Gene regulation in prokaryotes and eukaryotes Year 13

Enzymes Speed up the chemical reactions in living organisms. Are biological catalysts. Speed up the chemical reactions in living organisms. Without enzymes, the chemical reactions of life would proceed so slowly that life would be hardly possible. Are not used up or changed by the reaction.

What are enzymes made of? Chemically, enzymes are proteins. Because of the unique shape of each enzyme it is specific to a particular reaction – it will catalyse only one reaction. There are, therefore, thousands of different enzymes in any living organism.

Some definitions: Substrate: the chemicals an enzyme acts on. Active site: the part of the enzyme where the substrate binds and where the reaction occurs. The active site has a specific shape so only specific substrates can bind. Nomenclature: ase

How do enzymes work? 2 models: lock and key induced fit See page 95 Biozone

Metabolism Is all the chemical reactions that occur in the cell of an organism. Metabolism is made up of all the different processes an organism needs to maintain itself such as growth, energy, repair, and excretion. These processes are a complex network of metabolic pathways which are controlled by enzymes.

The importance of enzymes in metabolic pathways. A metabolic pathway is a series of “steps” from a starter molecule, resulting in the formation of a different end product. Many intermediate compounds can be formed in the pathway. Each step in the pathway is controlled by an enzyme. A faulty enzyme can cause metabolic disorders.

Metabolic pathways can be: anabolic: produce large molecules from smaller ones or catabolic: break large molecules into smaller ones.

Hydroxyphenylpyruvic acid Phenylalanine Enzyme A Thyroxine Tyrosine Melanin Enzymes Enzyme B Enzyme C Hydroxyphenylpyruvic acid Enzyme D Metabolism of phenylalanine Homogentisic acid Enzyme E Maleyacetoacetic acid Enzyme F Do exercises Page 97 and 98 CO2 and H2O

Control of gene expression in metabolic pathways Gene expression of enzymes in a metabolic pathway must be tightly controlled so the cell has the correct amount of each enzyme it requires. Control often occurs at transcription. Some genes are induced – they are only switched on in certain situations. Other genes are transcribed continuously because their products are always needed eg genes coding for respiratory enzymes.

Gene regulation Two types of genes: Structural genes – encode specific proteins Regulatory genes – control the level of activity of structural genes ie. Control structural gene expression.

Gene regulation in prokaryotes In prokaryotes, operons control the rate of transcription. An operon is a group of genes that work together and code for the enzymes regulating a particular metabolic pathway. Regulator gene Promoter Operator Structural gene A Produces the repressor RNA polymerase binding site Repressor binding site OPERON Structural gene B

Structure of the operon The operon in prokaryotes comprises a number of different features: Structural genes: code for particular enzymes in a metabolic pathway Promoter gene: recognition site for the RNA polymerase to bind to. Operator gene: controls the production of mRNA from structural genes.

See this movie on the Lac operon in E. coli for more detail INDUCTION If a substrate is uncommon the bacteria will not need the enzymes most of the time. So the repressor is usually attached. This prevents RNA polymerase from forming mRNA. Therefore: no enzymes. When the substrate molecule is present some of it acts as an inducer; it binds to the repressor, changing its shape so it can’t bind to the DNA. Transcription takes place. See this movie on the Lac operon in E. coli for more detail Inducer R R P O SG1 SG2 R

See this movie on the Tryp operon in E. coli for more detail REPRESSION When a substrate is normally present the enzyme should be normally operating. The only time this should stop is when the end product levels build up too much. The repressor cannot bind to the operator. Some of the excess product acts as an effector, which helps the repressor to bind. Transcription is stopped. See this movie on the Tryp operon in E. coli for more detail R R P O SG1 SG2 R

http://www. sumanasinc http://www.sumanasinc.com/webcontent/anisamples/majorsbiology/lacoperon.html

Lac Operon - induction: Lactose binds to the repressor protein. Lactose present- acts as an inducer. Lac gene off (normal state) Repressor can’t bind to the operator. Repressor molecule binds to operator and prevents transcription by RNA polymerase RNA polymerase binds . Lac gene on. Structural proteins made. Lactose all used up. Tryptophan operon - repression Tryptophan accumulates in excess. Some of it acts as an effector and activates the repressor molecule. Effector and repressor molecule bind to the operator gene and prevent transcription by RNA polymerase. Tryp gene on (normal state) RNA polymerase binds Tryptophan doesn’t bind to the repressor which then can’t bind to the operator. Tryptophan levels in cells decrease, no excess.

Gene regulation in prokaryotes - summary Genes for a metabolic pathway are linked together in operons with a common switch mechanism (operator). No introns – no RNA processing The structural genes undergo transcription and translation simultaneously. Regulation occurs by switching all genes of a pathway on or off.

Gene regulation in eukaryotes Genes for metabolic pathways in eukaryotic cells are separated, not grouped as operons. The genes for a metabolic pathway are switched on separately. Genes have introns that are removed in RNA processing. Eukaryotic genes have a relatively large number of control elements.

Regulatory DNA regions Eukaryotic genes have a promoter region upstream of the coding region, where RNA polymerase binds. There are 2 two types of regulatory sequences that effect transcription of the structural gene: 1) enhancer 2) silencer These are located upstream, downstream or within the gene (in introns).

Enhancer sequences These are non-protein-coding sections of DNA that help regulate transcription by binding proteins called transcription factors. Silencer sequences These are non-protein-coding sections of DNA that help regulate transcription by binding proteins called repressors.

Transcription factors Two types: 1) Activators – these are small proteins that bind to enhancer sequences or RNA polymerase. They cause an increase in transcription. 2) Repressors – these are small proteins that bind to silencer regulatory genes. They cause a decrease in transcription.

Transcription factors that bind to RNA polymerase Coding region of gene Promoter region of DNA RNA polymerase Transcription factors that bind to RNA polymerase Transcription factors (activators) that bind to the enhancer sequence Enhancer sequence of DNA

Role of Transcription Factors

Eukaryotic RNA polymerase cannot, on its own, initiate transcription. It depends on transcription factors to recognize and bind to the promoter. Transcription factors also bind to the enhancer sequence of DNA RNA polymerase Transcription factors that bind to RNA polymerase Transcription factors (activators) that bind to the enhancer Promoter region of DNA Enhancer sequence of DNA Coding region of gene

Activating Transcription

Transcription is activated when a hairpin loop in the DNA brings the transcription factors on the enhancer sequence (activators) in contact with the transcription factors bound to the RNA polymerase at the promoter. Protein-protein interactions are crucial to eukaryotic tanscription. The RNA polymerase can only produce a mRNA molecule once the complete initiation complex is assembled. Activators Transcription factors bound to RNA polymerase Transcription proceeds until a terminator sequence is encountered. Then transcription stops. Enhancer Promoter RNA polymerase Initiation complex

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DEFECTIVE GENES Cell division is tightly controlled. If a cells DNA becomes damaged a gene (p53) within the cell causes cell division to cease until it is repaired. Other genes (proto-oncogenes) allows cell division to begin. If DNA damage is irreparable or cells get too old they self destruct, called apoptosis. If damage occurs in either of the 2 genes mentioned above the cell will grow at an uncontrolled rate, or become effectively immortal. These cells cease to carry out normal functioning. If the damage is not too severe the cells may form a benign tumour. If many genes are affected the tumour is said to be cancerous. Lab manual page 99