Control Of Gene Expression PDF

**[Gene Defn.:]** The entire nucleic acid sequence that is necessary for the synthesis of functional gene product. More on Genes ============= Solitary Genes Think of a genome as a cookbook and genes as the recipes within the cookbook. Some genes don't have any copies, only appearing once in th...

**[Gene Defn.:]** The entire nucleic acid sequence that is necessary for the synthesis of functional gene product. More on Genes ============= Solitary Genes Think of a genome as a cookbook and genes as the recipes within the cookbook. Some genes don't have any copies, only appearing once in the genome. These genes are **solitary**. 25-50% of genes in higher eukaryotes are solitary. Gene Families ------------- - Other genes are really similar in sequence (highly homologous), but have slight variations. These are part of **gene families**. - These variations occur to fit different needs. For example, olfactory (smell) receptors have hundreds of related genes that each detect different smells - These genes are replicated in **"cis".** Meaning they're located really close together on the same strand of DNA. - Think of gene families as a bunch of pasta recipes that use the same ingredients but vary in the type of pasta you use. Promoters --------- **[What's a promoter]**? Imagine the promoter as the "On" switch for a gene. When you flip the switch, it lets the cell start reading the recipe (gene) and make its product (protein). The promoter is composed of core, proximal, and distal regions. **[Core region]**: It's right at the base of the switch (usually within 100bps 0f the start). You'll find usually (not always) **TATA, CAAT, and GACA boxes** here (for more on these, see end of the file), which are specific sequences that help kickstart the process. **[Proximal region:]** Region where a bunch of helpers, called **transcription factors**, attach. These helpers are needed to turn on the gene. - If genes are recipes then transcription factors (TF) are like chefs that start the cooking process. They attach to specific spots near the gene to signal that it's time to start "reading" it. - [Helping RNA Polymerase]: RNA polymerase is the enzyme that does the actual reading of the gene to make RNA, which will eventually become a protein. Transcription factors help RNA polymerase find the start of the gene and attach correctly. - [Turning Genes On or Off]: Some transcription factors activate a gene (helping it turn on), while others repress it (keeping it off or reducing its activity). So, depending on the transcription factors present, the gene may be read more or less, like adjusting the volume on a speaker. **[Distal region]**: May or may not be part of the switch, but it sometimes has **control elements** to help regulate the gene's activity, especially in complex organisms (eukaryotes). A close-up of a diagram Description automatically generated Exons and Introns ----------------- - **Eukaryotic genes (more complex cells) contain two parts:** - **Exons: These are the useful parts of the gene that actually get turned into protein.** - **Introns: These are non-useful parts within the gene that don't code for proteins. The cell will cut out introns when making a protein.** **In short, exons** are the "core instructions" in a gene, while **introns** are extra sequences that get removed but help regulate and diversify how proteins are made. Regulatory Sequences -------------------- Eukaryotic cells have more regulatory sequences that prokaryotic cells. Regulatory sequences are divided into: - **[Enhancers]** -- where activators bind, helping the gene make more of its product. - **[Silencers]** -- where repressors bind, reducing how much product the gene makes.  Operons Polycistronic Operons --------------------- - **Polycistronic** means that multiple genes are included in the same stretch of DNA, and they are all read and transcribed together as a single, continuous unit. - Each gene in this bundle codes for a single **polypeptide** (which will become part of a protein). In this context, each gene or coding unit is sometimes called a **cistron**. **[How It Works]** 1. **Single Promoter**: In an operon, all the genes share one **promoter**. When the cell activates this promoter, it starts reading and transcribing all the genes in that cluster as one long piece of mRNA. 2. **Continuous Pre-mRNA**: The cell makes a single, continuous mRNA strand that includes instructions for all the genes in the operon. 