Transcriptional Regulation

Transcription

1 Transcriptional Regulation Gene expression responds to environmental conditions. Some regulatory proteins are present at only 5 10 copies, whereas under certain conditions, the expression of these proteins can ramp up to about copies per cell. As such, in certain conditions like an abundance of glucose or an abundance of lactose, genes whose products (protein) aren t needed at that specific moment in time will be repressed. Model of Transcriptional Regulation: Lactose Metabolism In the absence of glucose and in the presence of lactose, the human body, and cells like E. Coli will up-regulate expression of an enzyme called ß Galactosidase to break down lactose into Galactose and glucose. Hence, we say that ß Galactosidase is induced by lactose. Figure 1 Hydrolysis of lactose (Weiss, 2016)

2 Lac Operon An operon is a unit of DNA that encodes proteins under a single form control. This means that a single control unit (like a light switch) regulates the expression of all the genes in that unit of DNA. When this operon is read, the resulting product is polycistronic mrna, which is subsequently translated into protein. Cis means adjacent, so polycistronic mrna refers to a stretch of RNA containing multiple genes next to each other. In the case of lactose metabolism, the gene responsible for producing ß Galactosidase is lacz, which forms part of the greater lac operon: Figure 2 Structure of the Lac Operon (Weiss, 2016) The gene that expresses this ß Galactosidase enzyme is thus regulated by factors: - Cis factors (Operator, Promoter Regions) Cis means adjacent, and these factors are components of the gene that sit on the DNA. It can be thought of a light switch on a wall that doesn t move - Trans factors (Repressor Gene) Trans factors can move throughout the cell and interact with DNA. It can be thought of the finger that controls the light switch Structural Genes Regulatory Region Repressor Gene (LacI) Technically not a part of Lac Operon Components of Lac Operon These are the genes that encode for the proteins required for lactose breakdown - LacZ: Encodes ß Galactosidase - LacY: Encodes Permease, which opens up the cell for lactose to enter - LacA: Encodes Transacetylase, which is also involved in metabolism Promoter Region: The sequence that RNA Polymerase recognises Operator Region: The control checkpoint that RNA polymerase can pass through or be blocked off. The operator region can be thought of as the light switch A gene encoding the lac repressor protein that can bind (or not) to the operator region to control transcription. This gene has its own promoter region, which allows for the constitutively low expression of the protein

3 When the structural genes are switched on, transcription of the genes gives rise to the three different enzymes: Figure 3 Expression of the enzymes in Lac Operon (Weiss, 2016) Regulation of Lac Operon: The LacI Repressor Gene The LacI regulator gene is not part of Lac Operon. Instead, it has its own promoter that constitutively expresses the repressor protein at low levels. Figure 4 The repressor protein has an operator and lactose binding site (Weiss, 2016)

4 Situation 1: No need for Lactose Metabolism In the absence of lactose or the presence of glucose (and hence without the need for lactose metabolism), the repressor binding protein binds to the Operator gene, preventing RNA polymerase from moving past the promoter region: Figure 5 Repressor prevents transcription in the absence of lactose (Weiss, 2016) Situation 2: Need for Lactose Metabolism In the presence of lactose, the repressor protein binds to lactose instead of the operator region, allowing RNA polymerase to bind to the promoter and freely transcribe: Figure 6 Repressor allows transcription in the presence of lactose (Weiss, 2016) In the diagrams is a dark blue region labeled L. This is the leader region, which is simply a blank space of DNA that separates the Operator Region and structural genes. Also, it is important to note that the Promoter and Operator Regions are not necessarily distinct and will sometimes have a region of overlap. Lac Operon expression thus depends on the inducer: - In the presence of lactose, the amount of mrna for the lac operon gene will increase and then fall in a bell shape. This fall is due to the eventual reduction in lactose as the body breaks it down, thus reducing the need for mrna - The amount of ß Galactosidase enzyme will increase and then plateau:

5 Figure 7 Lac Operon responds to the inducer (Weiss, 2016) The Role of CAP and camp for the Weak Promoter The promoter region is weak, meaning that RNA polymerase has difficulty binding to it even in the presence of lactose. Hence, there needs to be another factor that allows for the lac promoter to work better in the presence of lactose: Catabolite Activator Proteins (CAP) also known as CRP (camp Repressor Proteins) thus binds with cyclic AMP, which is produced during hunger, where glucose would be absent and the body would typically be breaking down lactose. This complex can then bind to the promoter region and allow RNA polymerase to bind more effectively Figure 8 CAP and camp help make the lac operon more efficient (Weiss, 2016)

6 Figure 9 camp is not produced when glucose is present (Weiss, 2016) Gratuitous Inducers Lactose is a normal inducer. Its presence induces the production of ß Galactosidase, which then breaks down lactose. In the laboratory, however, scientists often want to stimulate increased gene expression with an inducer that won t be broken down. Naturally, as a substance like lactose is broken down, the gene expression of the relevant enzymes decreases and the capacity to observe this expression is lost. Hence, artificially modified substrates like Isopropylthiogalactosidase (IPTG) are inducers that can induce ß Galactosidase expression, without being degraded. - These sort of inducers are called gratuitous inducers, which are not metabolized and thus remain at constant levels Other lab techniques that involve ß Galactosidase production include a disruption of the plasmid, cutting up the gene that encodes lac operon to prevent ß Galactosidase production. - This is commonly used when scientists have inserted a gene into a plasmid, and need to identify the cells that have the plasmid insert, and those without. They do this by inserting the lacz gene into the site of the insert. Plasmids with the gene insert thus block off LacZ, preventing ß Galactosidase production. When given a substrate called X Gal, the plasmids with the complete and uninterrupted LacZ turn blue as they break down lactose, while those with the interrupted LacZ remain white. Operator Region Similarities Whilst repressor proteins of any gene operon have a consensus binding sequence that is a best fit for the operator region that they are binding to, it is very rare for this sequence to be 100% complementary. In fact, proteins are very good at binding to the operator region even if there are discrepancies in nucleotide complementarity. As such, consensus sequences on operator regions aren t always the same. History of Lactose Metabolism: Jacob and Monod (1946) found that ß Galactosidase was only expressed when lactose was present in the medium. Their work also identified cis-acting elements (operator, promoter) and trans acting factors.

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