Biochemistry 1 PDF

LIST OF CONTENTS Subject Pages CARBOHYDRATE CHEMISTRY 2-10 LIPID OF BIOLOGICAL IMPORTANCE 11-23 AMINO ACIDS AND PROTEINS OF BIOLOGICAL 24-30 IMPORTANCE PROTEINS OF EXTRACELLULAR MATRIX 31-35 NUCLEOTIDES& NUCLEI...

LIST OF CONTENTS Subject Pages CARBOHYDRATE CHEMISTRY 2-10 LIPID OF BIOLOGICAL IMPORTANCE 11-23 AMINO ACIDS AND PROTEINS OF BIOLOGICAL 24-30 IMPORTANCE PROTEINS OF EXTRACELLULAR MATRIX 31-35 NUCLEOTIDES& NUCLEIC ACIDS 36-42 REPLICATION, TRANSCRIPTION & TRANSLATION 43-58 1  CARBOHYDRATE CHEMISTRY Carbohydrate, class of naturally occurring compounds and derivatives formed from them. composed mainly of molecules containing atoms of carbon (C), hydrogen (H), and oxygen (O) with similar formulas were found to have a similar ratio of hydrogen to oxygen. The general formula Cx(H2O)x is commonly used to represent many carbohydrates, which means “watered carbon.” Carbohydrates are probably the most abundant and widespread organic substances in nature, and they are essential constituents of all living things. Carbohydrates serve as energy sources and as essential structural components in organisms; in addition, part of the structure of nucleic acids, which contain genetic information, consists of carbohydrate. Classes of carbohydrates Although a number of classification schemes have been devised for carbohydrates, the division into four major groups— monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Monosaccharides The most common naturally occurring monosaccharides are D-glucose, D- mannose, D-fructose, and D-galactose among the hexoses and D-xylose and L- arabinose among the pentoses. In a special sense, D-ribose and 2-deoxy-D-ribose are ubiquitous because they form the carbohydrate component of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), respectively; these sugars are present in all cells as components of nucleic acids. D-Xylose, found in most plants in the form of a polysaccharide called xylan, is prepared from corncobs, cottonseed hulls, or straw by chemical breakdown of xylan. D-Galactose, a common constituent of both oligosaccharides and polysaccharides, also occurs in carbohydrate- containing lipids, called glycolipids, which are found in the brain and other nervous tissues of most animals. Galactose is generally prepared by acid hydrolysis (breakdown involving water) of lactose, which is composed of galactose and glucose. Since the biosynthesis of galactose in animals occurs 2 through intermediate compounds derived directly from glucose, animals do not require galactose in the diet. In fact, in most human populations the majority of people do not retain the ability to manufacture the enzyme necessary to metabolize galactose after they reach the age of four, and many individuals possess a hereditary defect known as galactosemia and never have the ability to metabolize galactose. D-Glucose ,the naturally occurring form, is found in fruits, honey, blood, and, under abnormal conditions, in urine. It is also a constituent of the two most common naturally found disaccharides, sucrose and lactose, as well as the exclusive structural unit of the polysaccharides cellulose, starch, and glycogen. Generally, D-glucose is prepared from either potato starch or cornstarch.D-Fructose, a ketohexose, is one of the constituents of the disaccharide sucrose and is also found in uncombined form in honey, apples, and tomatoes. Fructose, generally considered the sweetest monosaccharide, is prepared by sucrose hydrolysis and is metabolized by humans. According to the presence of aldehyde or ketone group, they are classified into aldoses and ketoses. 3  Glyceraldehydeis the simplest aldotriose and dihydroxyacetone is the simplest ketotriose. In the case of glyceraldehydes, there is one pair of enantiomers (D- and L- Glyceraldehyde). D-Glyceraldehyde differs from the L-form only in the penultimate C-atom (the one before the last), where the (─OH) group in the D- form is at the right, while in the L-form it is on the left. Dihydroxyacetone is a Examples of the monosaccharides are the aldopentoseribose, the aldohexoses (glucose and galactose) and the ketohexose fructose. Carbohydrates, such as fructose and glucose, with the same molecular formulas but with different structural arrangements and properties (i.e., isomers) can be formed by relatively simple variations of their spatial, or geometric, arrangements. This type of isomerism, which is called stereoisomerism, exists in all biological systems. Among carbohydrates, the simplest example is provided by the three-carbon aldose sugar glyceraldehyde. There is no way by which the structures of the two isomers of glyceraldehyde, which can be distinguished by the so-called Fischer projection formulas, can be made identical, excluding breaking and reforming the linkages, or bonds, of the hydrogen (―H) and hydroxyl (―OH) groups attached to the carbon at position 2. The isomers are, in fact, mirror images akin to right and left hands; the term enantiomorphism is frequently employed for such isomerism. The chemical and physical properties of enantiomers are identical except for the property of optical rotation. By definition, the carbon atom containing the aldehyde or keto group is called the anomeric carbon atom; similarly, carbohydrate stereoisomers that differ in configuration only at this carbon atom are called anomers. When a cyclic hemiacetal or hemiketal structure forms, the structure with the new hydroxyl group projecting on the same side as that of the oxygen involved in forming the ring is called the alpha anomer; that with the hydroxyl group projecting on the opposite side from that of the oxygen ring is called the beta anomer. Glucose can exist in both a straight-chain and ring form. The ring is formed by reaction the aldehyde group (or the ketonic group in ketoses) with an alcohol groups present in neighboring carbons. 