3. **One Transcription Unit**: Since it's all read and transcribed at once, the operon acts like a single **transcription unit**---meaning that the cell only needs to turn on one "switch" to transcribe all the genes in the operon A white text with black text Description automatically generated Basics of Gene Expression Regulation: ------------------------------------- - **Transcription Regulation:** Most gene expression control happens at the transcription level, where the production of mRNA from DNA is regulated. - **Translation Regulation:** Gene expression can also be controlled during translation, affecting how proteins are made at various stages. Lac Operon: Controlling gene expression at the level of transcription ===================================================================== Francois Jacob and Jacques Monod in 1962. Why Do Bacteria Use a Lac Operon?: ---------------------------------- - The **lac operon** helps bacteria break down lactose, which is a type of sugar. - Bacteria need carbon, usually from sugars, to survive. They often have different sugar options available in their environment. **[Choosing a Sugar Source:]** - Bacteria **prioritize** which sugar to use based on how easy it is to use. - **Glucose** is the easiest sugar because it can go straight into energy production (glycolysis) without needing extra work. - Other sugars, like lactose, require more effort to break down, so bacteria will use glucose first when it's available. **[When Glucose Runs Out:]** - Once glucose is used up, bacteria can switch to using lactose. - The lac operon helps regulate this switch by sensing the type and amount of sugar available.  Lac Operon: Layout ------------------ Consists of three genes necessary for the metabolism of lactose: β-Galactosidase: This enzyme hydrolyses lactose into glucose and galactose Lactose permease: Located in cell membrane and transfers lactose into the cell Thiogalactoside transacetylase: (transfers an acetyl group from acetyl-CoA to β-galactosides and is believed to have a role in cell detoxification). Also consists of a repressor gene/protein that sits in front of the operon. Blocking it when lactose isn't present A screenshot of a computer Description automatically generated Four Situations: ---------------- 1. **Glucose present, lactose absent**: No β-galactosidase made. 2. **Glucose present, lactose present**: No β-galactosidase made. 3. **Glucose absent, lactose absent**: No β-galactosidase made. 4. **Glucose absent, lactose present**: β-galactosidase is produced. ### When Lactose is Absent: 1. **No Lactose**: If there's no lactose around, the bacteria don't need to break it down. 2. **Regulator Gene Makes Repressor Protein**: The bacteria continuously make a repressor protein. Think of this protein like a guard that blocks the entrance to a gate (the operator site on the DNA). 3. **Blocking the Genes**: This repressor protein sits on the operator site, just downstream of the promoter site. - Because it's blocking the way, another important protein called RNA polymerase can't bind to the promoter site so no enzymes needed to break down lactose, like β-galactosidase. So, no β-galactosidase are produced.  ### When lactose is present: 1. A small amount of lactose is converted to allolactose. 2. Allolactose binds to a specific site (allosteric site) on the repressor protein causing it to change shape. 3. This conformational change means it can no longer bind to the operator site. 4. Now that it is not blocked by the repressor protein, RNA polymerase can bind to the promoter site, switching on the lac operon!!!! A black and red line with text Description automatically generated When Lactose AND Glucose Are Present ------------------------------------ Lac Operon is still not activated! **[Why?]** 1. No CAP (Catabolite Activator Protein) is present if there's glucose. **[Why does CAP matter?]** 1. It acts like a seatbelt for RNA polymerase. 2. RNA polymerase can reach the promoter site but without CAP it falls off.  A person in a green shirt Description automatically generated When Only Lactose is Present:  ### More on How CAP Works 1. **Glucose Levels Matter**: - When there's **lots of glucose** in the cell: - **ATP (energy)** is high (the cell has plenty of energy). - **cAMP (a signaling molecule)** is low (the cell doesn't need to signal for energy). - When there's **little or no glucose**: - **ATP is low** (the cell needs energy). - **cAMP is high** (the cell signals that it needs to find another sugar source, like lactose). 2. **Making the CAP-cAMP Complex**: - When glucose is low, **cAMP** levels go up, and cAMP will attach to the **CAP protein**. - This forms a new unit called the **CAP-cAMP complex**. 3. **Binding to the Lac Operon**: - The CAP-cAMP complex can then attach to a specific part of the **lac operon promoter** (the region where RNA polymerase starts). 4. **Bending the DNA**: - When the CAP-cAMP complex binds to the promoter, it **bends the DNA**. 