A transcriptional activating region with two contrasting modes of protein interaction

Abstract

A C-terminal segment of the yeast activator Gal4 manifests two functions: When tethered to DNA, it elicits gene activation, and it binds the inhibitor Gal80. Here we examine the effects on these two functions of cysteine and proline substitutions. We find that, although certain cysteine substitutions diminish interaction with Gal80, those substitutions have little effect on the activating function in vivo and interaction with TATA box-binding protein (TBP) in vitro. Proline substitutions introduced near residues critical for Gal80 binding abolish that interaction but once again have no effect on the activating function. Crosslinking experiments show that a defined position in the activating peptide is in close proximity to TBP and Gal80 in the two separate reactions and show that binding of the inhibitor blocks binding to TBP. Thus, the same stretch of amino acids are involved in two quite different protein-protein interactions: binding to Gal80, which depends on a precise sequence and the formation of a defined secondary structure, or interactions with the transcriptional machinery in vivo, which are not impaired by perturbations of either sequence or structure.

Figure 1

Activator and a reporter. (A) A Gal4 derivative comprising the N-terminal, 100-residue DNA binding domain (dark box) fused to the C-terminal, 42-residue activating region (the shaded box). (B) A chromosomally integrated reporter bearing five consensus 17-bp Gal4 binding sites 191 bp upstream of the gal1 promoter, which is fused to the LacZ reporter gene.

Figure 2

The effect of substituting cysteine for wild-type residues on activation and on TBP binding. (A) Reporter gene expression elicited by each of the cysteine substitution mutants as assayed in Gal80⁺ cells grown in galactose. (B) A GST pull-down experiment. ³⁵S-labeled Gal4 derivatives synthesized in vitro (lanes 1 and 3) and retained by immobilized GST–TBP (lanes 2 and 4). (C) The products of each cysteine derivative synthesized and ³⁵S-labeled in vitro. (D) Binding of Gal4 cysteine derivatives to immobilized GST–TBP. (E) Four surface plasmon resonance sensograms describing binding of immobilized Gal4 derivatives with yeast TBP as measured on a BIAcore machine (9, 21). In each case, the difference in RU values between the three plateaus at the points marked by the asterisks gives a value proportional to the relative binding affinity of the two proteins. The averaged ratio of TBP (ΔRU_TBP) retained by each Gal4 (ΔRU_GAL4) derivative is: wild-type, 2.02 ± 0.3; T858C, 1.84 ± 0.25; T859C, 1.45 ± 0.3; T860C, 2.06 ± 0.27.

Figure 3

The effect of substituting cysteine for wild-type residues on Gal80-mediated inhibition in vivo and interaction with Gal80 in vitro. (A) Reporter gene expression elicited by each of the cysteine substitution mutants of Fig. 1B as assayed in Gal80⁺ cells grown in raffinose. (B) Electrophoretic mobility super-shift assays performed with recombinant Gal80 and Gal4 derivatives. Gal4 derivatives were bound to a single DNA binding site at saturating concentrations of 10 nM (lanes marked −), and Gal80 was added in adjacent lanes (lanes marked +) at 15 nM concentration. At this inhibitor concentration, the two Gal4 mutants, F856C and T859C, show a 3- to 4-fold decrease in their affinity for the Gal80.

Figure 4

The effects of substituting proline for wild-type residues in Gal4 on activation, inhibition by Gal80, and on Gal80 synthesis. This figure shows reporter gene activity elicited by various proline mutants in cells grown in either galactose (A) or raffinose (B). In each case, a mutant bearing a cysteine substitution at the identical position was tested similarly in parallel. Immunoblot studies measured endogenous Gal80 levels in cells expressing either cysteine or proline mutants and grown in raffinose (C) or galactose (D).

Figure 5

Photoaffinity crosslinking of Gal4 to TBP and to Gal80. ¹²⁵I-APDP-Gal4 was incubated with purified Gal80 (lane 2), with purified yTBP (lane 3), and with both proteins (lane 4). In each case, after UV-induced crosslinking, the label was transferred by treatment with a reducing agent as described in ref. (the minor band in lanes 2 and 4 is a degradation product of Gal80).