCB::lacZ but not SCB::lacZ expression. (A) Wild-type (BY263) and stb1Δ (BY806) cells were transformed with the SCB::lacZ plasmid (pBA251, striped bars) or the MCB::lacZ plasmid (pBA487, solid bars). In MCB::lacZ assays, stb1Δ cells were also transformed with a 2μm STB1 (2μ STB1) plasmid (pBA1010) or a vector plasmid (pRS425). Strains were grown at 30°C to log phase, cell lysates were prepared, and β-galactosidase activity was measured. Depicted activity values represent the means of three experiments, and error bars indicate standard deviations for three experiments. (B) Stb1 localizes to MCB promoter elements. Wild-type (BY263), stb1Δ (BY806), mbp1Δ (BY551), and swi6Δ (BY185) strains containing a vector plasmid (pLGΔSS) (V) or an MCB::lacZ construct (pBA487) (M) were grown to mid-log phase and cross-linked with formaldehyde. Whole-cell extracts (WCE) were prepared (lanes 1 to 16) and subjected to immunoprecipitation (IP) using α-Stb1 affinity-purified antibodies (α-Stb1 IP) (lanes 17 to 24). PCR was performed on immunoprecipitated samples and on twofold serial dilutions (1× and 0.5×) of the WCE samples to amplify associated DNA. Primers used in this assay hybridized to the 3′ end of the URA3 gene and to sequences upstream of lacZ in both vector (pLGΔSS) and MCB::lacZ (pBA487) plasmids. (C) Stb1 does not localize to SCB promoter elements. Wild-type (BY263) strains containing a vector plasmid (pLGΔSS) or an SCB::lacZ construct (pBA251) were grown to mid-log phase and cross-linked with formaldehyde. Whole-cell extracts (WCE) were prepared (lanes 1 to 3 and 7 to 9) and subjected to immunoprecipitation (IP) using α-Stb1 affinity purified antibodies (α-Stb1-IP) (lanes 4 to 6 and 10 to 12). PCR was performed on immunoprecipitated samples and on twofold serial dilutions of the WCE samples to amplify associated DNA. The primers used to amplify associated DNA were the same as those described for panel B.

Growth characteristics of the stb1Δ bck2Δ double mutant. (A) Slow-growth phenotype of a stb1Δ bck2Δ mutant strain. Tenfold serial dilutions were prepared from wild-type (BY263), stb1Δ (BY805), bck2Δ (BY1110), and stb1Δ bck2Δ (BY1106) cultures, plated onto YEPD medium, and incubated at 30°C. (B) Morphology of wild-type, stb1Δ, bck2Δ, and stb1Δ bck2Δ strains. Wild-type (BY263), stb1Δ (BY805), bck2Δ (BY1110), and stb1Δ bck2Δ (BY1106) strains were grown to mid-log phase in rich medium. The cells were viewed with Nomarski optics and photographed. (C) DNA content as measured by FACS analysis of samples shown in panel B. The positions of cells with G₁ or G₂ DNA contents are indicated by 1N or 2N, respectively. The percentages of budded and unbudded cells are indicated below the FACS profile.

RNR1 rescues the lethality of a stb1Δ mec1 mutant. A stb1Δ mec1 strain (BY1830) containing a STB1-URA3 plasmid (pBA1220) and harboring either a STB1-LEU2 (pBA1010), MEC1-LEU2 (pBA1624), RNR1-LEU2 (pBA1625), or vector (pRS425) plasmid was grown in the absence (SD-LEU) or presence (SD-LEU + 5-FOA) of 5-FOA.

Interaction with Swi6 is required for Stb1 function. (A) Uninduced (lanes 1, 3, and 5) and IPTG-induced (lanes 2, 4, and 6) bacterial whole-cell extracts were prepared from cells expressing full-length STB1 (pBA1622; lanes 1 and 2), STB1ΔN (pBA1282; lanes 3 and 4) and STB1ΔC (pBA1623; lanes 5 and 6). Extracts were subjected to SDS-PAGE, and duplicate gels were transferred to nitrocellulose. One blot was probed with α-Stb1 antibodies (α-Stb1), while a second blot was probed with in vitro-translated [³⁵S]methionine Swi6 (³⁵S-Swi6), as indicated to the left of the photograph. (B) Anti-Stb1 Western blot analysis of extracts prepared from a stb1Δ mutant (BY806) harboring a p415 MET25 vector (lane 1) or a STB1 (pBA1044, lane 2), STB1ΔN (pBA1626, lane 3), or STB1ΔC (pBA1627, lane 4) plasmid. Cultures were grown to mid-log phase in medium lacking methionine. (C) Wild-type (wt) (BY263) and stb1Δ (BY806) cells were transformed with the MCB::lacZ plasmid (pBA487). The stb1Δ strain was also transformed with a vector (p415 MET25) or a STB1 (pBA1044) or STB1ΔN (pBA1626) plasmid. Strains were grown at 30°C to log phase in the absence of methionine, cell lysates were prepared, and β-galactosidase activity was measured. Depicted activity values represent the means of three experiments, and error bars indicate standard deviations for three experiments. (D) A stb1Δ swi4Δ (BY1693) mutant containing a SWI4-URA3 plasmid and harboring either a vector (p415 MET25) or a STB1-LEU2 (pBA1044) or STB1ΔN-LEU2 (pBA1626) plasmid was grown in the absence (SD-MET) or presence (SD-MET + 5-FOA) of 5-FOA. (E) Tenfold serial dilutions were prepared from a wild-type (BY263) and a stb1Δ cln3Δ (BY822) strain harboring a vector (p415 MET25) or a STB1 (pBA1044) or STB1ΔN (pBA1626) plasmid, plated onto medium lacking methionine, and incubated at 30°C.

