Signaling through regulated transcription factor interaction: mapping of a regulatory interaction domain in the Myb-related Bas1p.

Gene activation in eukaryotes is inherently combinatorial depending on cooperation between different transcription factors. An example where this cooperation seems to be directly exploited for regulation is the Bas1p/Bas2p couple in yeast. Bas1p is a Myb-related transcription factor that acts together with the homeodomain-related Bas2p (Pho2p) to regulate purine and histidine biosynthesis genes in response to extracellular purine limitation. We show that fusion of the two factors abolished adenine repression, suggesting that what is regulated by adenine is the Bas1p-Bas2p interaction. Analysis of Bas1p deletions revealed a critical domain (Bas1p interaction and regulatory domain, BIRD) acting in two-hybrid assays as an adenine-dependent Bas1p-Bas2p interaction domain. BIRD had a dual function, as an internal repressor of a centrally located Bas1p transactivation domain on the ADE1 promoter and as a Bas2p-dependent activator on the HIS4 promoter. This promoter-dependent behavior reflected a differential binding to the two promoters in vivo. On ADE1 Bas1p bound the promoter efficiently by itself, but required adenine limitation and Bas2p interaction through BIRD for derepression. On HIS4 efficient promoter binding and derepression required both factors and adenine limitation. We propose a promoter-dependent model for adenine regulation in yeast based on controlled Bas1p-Bas2p interactions through BIRD and exploited differentially by the two promoters.

Flexibility in the second half-site sequence recognised by the c-Myb R2 domain--in vitro and in vivo analysis.

The oncoprotein c-Myb is a transcription factor that recognises its specific target sequences through two subdomains. The R3-domain binds the first half-site, YAAC, and plays a dominant role in sequence recognition, while the homologous R2-domain interacts with a more loosely defined sequence in the second half-site. The difficulty in precisely defining a preferred second half-site sequence might reflect the flexible nature of R2 which only attains its fully folded structure upon binding to DNA, a process that might allow the protein to adapt to different half-site sequences. Here we report that shifting the most conserved base in the second half-site, the G6, into position 5 resulted only in a minor reduction of complex stability in vitro. From an analysis of a series of second half-site variants by EMSA and DMS-interference, we conclude that the preferred recognition sequence should be revised to read [YAACNG or YAACGN]. Modeling the structure of c-Myb R2R3 in complex with a GT half-site variant revealed specific interactions with G5. When second half-site variants were tested in vivo using a sensitive yeast effector-reporter system, both the TG and GT half-site variants were functional mediating c-Myb-dependent transactivation. Unexpectedly, we observed large differences between the best second half-site variants at low levels of c-Myb-effector, the GG variant being five- to fifteen-fold more active in vivo than the single-G half-sites, the GH or HG variants.

DNA-binding domain and recognition sequence of the yeast BAS1 protein, a divergent member of the Myb family of transcription factors.

The yeast BAS1 protein is a transcriptional activator with an amino-terminal domain homologous to the DNA-binding domain of the oncoprotein Myb containing three imperfect tryptophan-rich repeats. In contrast to Myb-related transcription factors from higher eukaryotes, where the second and third repeat constitutes a minimal independent DNA-binding domain, all three repeats of BAS1 were found to be necessary for sequence-specific DNA binding. Moreover, an active DNA-binding subdomain was obtained only if the first repeat was enlarged in the amino-terminal direction to include 3 tryptophans and a 23-amino acid insertion and if 55 amino acids carboxyl-terminal to the third repeat were included. The BAS1 DNA-binding site was analyzed in detail and found to cover 8-9 base pairs with no similarity to the Myb recognition element. The binding site included a conserved hexameric TGACTC motif, the methylation of which abolished BAS1 binding, as well as a 3-base pair extension that seemed to have a modulatory effect on BAS1 affinity and where binding was less affected by methylation.

Two functionally distinct half sites in the DNA-recognition sequence of the Myb oncoprotein.

The oncoprotein Myb is a sequence-specific DNA-binding protein with a pivotal function in the development and proliferation of hematopoietic precursor cells. A minimal DNA-binding domain composed of two tryptophan-rich repeats R2 and R3 is responsible for sequence recognition. Based on model building and mutational analysis, Myb was proposed to recognise its target through a double helix-turn-helix (HTH)-related motif using two recognition helices, one in R2 and one in R3. We found, by mutational analysis, that the DNA-binding site for c-Myb is functionally bipartite. While the first half site is dominant and absolutely required for binding, the second half site is only modulatory and mainly affects the half life of the complex. This bipartite nature of the binding site parallels the proposed bipartite structure of R2R3 with two HTH-related domains. Analysis of the DNA-binding site of R2R3 by missing-base interference-footprint analysis showed that the protein interacted with a 9-bp region. The same was found with a larger protein containing all three repeats. The effect of adding R1 was mainly to stabilise the complex. The borders of the complex, as revealed by exonuclease III footprinting, did not change due to the presence of R1. However, both borders became more refractory to the nuclease when R1 was present, but with a difference that suggested a specific orientation of the repeat domains relative to the DNA-binding site. We propose that the first half site is recognised by R3, while the second modulatory half site interacts with the R2 repeat.

The time courses of intracellular transport of some secretory proteins of rat liver are not affected by an induced acute phase response.

To study the effects of changed synthesis on intracellular transport of secretory proteins, an acute phase response was induced in rats. The synthesis and secretion of haptoglobin, complement C3, transferrin and albumin were then investigated by pulse labeling with [3H]leucine. Maximal increase in the syntheses of the positive acute phase proteins was observed after 24 h, amounting to an increase of nine, three and twofold for haptoglobin, C3 and transferrin, respectively. The synthesis of albumin decreased to a minimum after 48 h, reaching approximately one fourth of normal synthesis. The time courses for transit through rough endoplasmic reticulum and for secretion were determined after 36 h, and were found to be roughly unchanged for all four proteins despite the different changes in synthesis. The fraction of haptoglobin associated with the microsomal membrane was reduced during the acute phase response, but there was no significant change in membrane association as a function of time after labeling with [3H]leucine. It is concluded that the altered protein synthesis during an acute phase response in vivo has little effect on the time course of secretion of the proteins studied. Furthermore, the basal mechanisms for intracellular transport appear relatively unchanged during this condition.