5. **Activating Transcription**: - This bending helps RNA polymerase bind to the promoter, which allows the genes in the lac operon to be copied into mRNA. This process is called **transcription**. - However, this can only happen if the **lac repressor** isn't blocking the way (which would happen if lactose isn't present). A chart of different types of protein Description automatically generated Tryptophan (Trp) Operon ======================= Tryptophan or Trp is an essential amino acid required for protein production. In the absence of a ready source of this amino acid, then Trp must be made from scratch. The Trp operon is constitutively expressed, unlike the Lac operon which is activated/inducible. So.... How Is the Trp Operon Switched Off? ----------------------------------- If there's enough tryptophan around, the body doesn't need to make more. So, it switches off the operon. **When Trp is not present**: - The repressor protein is made by a regulatory gene that lies upstream of the Trp promoter. - The Trp promoter has a downstream operator site - When Trp is not present, the repressor protein is inactive, meaning it does not bind to the operator sequence. - Thus, the Trp Operon remains active.  **When Trp is present:** - Trp binds to the repressor protein causing a conformational change. - The repressor is now the perfect shape to bind to the operator sequence. - Now, the RNA polymerase that is bound to the promoter site can't move passed the operator sequence. - Therefore, transcription does not occur. A diagram of a cell division Description automatically generated Control of the Trp Operon Happens at a Translational Level ---------------------------------------------------------- ### Why is this possible? Translation Starts Early: - In bacteria, making proteins (translation) starts while the instructions (transcription) are still being made. - This is possible because bacteria don't have a protective nuclear membrane like humans do. ### Leader Sequence: The leader sequence is the first part of the mRNA that gets made when the Trp operon is turned on. It's crucial because it helps decide whether to keep making the protein or stop. #### The Four Domains: Within the leader sequence, there are four specific regions (or domains) called 1, 2, 3, and 4. These domains are like sections of a puzzle that can fit together in different ways. Each domain has sequences that are partially complementary, meaning they can stick together like magnets. This allows them to form structures known as hairpin loops. #### Hairpin Loops: Hairpin loops are structures formed when parts of the mRNA fold back on themselves. Here's how the domains can pair up: **Hairpin 1-2**: Domains 1 and 2 can stick together to form one hairpin. **Hairpin 3-4**: Domains 3 and 4 can stick together to form another hairpin. **Hairpin 2-3**: Domains 2 and 3 can form a different connection. Exclusive Pairing: It's important to note that not all hairpin loops can form at the same time. Depending on the conditions in the cell (like the presence of tryptophan), one pairing will occur over the others. **Termination Hairpin (3-4):** The 3-4 hairpin loop is particularly important because it acts as a termination signal for the ribosome (the protein-making machinery): When the 3-4 hairpin forms, it tells the ribosome to stop translation. This means that when the ribosome encounters this structure, it knows to stop making the protein right there. #### What Triggers Hairpin Formation? **Low Tryptophan**: If there isn't enough tryptophan available, the ribosome will slow down while trying to read the leader sequence. This gives more time for the 2-3 hairpin loop to form instead of the 3-4 hairpin. With the 2-3 hairpin formed, the ribosome continues to translate the mRNA and make the tryptophan-producing proteins. **High Tryptophan**: If there is plenty of tryptophan available, the ribosome moves quickly, doesn't stall, and allows the 3-4 hairpin to form. This stops translation because the ribosome gets the signal to stop.  The Results: ------------ - **Overall Control**: - The repressor can reduce the production of tryptophan by about **70 times** when there's enough tryptophan present. - The attenuation mechanism can reduce production by another **10 times** if tryptophan is high, making it a total control of about **700 times** over tryptophan production! Arabinose Induction in the pGLO Plasmid ======================================= What is the pGLO Plasmid? ------------------------- - The **pGLO plasmid** is a small, circular piece of DNA that can be introduced into bacteria (like E. coli) to give them new traits, like the ability to glow green under UV light. This glowing is due to a gene that makes a fluorescent protein, which is controlled by the presence of a sugar called arabinose. Key Components of Arabinose Induction: -------------------------------------- ### Promoter (pBAD): - The **pBAD promoter** is a special region of DNA that helps start the transcription (copying DNA to RNA) of the genes needed to use arabinose. ### AraC Gene: - The **araC gene** makes a protein called **AraC**. This protein is important for controlling whether the genes for arabinose metabolism are turned on or off. ### Two Promoters: - There are two promoters involved: **pBAD** and **pAraC**. They read the DNA in opposite directions and help regulate the expression of the genes. ### Operator Binding Sites: - There are two operator sites where the AraC protein can bind: **I1** and **O2**. These sites control whether RNA polymerase (the enzyme that makes RNA) can start working. ### Control by Glucose: - At a high level, the entire operon is controlled by **glucose** through another protein complex called **CAP/cAMP**. This means that when glucose is present, the operon is generally turned off, preventing transcription. A diagram of a line with red circles Description automatically generated In the Absence of Arabinose: ---------------------------- ### AraC Protein Binding: - When there is no arabinose around, the **AraC protein** attaches to two specific regions of the DNA called the **I1** and **O2 operator sites**. - This means that AraC is effectively grabbing onto the DNA in a way that influences how other proteins can interact with it. ### Dimerization: - **Dimerization** means that two AraC proteins stick together (form a dimer) while they are bound to the I1 and O2 sites. So, even though arabinose isn't present, the AraC proteins can still form this pair. ### Looped DNA Structure: - When AraC binds and dimerizes, it causes the DNA to loop around. This **looping** creates a structure that blocks other important proteins from doing their jobs. - Specifically, because of this loop, **RNA polymerase** cannot attach to the promoter region to start transcription. - Also, the **CAP/cAMP complex**, which helps activate transcription when glucose is low, also can't bind effectively to the DNA in this looped structure.  What Happens When Arabinose is Present: --------------------------------------- ### Arabinose Binding: - When arabinose enters the cell, it binds to the AraC protein. - This binding changes the shape of the AraC protein, allowing it to form a dimer (a pair of AraC proteins) while still attached to the **I1** site. ### CAP/cAMP Binding: - If glucose levels are low, the **CAP/cAMP complex** can also bind to a specific site on the DNA called the **CAP-binding site**. - This complex helps to stabilize the RNA polymerase on the promoter, enhancing transcription. ### Transcription Occurs: - With the changes in the binding of AraC and the presence of the CAP/cAMP complex, RNA polymerase can now access the **pBAD promoter**. - This allows the genes for using arabinose to be **transcribed**, meaning the cell can now use arabinose as a source of energy and produce the fluorescent protein. A diagram of a machine Description automatically generated What are Inducible and Repressible Systems? =========================================== These terms describe how certain genes are turned on (activated) or turned off (repressed) in response to specific signals or substances. Inducible Systems: ------------------ - **Inducible** means that a system can be turned on when something (an inducer) is present. ### How it Works: #### Activation (Induction): Inducible System - When an inducer (like a specific sugar or other molecule) is added, it can bind to a part of the system, usually the **promoter** or **operator**. - This binding allows the machinery (like RNA polymerase) to start making RNA from the gene, which means the gene is turned **on**. - If an inducible system requires an inducer to bind to the promoter/operator then this is positive - If an inducer is required to release from promoter operator system then the inducible system is negative #### Shutting Down (Repression): Repressible System - Repressor required to bind to the promoter/operator then this is negative - Repressor required to release from promoter operator system then this is positive  A white background with black text Description automatically generated Eukaryotic Promoters ==================== 1. **Diversity**: Eukaryotic promoters are varied and can have different structures and functions. 2. **Location**: Promoters typically sit upstream of the gene of interest (GOI), meaning they are found before the actual coding region of the gene. 3. **Regulatory Elements**: These elements can be found far away from the start of the gene (several kilobases). Because DNA can loop, these distant elements can still influence how a gene is expressed. - **Enhancers**: DNA sequences that can boost the activity of a promoter, helping to turn on gene expression. - **Silencers**: DNA sequences that can reduce or shut off gene expression. Do Eukaryotic Genes Contain Operons? ------------------------------------ - **Operons**: In prokaryotes (like bacteria), an operon is a cluster of genes that are transcribed together as a single mRNA and usually regulate similar functions. - **Eukaryotes**: Generally **do not** have operons. There might be a couple of rare exceptions, but they are not the norm. This is because eukaryotic genes are usually more complex, often coding for multiple proteins