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Results: 5

1.
Fig. 1.

Fig. 1. From: Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.

Interaction between R1-NTD and R1-CTD. (A) Schematic alignment of the S. cerevisiae Rnr1 and Rnr3 proteins with the E. coli R1. The thiyl radical-generating cysteine (Cys-439 in E. coli and Cys-428 in yeast) and the cysteine pair at the C-terminal end are shown. Both Rnr1 and Rnr3 have a CI region. R1-CTD refers to the entire C-terminal region including the CI and the CX2C motif. (B and C) Interaction between R1-CTD and R1-NTD in a yeast two-hybrid system. Plasmids expressing the Gal4 activating domain (ACT) domain alone (Vec) and ACT fusion proteins with full-length Rnr1 (R1-FL) and the C-terminal 765–888 region of Rnr1 (R1-CTD) were cotransformed in pairs with plasmids expressing the Gal4 DNA-binding domain (DBD) alone (Vec) and DBD fusion proteins with the full-length (R1-FL) and the N-terminal 778- and 768-residue regions of R1. Growth of cotransformants on SC-TrpLeu and SC-TrpLeuAde plates is shown in B. Activities of the LacZ reporter were measured in Miller units in C.

Zhen Zhang, et al. Proc Natl Acad Sci U S A. 2007 February 13;104(7):2217-2222.
2.
Fig. 2.

Fig. 2. From: Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.

The CX2C motif of the S. cerevisiae Rnr1 is essential for viability. (A) Schematic drawings of the substitutions and deletions introduced into the S. cerevisiae Rnr1. (B) Plasmid shuffle complementation assay. Shown is the 5-FOA plate after 2 days of incubation at 30°C for transformants of the RNR1 shuffle strain MHY784 (rnr1Δ rnr3Δ URA3CENRNR1) containing the TRP1CEN vector or test plasmid expressing (Myc)3-tagged wild-type and mutant Rnr1 proteins from the RNR1 promoter. (C) Comparison of protein abundance of different RNR1 alleles. The (Myc)3-Rnr1 proteins were detected on a Western blot by using the 9E10 antibody (α-Myc). Glucose-6-phosphate 1-dehydrogenase (G6PDH by α-Zwf1) was also probed on the same blot as a loading control. (D) FACS analysis of cells harboring the full-length (FL) and the CI-deleted (ΔCI) alleles of RNR1 from asynchronous (Asy) or synchronized cultures after release from an α-factor-mediated G1 arrest.

Zhen Zhang, et al. Proc Natl Acad Sci U S A. 2007 February 13;104(7):2217-2222.
3.
Fig. 3.

Fig. 3. From: Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.

Interallelic complementation between the catalytically inactive rnr1(C428S) and the CX2C-deficient rnr1(SX2S) mutant alleles. (A) Growth on a 5-FOA plate after 2 days of incubation at 30°C for transformants of the RNR1 shuffle strain MHY784 containing the following RNR1 plasmids: wild-type (WT), rnr1(C428S), rnr1(CX2C-to-SX2S), and rnr1(CX2C-to-SX2S) in combination with rnr1(C428S). (B) Comparison of plating efficiency of the rnr1Δ rnr3Δ double mutant carrying the indicated RNR1 alleles on the rich medium YPD. Cells from a log phase culture of each strain were measured for density by using a hemocytometer and diluted so that ∼300 cells were plated on each plate. All plates were incubated at 30°C for 2 days before comparison of colony formation. (C) Comparison of protein levels between full-length and CI-deleted Rnr1 mutant proteins. Protein extracts were prepared from cells harboring the indicated rnr1 mutant alleles. The HA-tagged Rnr1(C428S) and Rnr1(C428S, ΔCI), and (Myc)3-tagged Rnr1(SX2S) and Rnr1(ΔCI, SX2S) were detected on a Western blot by using anti-HA and anti-Myc antibodies, respectively. G6PDH (Zwf1) was probed on the same blot as a loading control.

Zhen Zhang, et al. Proc Natl Acad Sci U S A. 2007 February 13;104(7):2217-2222.
4.
Fig. 5.

Fig. 5. From: Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.