A model for Stb1-dependent regulation of MBF transcription. Our model predicts that Stb1 functions in an alternative pathway to activate G₁-specific transcription. Cln3-Cdc28 is the major activator of SBF- and MBF-dependent transcription, whereas Stb1 and Bck2 function as alternate activators of Start transcription. Unlike Cln3 and Bck2, Stb1 specifically regulates MBF. Prior to Start, Stb1 is predominantly unphosphorylated and interaction with Swi6 serves to activate MBF-dependent transcription (solid lines). Immediately following the time of maximal Start-specific transcription, Cln-Cdc28-dependent phosphorylation of Stb1 mediates dissociation of the Stb1-MBF complex, resulting in termination of MBF-specific transcription (dotted lines).

SWI4 is required for viability of a stb1Δ deletion strain. (A) stb1Δ (BY1298), swi4Δ (BY184), and stb1Δ swi4Δ (BY1693) mutants harboring a high-copy SWI4-URA3 plasmid (pBA314) were grown on SD-URA medium or on SD medium containing 5-FOA. (B) stb1Δ (BY1298), swi4Δ (BY184) and stb1Δ swi4Δ (BY1694 and BY1695) mutants containing a SWI4-URA3 plasmid and harboring either a SWI4-LEU2 (pBA417), MET25pr-STB1-LEU2 (pBA1044), or vector (pRS425) plasmid were grown in the absence (SD-MET) or presence (SD-MET + 5-FOA) of 5-FOA.

MBF-regulated genes are repressed in a stb1Δ swi4Δ double mutant. (A) Anti-Stb1 Western blot analysis of extracts prepared from a stb1Δ swi4Δ strain harboring a MET25pr-STB1 plasmid (BY1695; lanes 1 to 5), a wild-type (wt) strain (BY263; lane 6), and a stb1Δ strain (BY806; lane 7). Log-phase stb1Δ swi4Δ strain plus MET25pr-STB1 cultures were grown in the absence of methionine (lane 1) and subsequently grown in medium supplemented with 5 mM methionine (lanes 2 to 5). Wild-type and stb1Δ cultures were also grown in medium supplemented with 5 mM methionine (lanes 6 to 7). (B) A stb1Δ swi4Δ strain containing a MET25pr-STB1 construct (BY1695) was grown to mid-log phase in the absence of methionine. Cultures were then diluted and grown in the absence or presence of 5 mM methionine. cDNAs derived from cultures grown in the absence of methionine were labeled with Cy3 fluor, while cDNAs from cultures grown in 5 mM methionine were labeled with a Cy5 fluor. The results for a subset of genes whose expression levels changed at least threefold are shown. Green indicates methionine-dependent repression, while red indicates genes induced in the presence of 5 mM methionine. The reciprocal experiment was also performed (data not shown). (C) RNR1 is repressed in a stb1Δ swi4Δ double mutant. Wild-type (wt) (BY263), stb1Δ (BY806), swi4Δ (BY184), and stb1Δ swi4Δ (BY1695) mutants (containing a MET25pr-STB1 plasmid) were grown to mid-log phase in the absence of methionine (t = 0). Strains were subsequently grown in minimal medium containing 5 mM methionine. Aliquots were taken at 4- and 8-h time points. Total RNA was isolated from cells and probed with radiolabeled RNR1 and ACT1. The histogram depicts quantitation of the Northern blot. The RNR1 signal was quantified by phosphorimager analysis, and the values were normalized to the ACT1 loading control before plotting.

Stb1 localizes to the RNR1 promoter. Wild-type (wt) (BY263), stb1Δ (BY806), mbp1Δ (BY551), and swi6Δ (BY185) strains were grown to mid-log phase and cross-linked with formaldehyde. Cross-linked and non-cross-linked (NX) whole-cell extracts (WCE) were prepared and subjected to immunoprecipitation (IP) with α-Stb1 antibodies (Stb1-IP). PCR was performed on serial dilutions of the WCE samples (lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18) and on immunoprecipitated samples (lanes 4 to 5, 9 to 10, 14 to 15, and 19 to 20) to amplify associated DNA. Primers used for multiplex PCR were designed to flank the PHO5 promoter or the MCB elements of the RNR1 promoter.

Cln1- and Cln2-dependent phosphorylation of Stb1 inhibits the Swi6-Stb1 interaction in vitro. Bacterially expressed GST-Stb1 was phosphorylated in vitro with Cdc28-Cln1 (lane 1) or Cdc28-Cln2 (lane 4) purified from insect cells. Phosphorylated Stb1 (lanes 1 and 4) and unphosphorylated Stb1 (lanes 2 and 5) were subjected to SDS-PAGE. Duplicate gels were transferred to nitrocellulose. One blot was probed with α-Stb1 antibodies (α-Stb1). A second blot was probed with in vitro-translated [³⁵S]methionine Swi6 (³⁵S-Swi6).