4 The rings can open and re-close, allowing rotation to occur about the carbon bearing the reactive carbonyl yielding two distinct configurations (α and β). The carbon about which this rotation occurs is the anomeric carbon and the two forms are termed anomers. In the α configuration the hydroxyl attached to the anomeric carbon to the right. In the β configuration the hydroxyl attached to the anomeric carbon to the left. Disaccharides Disaccharides are a specialized type of glycoside in which the anomeric hydroxyl group of one sugar has combined with the hydroxyl group of a second sugar with the elimination of the elements of water. Although an enormous number of disaccharide structures are possible, only a limited number are of commercial or biological significance. Lactose is one of the sugars found most commonly in human diets throughout the world; it constitutes about 7 percent of human milk and about 4–5 percent of the milk of mammals such as cows, goats, and sheep. Lactose consists of two aldohexoses—β-D-galactose and glucose— in a β-(1,4) glycosidic bond. 5 Maltose is biologically important because it is a product of the enzymatic breakdown of starches during digestion., is composed of 2 glucose monomers in an α-(1,4) glycosidic bond. Sucrose, or common table sugar, is a major commodity worldwide. is composed of glucose and fructose through an α-(1,2)-β-glycosidic bond. 6 Polysaccharides Polysaccharides, or glycans, may be classified in a number of ways; the following scheme is frequently used. Homopolysaccharides are defined as polysaccharides formed from only one type of monosaccharide. Homopolysaccharides may be further subdivided into straight-chain and branched-chain representatives, depending upon the arrangement of the monosaccharide units. Heteropolysaccharides are defined as polysaccharides containing two or more different types of monosaccharides; they may also occur in both straight-chain and branched-chain forms. In general, extensive variation of linkage types does not occur within a polysaccharide structure, nor are there many polysaccharides composed of more than three or four different monosaccharides; most contain one or two Homopolysachariedes starch refers to a group of plant reserve polysaccharides consisting almost exclusively of a linear component (amylose) and a branched component (amylopectin). Both forms of starch are polymers of α-D-Glucose units, linked by α-glucosidic linkages. The linkage is α-1,4glucosidic linkage in amylose and amylopectin. Branches of about 30 α-D-glucose units are attached with α- 1,6glucosidic linkage at the branching point. The amylopectin component of starch is structurally similar to glycogen in that both are composed of glucose units linked together in the same way, but the distance between branch points is greater in amylopectin than in glycogen, and the former may be thought of as occupying more space per unit weight. Glycogen, which is found in all animal tissues, is the primary animal storage form of carbohydrate and, indirectly, of rapidly available energy. The distance between branch points in a glycogen molecule is only five or six units, which results in a compact treelike structure. The ability of higher animals to form and break down this extensively branched structure is essential to their well-being.. It is a homopolymer of glucose inα-1,4glucosidic linkage; it is also highly branched, with α-(1,6)branch linkages occurring every 8-10 residues. 7  Cellulose forms the principal part of the cell wall of plants. It is the major component of wood paper. It is found in a pure form in cotton. It occurs as bundle of fibers in nature. It is formed of a long non-branched chain of D-glucose units connected by β- 1,4glucosidic linkage. It is non-hydrolysable by amylase because it contains a β- 1,4glucosidic linkage. It can be hydrolyzed by strong acids or by cellulose (present in some bacteria). The presence of cellulose in diet is important as it increases the bulk of food, which stimulates intestinal contractions and prevents constipation. Heteropolysaccharides These are polysaccharides which are formed of more than one type of monosaccharide unit. They include glycosaminoglycans (GAGs) Glycosaminoglycans (GAGs) They are negatively charged unbranched heteropolysaccharides. They are composed of repeating units of disaccharides that include acidic and amino sugars. GAGs are found in animals and bacteria but are absent in plants. The amino sugar in GAGs is either N-acetylglucosamine or N-cetylgalactosamine. And the acidic sugar is usually a uronic acid (like glucuronic acid). It’s composed of amino sugar and uronic acids. Except for hyaluronic acids, all GAGs are sulfated, either as O-esters or N- sulfate.Except for hyaluronic acids, all GAGs are covalently attached to some proteins forming proteoglycans.They are important constituents of extracellular matrix. 8  Variations in the type of monosaccharides and the presence or absence of modification by sulfation results in the different major categories of GAGs, including hyaluronic acid, heparin/heparan sulfate, chondroitin sulfate/dermatan sulfate, and keratan sulfate. The molecular structure of each of the major categories appears below. GAGs are highly negatively charged molecules due to the presence of the carboxyl and/or sulfate groups. Their molecules bind large volumes of water, forming a gel- like matrix that supports cellular and fibrous components of connective tissue. At the same time, the rigidity of GAGs provides structural integrity to cells. GAGs have an extended conformation that raises the viscosity of the solution. This produces the slippery consistency of mucous secretions and lubricating property of synovial fluid. GAGs provide the synovial fluid the shock-absorbing property. When a GAG solution is compressed, water is squeezed out and the GAG molecules occupy a smaller volume. When the compression is released, the GAG molecules regain their original hydrated volume. The negatively charged carboxylate and sulfate groups on the proteoglycan bind positively charged ions and form hydrogen bonds with trapped water molecules, thereby creating a hydrated gel. This gel: Provides flexible mechanical support for the ECM. Acts as a filter that allows the diffusion of ions (e.g., Ca2+), H2O, and other small molecules but slows diffusion of proteins and movement of cells. Acts as a lubricant in synovial fluid. Is compressible: when a GAG solution is compressed, water is squeezed out and GAGs occupy a smaller volume. When the compression is released, their molecules regain their original hydrated size. This gives GAGs solutions the shock absorbing properties and explains their role as shock absorbents in joints and making the eyeball resilient. Hyaluronic Acid Hyaluronic acid (HA) has the simplest structure of all GAGs and does not require additional sulfation of functional groups in the Golgi apparatus as do the other GAGs. Instead, the structure consists of sequentially bound glucuronic acid and N-acetylglucosamine residues. These monosaccharide building blocks are synthesized in the cell cytoplasm and are recruited to the plasma membrane by diffusion for HA synthesis. After synthesis within the plasma membrane, HA gets secreted from the cell into the extracellular space unmodified. 