Question

In our discussion of the regulation of gene expression in eukaryotes, we covered the Gal1 locus of yeast.

(a) In the space below, draw out the general cis and trans elements of the yeast Gal1 locus in the presence of Galactose (Gal1 gene is expressed, +Galactose).

(b) What is one aspect of the regulation of Gal1 expression (in the activated or repressed state) that is conceptually distinct from the regulation of the prokaryotic lac operon in E.coli?

(c) In class, we talked about a series of experiments called the activator-bypass experiments. In one set of experiments, a set of cis/trans elements called LexA were used that are not normally even in the yeast genome. These elements were used to test a hypothesis about the role of the enhancer/activator's function in promoting gene expression. What was the hypothesis/model that was being tested? (You can write your hypothesis as a question or a statement.)

(d) What was the overall conclusion about the role of activator proteins from this series of bypass experiments?

Answer 1

GAL1 is a gene in Saccharomyces that codes for Galactokinase protein which phosphorylates galactose to galactose-1-phosphate. The galactose structural genes (GAL1, GAL10, GAL7, GAL2) are co-ordinately regulated at the transcriptional level in response to galactose by Gal4, Gal80, and Gal3. The GALI and GAL10 genes are adjacent and are transcribed in opposite directions. with approximately. 600 base pairs of DNA separating their transcription initiation sites. 600-base-pair region of DNA carries sequences responsible for the regulation of GALI and GAL10. This 608 basepair region is called Upstream Activation Sequence (UAS) and is required for the regulation of both the genes.

Within 60 nucleotides of the transcriptional start sites are two short homologous regions with similarities to a TATA box. A TATA box is a DNA sequence that indicates where a genetic sequence can be read and decoded. It is a type of promoter sequence, which specifies to other molecules where transcription begins.

This is how the GAL1 locus is maintained by the presence of Glucose, raffinose or Galactose

Regulation of the GAL1 promoter. In the presence of glucose, transcription is repressed because repressor proteins bind to regulatory sites in the DNA and to the Gal4p transcriptional activator. Glucose repressionis relieved in the presence of raffinose, but Gal4p remains inactive. Gal4p activates transcription in the presence of galactose due to the removal of the Gal80p protein.

Reference
1) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC369078/pdf/molcellb00153-0229.pdf

2) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC368932/pdf/molcellb00150-0020.pdf

b) The conceptual difference between GAL1 expression in yeast and lac operon in E.coli is that the lac operon is operated by only a single promoter whereas each structural gene of yeast in Gal1 expression has its own different promoters.

c and d) In S.cerevisiae, activators (Act) function by contacting components of the holoenzymes. The putative targets include TATA-box binding protein (TBP), the mediator, general transcription factors such as TFIIB and TFIIH, and TAFs. The functional consequences of all these interactions are the recruitment of the holoenzyme and/or isomerization from an inactive to an active form.

The requirement for activators can be bypassed by fusing different components of the holoenzyme to a DNA-binding domain such as that of LexA.

One prediction of the recruitment hypothesis was that targeting GAL11 to a promoter by a LexA-GAL11 fusion would target the holoenzyme, thereby circumventing the requirement for upstream activators. Indeed, a LexA-GAL11 fusion protein resulted in high levels of transcription in vivo from reporter templates bearing only upstream LexA-binding sites and a TATA box. The ability of both LexA-GAL11 and LexA-TBP to bypass activators is consistent with the idea that TBP and GAL11 are part of the holoenzyme in the cell, a supposition further supported by the finding that TBP co-purifies with the holoenzyme during early stages of its isolation in vitro. A single-step model would imply that, in some instances, a single molecule of a potent activator could stimulate transcription from a high-affinity binding site in yeast, a prediction borne out using GAL4 and GAL4-VP16

Reference
1) https://www.sciencedirect.com/science/article/pii/S0960982295002016