or functions. Transcription Units and Alternative Splicing ============================================ - **Transcription Units**: In eukaryotes, a single gene can produce multiple types of mRNA, leading to different protein products. This complexity allows for more varied gene regulation. - **Alternative Splicing**: This process allows a single gene to be spliced in different ways, leading to different protein forms (isoforms). - **Mechanism**: During the splicing of pre-mRNA, some exons (coding regions) may be included or excluded, creating different versions of the mRNA. - **Importance**: This mechanism is crucial in many biological processes, allowing for flexibility and adaptability in gene expression, especially in different cell types or developmental stages. How Drosophila (Fruit Fly) Decides if It\'s Male or Female X and Autosomes: --------------------------------------------------------------------------- Fruit flies have sex chromosomes (X) and other chromosomes (autosomes). The ratio of X chromosomes to autosomes helps the fly decide if it will be male or female. If there are more X chromosomes, the fly is female. If there\'s only one X, the fly is male. ### Sxl Protein: - There's a protein called Sxl (short for sex-lethal). - In females, Sxl is active and helps control how other genes are spliced (cut and pasted together). - In males, Sxl isn't made, so the splicing process goes back to normal, producing a broken version of the protein that doesn't work. ### Tra Protein: - Another important player is the Tra protein (which stands for transformer). - In females, Tra is made because Sxl is active. This Tra protein helps produce a female version of another protein called Dsx (double-sex). - The female Dsx protein turns off genes that would make male traits. So, it helps keep the fly female. - In males, Tra isn't made (because Sxl is not active), so the fly makes the male version of Dsx instead. - The male Dsx protein turns off genes that would make female traits, ensuring the fly develops as male. ### How It Works: - Active Sxl helps create Tra in females, which in turn helps make the female Dsx protein. - In males, without Sxl, there's no Tra, so the male Dsx is produced instead. - This process of making different proteins depending on whether the fly is male or female is called alternative splicing. - In Simple Terms: - The sex of fruit flies is decided by how many X chromosomes they have. - If there\'s one X, they become males; if there are two, they become females. - The Sxl protein is like a switch that helps control other proteins. If Sxl is on (females), they make the Tra protein, which helps keep them female. If Sxl is off (males), they don't make Tra, which leads to male development. - This is all about how the cells read their genetic instructions and make the right proteins to determine whether the fly looks and acts male or female. Summary: ======== - **Eukaryotic promoters** are complex and can be influenced by elements that are far away, such as enhancers and silencers. - **Operons** are not common in eukaryotes due to their more complex genes that often have multiple protein outputs. - **Alternative splicing** allows eukaryotic cells to generate multiple protein forms from a single gene, playing a key role in processes like Drosophila sex determination, where it helps determine whether the fly develops as male or female based on splicing regulation. **What Are These \"Boxes\"?** - **TATA, CAAT, and GACA boxes** are specific **DNA sequences** found near the beginning of many genes in eukaryotic cells (like those in humans). - These boxes are called "boxes" because they are short, repeated sequences that are often illustrated as small, rectangular sections on DNA diagrams. **What Do They Do?** - Think of these boxes as **signposts or landmarks** that tell the cell, "Start reading here!" - When a cell needs to make a specific protein, it starts by making a copy of the gene's instructions (transcription). But to do this, the cell's machinery needs to know where to begin. - **TATA, CAAT, and GACA boxes** help the cell\'s machinery, particularly an enzyme called **RNA polymerase**, find the starting point of the gene. **How Each Box Helps** 1. **TATA Box**: - This is the most famous one, usually found about 25-30 bases before the start of the gene. - It helps the RNA polymerase enzyme bind to the DNA so it can start copying the gene. Think of it as a "landing pad" for the enzyme. 2. **CAAT Box**: - This box is often found a bit further upstream (before the TATA box) and helps boost the gene's expression (how much of the protein is made). - It's like an additional sign that says, "Make sure to start here and make plenty!" 3. **GACA Box**: - Though it's less common, this box can help fine-tune how the gene is expressed in certain contexts or cells. - It provides extra signals to help the cell's machinery read the gene accurately.