9 - Found in synovial fluid of joints and vitreous humor of the eye Heparan Sulfate/Heparin Heparan sulfate (HS) and heparin (Hep) contain repeating disaccharide units of N-acetylglucosamine and hexuronic acid residues. The hexuronic acid residue glucuronic acid is seen in heparan sulfate, while iduronic acid is present in heparin. Sulfation of the various hydroxyl groups or the amino group present on the glucosamine compound of HS/Hep determines its ability to interact with various proteins, cytokines, and growth factors Heparin represents the earliest recognized biological role of GAGs for its use as an anticoagulant. The mechanism for this role involves its interaction with the protein antithrombin III (ATIII). The interaction of heparin with ATIII causes a conformational change in ATIII that enhances its ability to function as a serine protease inhibitor of coagulation factors. Chondroitin Sulfate/Dermatan Sulfate Chondroitin sulfate (CS) and dermatan sulfate (DS) are similar in structural composition to HS. Their disaccharide repeat consists of N-acetylgalactosamine and hexuronic acids – iduronic acid in DS and glucuronic acid in CS. They are tethered to a PG protein core via the same serine residue and tetrasaccharide linker as HS. Similar to HS/Hep, the sulfation pattern of CS/DS that takes place in the Golgi apparatus determines the biological activity of the resulting compound. - Dermatan Sulfate Found in skin ,Chondroitin sulfate is - Most prevalent GAG in the body being widely spread in cartilage, tendons, and ligaments. Keratan Sulfate Keratan sulfate (KS) contains the disaccharide repeat consisting of galactose and N-acetylglucosamine. Sulfation patterns may be present on either unit of the disaccharide repeat of KS with increased frequency on the N-acetylglucosamine residue. Keratan sulfate has been well-studied for its functional role in both the cornea and the nervous system. The cornea comprises the richest known source of keratan sulfate in the body, followed by brain tissue. The role of keratan sulfate in the cornea includes regulation of collagen fibril spacing that is essential for optical clarity, as well as optimization of corneal hydration during development based on its interaction with water molecules. 10  LIPID OF BIOLOGICAL IMPORTANCE Lipids are a heterogeneous group of water-insoluble (hydrophobic) organic molecules including fats, oils, waxes, steroids and other compounds. Because of their insolubility in aqueous solutions, body lipids are generally found compartmentalized, as in the case of membrane-associated lipids or droplets of triacylglycerol in adipocytes, or transported in plasma in association with protein, as in lipoprotein particles or on albumin. Lipids are a major source of energy for the body, and they also provide the hydrophobic barrier that permits partitioning of the aqueous contents of cells and subcellular structures. Lipids serve additional functions in the body (for example, some fat-soluble vitamins have regulatory or coenzyme functions, and the prostaglandins and steroid hormones play major roles in the control of the body’s homeostasis). Not surprisingly, deficiencies or imbalances of lipid metabolism can lead to some of the major clinical problems encountered by physicians, such as atherosclerosis, diabetes, and obesity. II. Lipids are organic compounds, which have the following common properties, they are esters of fatty acids or substances associated with them in nature and Most of them are insoluble in water but soluble in fat solvents (non polar solvents) e.g.: benzene, chloroform, acetone and ether. LIPIDS ARE CLASSIFIED AS SIMPLE OR COMPLEX. Simple lipids: Esters of fatty acids with various alcohols. a. Fats: Esters of fatty acids with glycerol. Oils are fats in the liquid state. b. Waxes: Esters of fatty acids with higher molecular weight monohydric alcohols. 2. Complex lipids: Esters of fatty acids containing groups in addition to an alcohol and a fatty acid. a. Phospholipids: Lipids containing, in addition to fatty acids and an alcohol, a phosphoric acid residue. They frequently have nitrogencontaining bases and other substituents, eg, in glycerophospholipids the alcohol is glycerol and in sphingophospholipids the alcohol is sphingosine. b. Glycolipids (glycosphingolipids): Lipids containing a fatty acid, sphingosine, and carbohydrate c. Other complex lipids: Lipids such as sulfolipids and aminolipids. Lipoproteins may also be placed in this category. 