A model for the role of R1-CTD in regeneration of R1 and its inhibition by Sml1. (a–d) Steps of R1 active-site regeneration by means of an interchain cross-talk between the C terminus of one R1 monomer and the active site of its neighboring monomer. For simplicity, only one active site and one CX2C motif are highlighted for each pair of monomers. (a) The cysteine residue shown at the top of the R1 active site is converted to a transient thiyl radical through electron relay from the tyrosyl radical located in R2 (8). At the completion of each turnover cycle, a disulfide bond is formed between the conserved redox-active cysteine pair at the active site, thereby inactivating the enzyme (R1ox, the oxidized form). (b–d) Rereduction of the R1 active site is mediated by the C-terminal CX2C motif that shuttles reducing equivalents from thioredoxin (Trx) or glutaredoxin (Grx) through two disulfide-exchange steps, resulting in an active enzyme (R1red, the reduced form). (e) Sml1 competes against R1-CTD in the association with R1-NTD and thus interferes with the regeneration of the active site.

Zhen Zhang, et al. Proc Natl Acad Sci U S A. 2007 February 13;104(7):2217-2222.
5.
Fig. 4.

Fig. 4. From: Role of the C terminus of the ribonucleotide reductase large subunit in enzyme regeneration and its inhibition by Sml1.

Interaction of the R1-NTD with both Sml1 and R1-CTD. (A) Increased interaction of Sml1 with R1-NTD relative to R1-FL. Plasmids expressing the Gal4 ACT domain alone (pACT) and the ACT-Sml1 fusion protein (pACT-SML1) were cotransformed in pairs with plasmids expressing the Gal4 DBD fusion proteins with the full-length Rnr1 (Rnr1-FL) and the R1-NTD (residues 1–778). Activities of the LacZ reporter were measured in Miller units. (B) Overexpression of Sml1 compromises the interaction between R1-NTD and R1-CTD. Cells harboring pDBD-R1NTD and pACT, or pDBD-R1NTD and pACT-R1CTD were transformed with a plasmid expressing a TDH3 promoter-driven SML1 or the vector control. Activities of the LacZ reporter were measured in Miller units. (C) Substitutions of the WE 688–689 sequence motif in R1-NTD alter its interaction with both Sml1 and R1-CTD. (Upper) Sequence alignment of the region including the S. cerevisiae Rnr1 Tyr-688 and Glu-698 residues among the R1s of E. coli, human, and S. cerevisiae. (Lower) Two-hybrid interactions assayed with LacZ activities between the DBD-R1NTD fusion proteins containing the wild-type sequence (WE) or the WE-to-AD mutation, and ACT vector (Vec) and ACT fusion proteins with Sml1 or the R1-CTD (residues 765–888). (D) The WE-to-AD mutation at residues 688–689 of Rnr1 causes SML1-dependent lethality. Shown is the 5-FOA plate after 2.5 days of incubation at 30°C for transformants of the shuffle strains MHY784 (Upper) and MHY802 [rnr1Δ rnr3Δ sml1Δ URA3CENRNR1 (Lower)], all containing a TRP1CEN plasmid that carries the wild-type (WE) or the WE-to-AD mutant alleles of RNR1. (E) Overexpression of Rnr1 increases the Sml1 protein level. The endogenous Sml1 protein levels of wild-type cells harboring a plasmid expressing a TDH3 promoter-driven RNR1 or the vector control (vec) were probed on a Western blot. G6PDH (Zwf1) was probed on the same blot as a loading control. (F) The rnr1(WE-to-AD) mutant allele stabilizes the Sml1 protein after hydroxyurea (HU) treatment. Wild-type cells bearing an extra copy of the wild-type RNR1 or the rnr1(WE-to-AD) mutant allele, both under the control of the RNR1 promoter, were treated with 150 mM hydroxyurea, and the endogenous Sml1 levels were probed on a Western blot at the indicated time points after the addition of hydroxyurea. G6PDH (Zwf1) was probed on the same blot as a loading control.

Zhen Zhang, et al. Proc Natl Acad Sci U S A. 2007 February 13;104(7):2217-2222.

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