Textbook assignment: Chapter 19, pages 556-566. Note that the full story of regulation of the GAL4 protein is not included in our textbook, although the protein itself and its basic function as a transcription factor are discussed on pages 548-549. For the rest of the story, you will need to rely on these notes.
Major concepts:
Galactose metabolism in yeast: In order for the yeast, Saccharomyces cerevisiae to utilize galactose, it must activate a set of four genes that remain inactive when galactose is not present. The genes and the funcitons of their enzymatic products are as follows:
GAL1: Converts galactose to galactose-1-phosphateGAL2: Galactose permease, facilitates entry of galactose into the cells
GAL7: Converts galactose-1-phosphate to UDP-galactose
GAL10: Converts UDP-galactose to UDP-glucose.
UDP-glucose then feeds into a normal pathway of glucose utilization, with the first step being conversion to glucose-1-phosphate.
Upstream activating sequences: Three of the genes whose transcription is induced by the presence of galactose, GAL1, GAL7, and GAL 10, are clustered close together on chromosome II. However, they do not form an operon. Each is transcribed separately as a monocistronic mRNA. A galactose-specific upstream activating sequence (UASG) that serves as a binding site for a galactose-specific transcription enhancing factor (the GAL4 gene product) is associated with the promoter for each of these genes. The binding domain is a 17 bp palindrome, which is repeated four times in the UAS.
Bidirectional transcription: The GAL7 promoter and its UAS have no unusual features. However, the GAL1 and GAL10 genes are transcribed in opposite directions from divergent promoters and UAS sequences that are located between the transcriptional start sites for the two genes. In the usual depiction of the gene cluster, GAL1 is shown as transcribing to the right and GAL10 to the left. GAL7 is located further to the left and also transcribes to the left.
<--GAL7-promoter-UAS............<--GAL10-promoter-UAS...UAS-promoter-GAL1-->
Requirement for GAL4 protein: Transcription of all three of these genes (and also of the GAL2 gene, which is on a separate chromosome) requires the binding of GAL4 to the UAS to be activated above a very low basal level. The GAL4 protein has the expected properties of a transcription activating factor. These include a DNA-binding domain that is specific for UASG, and two separate transcriptional activation domains that must both be intact for full activity. However, the GAL4 protein does not interact directly with galactose. Instead, it has a binding site for the GAL80 protein, and is maintained in an inactive condition when galactose is not present by being bound to that protein.
Regulatory role of GAL80 protein: The GAL80 gene product is the only protein involved in induction of the lactose utilization genes that is capable of direct interaction with galactose. In a sense, the GAL80 protein functions as a repressor, but it does not do so by binding an operator site. Insterad, in the absence of galactose, the GAL80 protein binds to the GAL4 transcriptional activator protein and blocks its ability to interact with the UASG and enhance transcription of the lactose utilization genes. When galactose enters the cell, it binds to the GAL80 protein and causes it to undergo an allosteric change, which in turn causes dissociation of the GAL 80 protein from the GAL4 protein and allows the GAL4 protein to function as a positive-acting transcription factor for the galactose utilization genes. The increase in transcription is presumed to be accomplished by an interaction between the activation domains of GAL 4 and the RNA polymerase II initiation complex, as depicted in Figure 19.13 for classical enhancers.
Summary: In the absence of galactose, GAL80 binds to GAL4 and prevents it from acting as a transcription-enhancing factor for GAL1, GAL7, and GAL10. When galactose is present, it binds to GAL80, causing it to dissociate from GAL4. GAL4 then binds to the UASG associated with each of GAL1, GAL7, and GAL10 and stimulates transcription of all three of these genes.
For another view of the GAL80/GAL4 regulatory system, you may want to look at the online notes provided for Dr. Dutcher's section of MCDB 2150 last spring. Click here to see notes
Gene amplification: In ordinary discussions of gene expression, we generally assume that the number of copies per genome of the gene is question remains constant. While this is usually the case, there are important exceptions, as discussed on pages 556-558 of the textbook. (You may also want to refer back to previous mentions of these phenomena on pages 245, and 341). Four major examples are discussed briefly.
Amplification of ribosomal RNA coding genes: Amphibian embryos develop from rather large eggs that support a large amount of protein synthesis over a relatively short period of time. This requires accumulation of a huge number of ribosomes in the egg, which in turn requires a very high rate of transcription of ribosomal RNA. The mechanism that is used to achieve such synthesis is extrachromosomal amplification of the DNA sequences that the ribosomal RNA is transcribed from.
Amplification of genes coding for chorion proteins in Drosophila eggs: Amplification of genes coding for egg shell proteins occurs during oogenesis in Drosophila, with the region of amplification extending well beyond the immmediate limits of the genes that are directly involved.
Chromosomal puffs: In some species of insects, the polytene chromosomes exhibit "puffs", in which particularly active genes are amplified beyond the extent of amplification of the entire genome in the polytene chromosomes (Figure 19.21).
Drug-resistant tumors: Gene amplification does not appear to be a normal developmental phenomenon in mammals. However, aneuploid tumor cells sometimes respond to chemotherapeutic drugs by greatly amplifying the genes whose products are inhibited by the drugs, sometimes overwhelming the ability of the drugs to control the malignant growth. In some cases, the amplificaiton products are seen as extrachromosomal structures that have come to be called double minute chromosomes.
Alternative splicing: Nearly all eukaryotic genes contain introns that are "spliced out" during processing of the initial transcript to generate the final mRNA. One fairly common mechanism for altering the properties of the final protein product is to modify the pattern of splicing, such that certain exons are included in some versions of the protein and excluded from others. Often this phenomenon is controlled in a tissue specific manner. In an extreme case, 64 different alternatively spliced forms of the muscle protein troponin T have been identified. Similarly, there are 10 different forms of alpha tropomyosin. In some cases, different peptide sequences with different biological functions may be included or excluded in different tissues. An example of this is shown in Figure 19.23, where tachykinen K may or may not be produced.
Sex determination in Drosophila: The cascade of regulatory mechanisms that determines whether a Drosophila develops as a male or a female includes several steps of directed alternative splicing of mRNAs for regulatory proteins that ultimately determine which pathway is followed (See figures 19.24 and 19.25). This pathway will be examined in detail in MCDB 4650.
Message stability: Another interesting control over gene expression is achieved by changes in massage stability. A message with a long half-life will be translated far more times before it is degraded than a message with a short half life. The textbook describes a scheme of translation-coupled control over message stability, in which presence of adequate amounts of the proteins that are being translated triggers a special ribonuclease activity that destroys the message for those proteins (Figure 19.26). In addition to mechanisms of this sort, there are also inherent differences in message stability, determined by the presence or absence of particular sequences in the messages (normally found in the 3'-untranslated region).
Bigger picture: As summarized briefly in Figure 19.1, this brief summary has not even come close to examining all of the possible points in the flow of information from a gene to a final functional gene product at which controls can be exerted. As you progress through the MCDB major, you will encounter many more of these regulatory mechanisms and gain an increased appreciation of just how complex eukaryotic gene regulatory mechanisms actually are (and perhaps be thankful that you did not have to learn all of them in this course).