3. Precursor and derived lipids: These include fatty acids, glycerol, steroids, other alcohols, fatty aldehydes, and ketone bodies, hydrocarbons, lipid-soluble vitamins, and hormones. Because 11 they are uncharged, acylglycerols (glycerides), cholesterol, and cholesteryl esters are termed neutral lipids. Fatty Acids Fatty acids occur mainly as esters in natural fats and oils but do occur in the unesterified form as free fatty acids, a transport form found in the plasma. Fatty acids that occur in natural fats are usually straight-chain derivatives containing an even number of carbon atoms. The chain may be saturated (containing no double bonds) or unsaturated (containing one or more double I- Saturated Fatty Acids (SFA): Saturated fatty acids may be envisaged as based on acetic acid (CH3COOH) as the first member of the series in which CH2 is progressively added between the terminal CH3 and COOH groups. Examples Other higher members of the series are known to occur, particularly in waxes. A few branched-chain fatty acids have also been isolated from both plant and animal sources. CH₃- (CH₂) n- COOH Where n = Total number of carbons – 2 A- Short chain fatty acids include: - Acetic acid (C2) - Butyric acid (C4) - Caproic acid (C6) - Caprylic acid (C8) - Capric acid (C10) B- Long chain fatty acid, the most common include mainly: - Palmitic acid (C16) - Stearic acid (C18) - Arachidic acid (C20) - Lignoceric acid (C24) The most important fatty acids include: - Palmitic and stearic acids which are widely distributed in animal fats. - Palmitic acid is the commonest fatty acid in human tissues. II- Unsaturated Fatty Acids (USFA): They contain one or more double bonds. Most of the double bonds present in USFA are of the Cis type and they are liquid at room temperature. USFA containing Trans double bonds are linear and solid at room temperature. 12  In case of poly unsaturated fatty acids (PUFA), each two double bonds are separated by a methylene group (CH₂) as follows: -CH=CH-CH₂-CH=CH- CH₂- : - Palmitoleic acid (16:1:ω7) CH₃ (CH₂)₅ CH=CH (CH₂)₇.COOH - Oleic acid (18:1:ω9) CH₃ (CH₂)₇ CH=CH (CH₂)₇.COOH - Nervonic acid (24:1:ω9) CH₃ (CH₂)₇ CH=CH (CH₂)₁₃.COOH - α Linolenic acid (18:3 CH₃.CH₂.(CH=CH.CH₂)₃.(CH₂)₆.COOH - Timnodonic acid (20:5) CH₃.CH₂.(CH=CH.CH₂)₅.(CH₂)₂.COOH - Cervonic acid (22:6) CH₃.CH₂. (CH=CH.CH₂)₆.(CH₂)₁.COOH - Linoleic acid (18:2), CH₃.(CH₂)₄.(CH=CH.CH₂)₂.(CH₂)₆.COOH - γ- Linolenic acid (18:3) CH₃.(CH₂)₄.(CH=CH.CH₂)₃.(CH₂)₃.COOH - Arachidonic acid (20:4) CH₃.(CH₂)₄.(CH=CH.CH₂)₄.(CH₂)₂.COOH Nutritional classification of fatty acids: Nutritional classification of fatty acids: Essential Fatty Acids: They are not synthesized in our body, so it is essential to take them in diet. They include α linolenic and linoleic acids. Arachidonic acid is synthesized in our bodies from linloleic but in its absence, arachidonic acid might be considered as an essential fatty acid. Deficiency of essential fatty acids produces Fatty liver and sterility in adults. Impaired growth and dermatitis in infants.Sources of PUFA: They are present mainly in fish and vegetable oils e.g.: maize, cottonseed, linseed, olive, sun flower and soya been oils. Non Essential Fatty Acids: They include all other 13 fatty acids because they are formed in our body in good amounts mainly from carbohydrates. It is not essential to take them in diets. Eicosanoids Eicosanoids: These compounds, derived from eicosa- (20-carbon) polyenoic fatty acids, comprise the prostanoids, leukotrienes (LTs), and lipoxins (LXs). Prostanoids include prostaglandins (PGs), prostacyclins (PGIs), and thromboxanes (TXs). they have important physiologic and pharmacologic activities. They are synthesized in vivo by cyclization of the center of the carbon chain of 20-carbon (eicosanoic) polyunsaturated fatty acids (eg, arachidonic acid) Classification: There are multiple subfamilies of eicosanoids, including most prominently the prostaglandins, thromboxanes, leukotrienes, lipoxins, resolvins,and eoxins. For each subfamily, there is the potential to have at least 4 separate series of metabolites, two series derived from ω-6 PUFAs (arachidonic and dihomo- gamma-linolenic acids), one series derived from the ω-3 PUFA (eicosapentaenoic acid), and one series derived from the ω-9 PUFA (mead acid). This subfamily distinction is important. Mammals, including humans, are unable to convert ω-6 into ω-3 PUFA. In consequence, tissue levels of the ω-6 and ω-3 PUFAs and their corresponding eicosanoid metabolites link directly to the amount of dietary ω-6 versus ω-3 PUFAs consumed. on other way They are classified into 2 main groups: Cyclic compounds (prostanoids): as (Prostaglandins (PG). - Prostacyclins (PGI). - Thromboxanes (TX). Acyclic compounds: (Leukotrienes (LT). - Lipoxins (LX)). 1. Prostaglandins: They are derivatives of the C20 hypothetical compound termed prostanoic acid. They were first discovered in the secretion of prostatic gland hence, the name prostaglandins. However, they are discovered in most human tissues both in males and females. All prostaglandins have a cylopentane ring in the middle of the molecule (from C₈ to C₁₂).There are many types of prostaglandins e.g.: PGA, PGB, PGE, PGF, PGG & PGH. In human tissues the most important members are: PGE: ether soluble and contains a ketone group at C₉. PGF: soluble in phosphate buffer and contains hydroxyl group at C₉. 14 Actions and functions of prostaglandins: PGE₂: Prostaglandins are hormone-like substances that affect several bodily functions, including inflammation, pain and uterine contractions. Healthcare providers use synthetic forms of prostaglandins to treat several conditions. They also use medications to block the effects of prostaglandins. It produces vasodilatation, smooth muscle relaxation and bronchodilation and relief of bronchial asthma. PGF₂α: It produces vasoconstriction and smooth muscle contraction. 2. Thromboxanes (TX): Thromboxane is a member of the family of lipids known as eicosanoids. The two major thromboxanes are thromboxane A2 and thromboxane B2. The distinguishing feature of thromboxanes is a 6-membered ether-containing ring. Thromboxane is named for its role in blood clot formation (thrombosis). They are characterized by the presence of an oxane ring (containing 2 oxygen atoms) e.g. TXA₂ & TXB₂. They are formed by platelets. Thromboxane-A synthase, an enzyme found in platelets, converts the arachidonic acid derivative prostaglandin H2 to thromboxane. People with asthma tend to have increased thromboxane production, and analogs of thromboxane act as bronchoconstrictors in patients with asthma 3. Prostacyclins (PGI): They contain an additional ring in their structure e.g. PGI₂ which is an antagonist of TXA₂. Prostacyclin (also called prostaglandin I2 or PGI2) is a prostaglandin member of the eicosanoid family of lipid molecules. It inhibits platelet activation and is also an effective vasodilator. 15 Action: PGI2 produces vasodilatation, prevents platelet aggregation and thrombus formation. 