Conserved Mechanisms Of Transcriptional Regulation From Yeast To Mammals

In this chapter we consider gene regulation in organisms ranging from single-ceiled yeast to mammals. All these organisms have both the more elaborate transcriptional machinery and the nucleosomes and their modifiers typical of eukaryotea. So it is not surprising that many of the basic features of gene regulation are the same in all enkaryotes. As yeast are the most amenable to a combination of genetic and biochemical dissection, much of the information about how activators and repressors work comes from that organism. Important for the conclusions drawn from this work, when expressed in a mammalian cell, a typical yeast activator can stimulate transcription. This is tested using a reporter gene. The reporter gene consists of binding sites for the yeast activator inserted upstream of the pro-rooter of a gene whose expression level is readily measured (as we discuss below}.

We will see that the typical eukaryotic activator works in a manner similar to the simplest bacterial case: it has separate DNA binding and activating regions, and activates transcription by recruiting protein complexes to specific genes. In contrast, repressors work in a variety of ways, some different from anything we encountered in bacteria. These include examples of what is called gene silencing, in which modification to regions of chromatin keep genes in sometimes large stretches of DNA switched off.

Despite having so much in common, not all details of gene regulation are the same in all eukaryotes. Most importantly, as we have mentioned, a typical yeast gene has less extensive regulatory sequences than its multicellular counterpart. So we must look to higher organisms to see hnw the basic mechanisms of gone regulation are extended to accommodate more complicated cases of signal integration and combinatorial control. Regulation at later stages of gene expression—transcript elongation, RNA splicing and translation — are deall with later in the chapter.

Activators Have Separate DNA Binding and Activating Functions

In bacteria we saw that a typical activator, such as CAR has separate DNA binding and activating functions. We described die genetic demonstration of this: positive control (ur pc) mutants bind DNA normally, but activation domain activation domain

DNA-binding domain

DNA-binding site

FIGURE 17-2 Gal4 bound to its site on DNA. The yeast activator Ga(4 binds as a dinner to a 17 bp site on DNA. The DNA-bmding domatn of the protein is separate from the region of the prolan containing the activating region (the activation domain).

DNA-binding domain

DNA-binding site

FIGURE 17-2 Gal4 bound to its site on DNA. The yeast activator Ga(4 binds as a dinner to a 17 bp site on DNA. The DNA-bmding domatn of the protein is separate from the region of the prolan containing the activating region (the activation domain).

are defective in activation. Eukaryotic activators have separate DNA binding and activating regions as vvcli. Indued, in that case, the two surfaces are very often on separate domains of the protein.

We take as an example the most studied eukaryotic activator, Gal4 (Figure 17*2). This protein activates transcription of the galactose genes in the yeast S. cerevisiae. Those genes, like their bacterial counterparts, encode enzymes required for galactose metabolism. One such gene is called CALÍ. Gal4 binds to four sites located 275 bp upstream of GAL1 (Figure 17-3). When bound there, in the presence of galactose, Gal4 activates transcription of the GALi gene 1,000-fold.

The separate DNA binding and activating regions of Gat4 were revealed in two complementar)' experiments. In one experiment, expression of a fragment of the GAL4 gene—encoding the N-terminal third of Iho activator—produced a protein that bound DNA normally but did not activate transcription. This protein contained the DNA-binding domain but lacked the activating region and was, therefore, formally comparable to the pc mutants of bacterial activators (Figure 17-4a).

In a second experiment, a hybrid gene was constructed that encoded the C-terminal three-quarters of Gal4 fused to the DNA-binding domain of a bacterial repressor protein, LexA, The fusion protein was expressed in yeast together with a reporter plasmid hearing LexA binding sites upstream of the GALl promoter. The fusion protein activated transcription of this reporter (Figure 17-4b). This experiment shows thai activation is not mediated by DNA binding alone, as it was in one of the alternative mechanisms we encountered in bacteria—activation by MerR. Instead, the DNA-binding domain serves merely to tether the activating region to the promoter just as in the most common mechanism we saw in bacteria (Chapter lü).

Many other eukaryotic activators have been examined in similar experiments and whether from yeast, flies, or mammals, the same story typically holds: DNA-binding domains and activating regions are separable. In some cases they are even carried on separate polypeptides: one has a DNA-binding domain, the other an activating region, and they form a complex on DNA. An example of this is the herpes virus activator VP16, which interacts with the Octl DNA-binding protein found in cells infected by that virus. Another example is the Drosaphila activator Notch, described in the next chapter. The separable nature of DNA binding and activating regions of eukaryotic activators is the basis for a widely used assay to detect protein-protein interactions (see Box 17-1, The Two Hybrid Assay), id

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