4. Leukotrienes (LT): They are acyclic compounds and characterized by presence of 3 conjugated double bonds. They are secreted from leukocytes, platelets and mast cells. Leukotrienes are a family of eicosanoid inflammatory mediators produced in leukocytes by the oxidation of arachidonic acid (AA) and the essential fatty acid eicosapentaenoic acid (EPA) by the enzyme arachidonate 5-lipoxygenase Leukotrienes use lipid signaling to convey information to either the cell producing them (autocrine signaling) or neighboring cells (paracrine signaling) in order to regulate immune responses. The production of leukotrienes is usually accompanied by the production of histamine and prostaglandins, which also act as inflammatory mediators. Action: - Increase vascular permeability. - Components of slow-reacting substances of anaphylaxis (SRS-A) which are severe allergic reactions. Leukotrienes act principally on a subfamily of G protein-coupled receptors. They may also act upon peroxisome proliferator- activated receptors. Leukotrienes are involved in asthmatic and allergic reactions and act to sustain inflammatory reactions. Several leukotriene receptor antagonists such as montelukast and zafirlukast are used to treat asthma 16 5. Lipoxins (LX): They are also acyclic compounds containing three conjugated double bonds but they contain more oxygen than (LT). They are secreted from arterial walls. Action: They are anti-inflammatory compounds. I- Simple Lipids They are esters of fatty acids with alcohols, according to the types of alcohols there are two main sub-groups: Neutral fats or triacylglycerols (TAG): Neutral fats, also known as true fats, are simple lipids that are produced by the dehydration synthesis of one or more fatty acids with an alcohol like glycerol. Waxes: Natural waxes may contain unsaturated bonds and include various functional groups such as fatty acids, primary and secondary alcohols, ketones, aldehydes and fatty acid esters. Synthetic waxes often consist of homologous series of long-chain aliphatic hydrocarbons (alkanes or paraffins) that lack functional groups. Neutral fats (Triacylglycerol or Triglycerides): They contain glycerol alcohol, which is colorless, odorless and has a sweet taste. It is liquid and soluble in water. The fatty acids present in TAG are usually of different types (mixed TAG). Neutral fats are classified into two sub-groups: Oils: They are liquid at room temperature due to their high content of USFA and Solid fats: They are solid at room temperature due to their high content of long chains SFA. 17 Addition of hydrogen (reduction or hardening): It is the addition of hydrogen through the double bonds to convert USFA into SFA. So the liquid oils will be converted into solid fats or margarine and hence the name (hardening). II- Conjugated Lipids (Compound Lipids) They contain fatty acids, alcohols and other groups. According to the type of the attached group they are classified into: 1- Phospholipids: Containing phosphate radicals. 2- Glycolipids: Containing carbohydrate radicals. 1- Phospholipids They are classified according to the alcohol present into two main sub-groups: A- Glycerophospholipids: Containing glycerol. B- Sphingomyelin: Containing sphingosine (sphigol). A- Glycerophospholipids(Glycerophosphatides): They are phospholipids containing glycerol. They are derivatives of phosphatidic acid and include the following types: 1.Phosphatidic acid (Diacylglycerolphosphate): On hydrolysis: It gives one glycerol, one saturated fatty acid (usually at position 1), one unsaturated fatty acid (usua-lly at position 2), and phosphoric acid. 2. Lecithin (phosphatidyl-choline): It is formed of phosphatidic acid and choline. It is usually present in the cell membrane specially in the liver, lung and brain. It is also present in blood plasma. 3. Cephalin (phosphatidyl-ethanolamine): It is formed of phosphatidic acid and ethanolamine. It is present in the cell membranes and blood plasma. 4. Phosphatidyl serine: It is formed of phosphatidic acid and serine amino acid. It is present in cell membranes. 5. Phosphatidyl inositol (lipositol): It is formed of phosphatidic acid and inositol. It is present in cell membranes. Phosphatidyl inositol 4,5 bisphosphate (PIP₂) 18 acts as secondary messenger in the process of intracellular signal transduction (explained later on). 6. Phosphatidyl glycerol: It is formed of phosphatidic acid and glycerol. 7- Cardiolipins (diphosphatidyl-glycerol): They are formed of two molecules of phosphatidic acid connected by a molecule of glycerol. So, they contain 4 FA, 3 glycerol and 2 phosphates. They form an important component of inner mitochondrial membrane. 19 Hydrolysis of glycerophospholipids They are hydrolyzed by group of enzymes termed phospholipases - Phospholipase A1 (PLA1): separates SFA from position 1. - Phospholipase A2 (PLA2): separates USFA from position 2. Lysophospholipids: are formed by the action of phospholipase A2 on lecithin or cephalin to form lysolecithin and lysocephalin. Phospholipase A2 removes the fatty acid in position 2. Snake venom toxins contain lecithinase enzyme with PLA2activity, when injected into blood it converts phospholipids present in cell membranes of RBCs into lysophospholipids which produce hemolysis of RBCs. This causes death if not treated by antitoxins. B- Sphingomyelin: This type is present in cell membranes specially of the lungs and brain mainly in the myelin sheath. It contains sphingosine (sphingol) which is 18 carbon amino alcohol. Fatty acids are linked to sphingosine by an amide bond to form ceramide, which is connected to phoshocholine to form sphingomyelin. Importance and functions of phospholipids: The main function of phospholipids is to act as a barrier in the cell. In the cell, the phospholipids form a bilayer which allows some molecules to pass through and prevents others from passing through. Other functions of phospholipids in the body include: Regulating cellular processes Assembling lipoproteins Emulsifying cholesterol and bile acids Helps the body absorb fat Acting as a lubricant to help joints move smoothly - Phospholipids are amphipathic molecules that contain non-polar groups (fatty acid side chains) and polar groups (phosphate, serine, ethanolamine, glycerol, 20 choline and inositol) they form micelles in water.They are good emulsifying factors, important for digestion and absorption of dietary fats. - They are good hydrotropic substances; they prevent deposition of cholesterol as cholesterol stones (biliary calculi). Also they are important constituents of lipid bilayer in cell membranes.They are important constituents of plasma lipoproteins. - Lung surfactant is formed mainly of dipalmitoyl-lecithin, the lack of which is responsible for respiratory distress syndrome in premature infants.They provide arachidonic acid for synthesis of eicosanoids. - They are essential for blood clotting, as they provide the platelet activating factor (PAF), which is a choline plasmalogen that contains palmitoyl alcohol at position 1 and acetic acid at position 2. - Intracellular signal transduction: Receptor interaction with specific ligand at cell membrane produces activation of G-proteins that produce activation of phospholipase C. Phospholipase C converts phosphatidylinositol-4,5- bisphosphate (PIP2) into inositol-trisphosphate (IP3) and diacylglycerols (DAG). IP3 and DAG act as second messengers. IP3 increases release of intracellular Ca2+ form intracellular storage sites. DAG is capable of activating protein kinase C (PKC) which produces phosphorylation of certain proteins. Both effects are responsible for producing the specific cellular response. Many chemical transmitters (e.g. acetylcholine, histamine and serotonin), hormones (e.g. vasopressin and α-1 receptors for epinephrine and norepinephrine) and growth factors act through activation of phospholipase C. III- Derived Lipids They are produced by hydrolysis of either simple or conjugated lipids or they are associated with lipids in nature. They include the following: 1. Fatty acids. 2. Alcohols. 3. Steroids. 4. Carotenoids. 5. Fat soluble vitamins: as vitamins A, D, E & K. 21 Steroids They are compounds containing steroid nucleus. This nucleus is composed of four fused rings with 17 carbon atoms. It is named as cyclo-pentano-perhydro- phenanthrene ring (CPPP). Classification of steroids They include the following groups: 1- Sterols. 2- Bile acids. 3- Steroid hormones. Sterols Bile Acids They contain 24 carbon atoms and they are classified into: Primary bile acids: they are formed in the liver from cholesterol and they include: Cholic acid: 3,7,12 trihydroxycholanic acid. It is the main bile acid present in bile. Chenodeoxy cholic acid: 3,7 dihydroxycholanic acid. Secondary bile acids: they are formed of primary bile acid in large intestine. By the action of 7 α- dehydroxylase enzyme which removes the hydroxyl group at C7. They include: Deoxycholic acid: 3,12 dihydroxycholanic acid. Lithocholic acid: 3 monohydroxycholanic acid, which is the least soluble 22 Bile salts: They are formed by conjugation of cholic acid with glycine (80%) or taurine (20%) then they are excreted by liver in bile as sodium glycocholate or sodium taurocholate. Bile salts pass to the intestine where they are reabsorbed and return back to the liver to be excreted again in bile (entero-hepatic circulation). Importance of bile salts: Conversion of cholesterol to bile salt is an important mechanism for removal of excess cholesterol from blood. They are good emulsifying factors important for digestion and absorption of fats.They prevent precipitation of cholesterol in the bile as cholesterol stones.They stimulate liver cells to secrete more bile (choleretic effect). 23  AMINO ACIDS and protein OF BIOLOGICAL IMPORTNCE Proteins are organic compounds with a high molecular weight. Twenty different amino acids found in protein structure, all are amino acids, except proline is imino acid. These amino acids are organic acids, which have one or more substituted amino group (NH2). Basic structure for amino acids COO- COOH carboxyl end + H3N – C – H amino endH2N – C – H - Ionization of amino acids at carbon R side chain (functional group) R physiological pH 7.4 Classification of Amino Acids Amino acids can be classified according to: their chemical structure "Chemical classification”, according to” polarity” of the side chain, according to their nutritional importance "Nutritional classification" or according to their metabolic fate "Metabolic classification". I - Chemical classification Although about 300 amino acids occur in nature, only 20 form the primary structure of all proteins. Only L,α-amino acids which are present in proteins from all forms of life i.e. plant, animal or microbial. These amino acids are classified into: aliphatic amino acids, aromatic amino acids, and heterocyclic amino acids. Amino Acids with Aliphatic Side Chain These amino acids have no rings in their side chains. According to the number of both NH2 groups and COOH groups they are further subdivided into:Aliphatic neutral amino acids, which contain one NH2 group and one COOH group (monoamino-monocarboxylic acids). Aliphatic acidic amino acids, which contain one NH2 group and two COOH groups (monoaminodicarboxylic acids). Aliphatic basic amino acids, which contain one COOH and more than one NH 2 groups (diaminomonocarboxylic acids).Aliphatic neutral amino acids include: 24 - Glycine: -aminoaceticacid.,Alanine: α-amino propionic acid ,Valine: α- amino, β-methyl butyric acid, Leucine:α amino isocaproic acid.Isoleucine: β- methyl,β -ethyl, α-amino propionic acid or β-methyl, α-amino valericacid,Serine: α-amino, β-hydroxy propionic acid,Threonine: α-amino,β - hydroxy butyric acid.Cysteine: α-amino,β-thiopropionic acid Cystine or "dicysteine": It is formed of two cysteine molecules by removal of 2 hydrogen atoms and formation of a disulfide bond. Methionine: γ-methyl thiol, α-amino butyric acid. IAliphatic acidic amino acids and their amides include: Aspartic acid: α-amino succinic acid.,Asparagine: amide of aspartic acid Glutamic acid: α-amino glutaric acid. Glutamine: amide of glutamic acid IAliphatic basic amino acids include:Lysine: α,-diamino caproic acidArginine: amino, -guanidovaleric acid. B- Amino Acids with Side Chains Containing Aromatic Rings: These are α-amino acids which contain benzene ring in their side chains and include:Phenylalanine: -amino, -phenylpropionic acid. Tyrosine: P- hydroxyphenylalanine.Tryptophan: -amino, -indole propionic acid. C- Amino Acids with Side Chains Containing Heterocyclic Rings These are amino acids which contain heterocyclic ring in their side chains. They include histidine, trypyophan (aromatic and heterocyclic amino acid) and praline (imino acid) they include:Histidine:-amino, -imidazole propionic acid. Proline: 2-pyrrolidine carboxylic acid. N.B. Cystine, hydroxyproline and hydroxylysine are formed after synthesis of proteins from cysteine, proline and lysine respectively. II. Classification of Amino Acids According to” Polarity” of Side Chain Polar charged (amino acids with charged R groups)Basic amino acids with positively charged side chain: lysine, arginine and histidineAcidic amino acids with negatively charged side chain: aspartic and glutamic acids Polar uncharged (amino acids with uncharged polar R groups) Hydroxy amino acids, as serine, threonine, tyrosine, hydroxyproline and hydroxylysine.SH group containing amino acids as cystiene Asparagine and glutamine having amide group Non polar none charged( amino acids with uncharged R groups)All amino acids with hydrophobic side chain e.g. alanine, valine, leucine and isoleucine 25 III - Nutritional Classification of Amino Acids According to their nutritional significance, amino acids in general can be classified into three main categories: Essential amino acids :These are amino acids which cannot be synthesized in the body and have to be supplied in diet, otherwise, manifestations of protein deficiency will appear. They are 8 amino acids.Threonine ,LeucineIsoleucine Valine Methionine -Lysine Phenylalanine Tryptophan Semi-essential: they are formed at a rate enough for adults but not for growing animals and include 2 only. HistidineArginine Non-essential amino acids: These are amino acids which can be synthesized in the body from intermediates of many metabolic processes. Their dietary shortage is not associated with manifestations of protein deficiency.They include the remaining 10 amino acids; Glycine, Alanine, Serine, Cysteine, Aspartate, Asparagine, Glutamate, Glutamine, Tyrosine and Proline. A - General Properties: Amphoteric property "Protonic equilibrium" : Amino acids can react both with acids and bases, so they are ampholytes. In acidic medium: They are positively charged (R-NH3+). In alkaline medium: They are negatively charged (R-COO-). At Iso Electric Point (IEP): They form dipolar ions (Zwitterions) which are at pH 6.02 for all monoamino-monocarboxylic amino acids. In this form, the amino acid cannot migrate in electric field. R – CH – COOH HCl R – CH – COO- NaOH R – CH – COO- Na+ + H2O NH3+Cl- NH3+ NH2 Acidic medium Zwitterion (IEP) Alkaline medium So, amino acids are present in three forms according to the pH of the solution and the uncharged form is not present at any pH. PROTEINS OF BIOLOGICAL IMPORTANCE Proteins are formed of amino acids linked together by peptide bonds. The term polypeptides mean the presence of large number of peptide bonds. Oligopeptides contain from 2 to 10 amino acids. Polypeptides contain from 11 to 49 amino acids, Protein molecule is formed of 50 or more amino acids. The molecular weight of proteins ranges from 5000 to several millions. Proteins constitute 50% or more of organic molecules of dry weight of any cell. All proteins contain carbon, hydrogen, nitrogen and oxygen and nearly all 26 contain sulfur. Some proteins contain additional elements e.g. phosphorus, iron, zinc, manganese and copper. Biological importance and function of proteins: Provide the body with essential amino acids, nitrogen and sulfur Enzymes are mainly protein in nature. Glutathione:- Tripeptide formed of 3 amino acids (glycine, glutamate and cysteine).- Functions: antioxidant, coenzyme and plays an important role in amino acid transport. Glucagon- Peptide hormone formed of 29 amino acids produced by the pancreas.-Functions- Increases plasma glucose level (hyperglycemic agent). Insulin- Protein hormone formed of 51 amino acids produced by the pancreas. Functions - Decreases plasma glucose level (hypoglycemic agent).The conformation of proteins Different Levels of Protein Structure Proteins in its native state are characterized by its three dimensional structure, (primary. secondary and tertiary). Proteins which are formed of two or more polypeptide chains have additional quaternary structure. Structure of proteins can be divided into four orders: 1 - Primary Structure: It is the sequence of amino acids and the location of disulfide bond in the polypeptide chain. Each polypeptide chain is formed of -amino acids united by peptide bonds. Bonds responsible for the maintenance of primary structure are mainly peptide bonds and disulfide bonds. Both of them are covalent bondsThe chain starts on the left side by amino acid number 1, which contains a free amino group (terminal amino group) and termed the N-terminus amino acid. On the right side, at the end, the polypeptide chain contains an amino acid with a free carboxylic group (terminal carboxylic group) and termed C-terminus amino acid.Even change of a single amino acid in the linear order of polypeptide chain may lead to profound physiologic effects. The genetic information present in DNA control the primary structures of proteins, which determine the secondary and tertiary structures that are essential for functions of proteins. 2 - Secondary structure: It includes mainly two forms, α-helical or β-pleated sheets (regular secondary structure). Regions of protein structure which are not present in  helix or β pleated sheets are said to be present in random coil. Other forms also exist, loop regions β bends and disordered regions. Regular secondary structure There are two main types of ordered secondary structure observed in proteins. 27  1. () -Helix In an alpha-helix, the protein chain is coiled like a loosely-coiled spring. The "alpha" means that if you look down the length of the spring, the coiling is happening in a clockwise direction as it goes away from you. 2. β -Pleated Sheet In a beta-pleated sheet, the chains are folded so that they lie alongside each other. The folded chains are again held together by hydrogen bonds involving exactly the same groups as in the alpha-helix. 3. Tertiary structure: The tertiary structure of a protein is a description of the way the whole chain (including the secondary structures) folds itself into its final 3-dimensional shape. Amino acids which are very distant in the primary structure might be close in the tertiary one because of the folding of the chain. This structure is maintained by four types of interactions. A. Hydrophobic interactions The non-polar side chains of neutral amino acids tend to associate together in proteins. These are called as hydrophobic interactions. They play significant role in maintaining tertiary structure. B. Electrostatic bonds These bonds are formed between oppositely charged groups of amino acid side chains. For example, the -amino groups of lysine is positively charged and second (non-α) carboxyl group of aspartic acid is negatively charged at physiological or body pH. These interact electrostatically to stabilize tertiary structure of protein. They are also called as salt bridges or ionic bonds. C. Internal hydrogen bonds Amino acid side chains are involved in the hydrogen bond formation. Hydroxyl group of some amino acids e.g. serine, threonine, the amino groups and carbonyl oxygen of glutamine and aspargine, the ring nitrogen of histidine participate in internal hydrogen bond formation. D. Van der Waals interactions These are the weak interactions between uncharged groups of protein molecule. They also contribute to the stability of proteins. E- Disulfide Bonds They are present in many proteins e.g. keratin and insulin. 4. Quaternary structure : It is the overall 3-dimensional structure of the entire protein.Quaternary structure only is defined for proteins with more than one polypeptide chain... Hemoglobin has quaternary structure due to association of two alpha globin and two beta globin chains. Many proteins do not have the quaternary structure and function as monomers 28 Fibrous and Globular Proteins: Two types of proteins are distinguished, according to their overall dimensions: 1 - Fibrous proteins: These have an axial ratio greater than 10 e.g. keratin and collagen. 2- Globular proteins: These have an axial ratio (length / width) of less than 10 e.g. insulin and myoglobin. General Properties of Proteins 1. Solubility: Most proteins are soluble in water forming colloidal solutions. Some proteins are insoluble in water, e.g. keratin. The hydrophobic amino acids tend to repel the aqueous environment and, therefore, are mainly located in the interior of proteins. This class of amino acids does not ionize nor participate in the formation of hydrogen bonds. The hydrophilic amino acids tend to interact with the aqueous environment, are found on the exterior surfaces proteins.This class often involved in the formation of hydrogen bonds 2. Amphoteric Properties: - Proteins contain free amino groups and free carboxylic groups. - Proteins are positively charged in acidic medium and negatively charged in alkaline medium. - At a certain pH, the protein molecule carries equal positive and negative charges. This is called the Iso-Electric Point (I.E.P.) of the protein. At this pH the protein is least soluble and cannot migrate in an electric field 3-Denaturation of Proteins - Denaruration is disruption of the organization (secondary, tertiary and quaternary structures) of the protein molecule. - Denaturation is due to destruction of the non-covalent bonds holding a protein in its correct conformation by heat, pH, and salt concentration. - The peptide chains become unfolded and irregularly arranged. - The primary structure remains intact. Denaturation Renaturation Refolding Native form Native form Random coil Effects of denaturation: 1. Decreased solubility due to appearance of hydrophobic R groups. 2. Increased viscosity due to expansion of the molecule. 29  3. Increased digestibility by proteolytic enzymes due to unfolding of polypeptide chain. 4. Loss of biological activity, e.g. enzymatic, hormonal & immunologic properties. Sometimes these effects are reversible and the protein can resume its normal conformation once these are removed. 30  PROTEINS OF EXTRACELLULAR MATRIX Extracellular matrix is the connective tissue (CT) surrounding the cells and organs. Extracellular Matrix (ECM) contains 3 types of proteins: A- Structural proteins e.g. collagen and elastin. B- Specialized proteins as fibronectin, laminin and fibrillin. C- Proteoglycans: a core of proteins (5%) to which long chains of glycosaminoglycans (GAGs) are attached. 1-Collagen: Glycine Usually proline Usually hydroxyproline Typical Sequence in -Chain Triple helix of tropocollagen molecule It is formed by C.T cells (fibroblasts) and is present in skin, bones, cartilages, tendons, lungs, liver, vessels and cornea. It forms 25% of total body proteins. Amino acid composition: The α-chain contains the repeating triplet sequence Gly-X-Y, where Gly is glycine, which is present in every third position. - Proline is commonly found in the X position and hydroxyproline in the Y position of the Gly-X-Y triplets. The X position is sometimes occupied by lysine. The Y position is sometimes occupied by hydroxylysine. The number of hydroxylysine varies for different types of collagen. Glucose or galactose are attached to hydroxylysine residues (so collagen is considered as a glycoprotein). N.B. Collagen is nondigestible (of low biological value). Boiling changes it into gelatin; which is soluble and digestible. Structure of collagen: Tropocollagen is the building unit.It is a long protein (300-nm) and thin (1.5-nm- diameter).It consists of three coiled peptides [α-Chains]. Each chain is a left handed helix. The presence of proline prevents right handed helix formation.The three left handed peptide chains are tightly twisted to form a right handed superhelix (tropocollagen molecule). Collagen has a very firm structure due to: 31 -Each turn contains 3 amino acid residues (makes a tight helix), normal proteins contain 3.6 a.a. per turn.The presence of glycine with its short side chain makes the polypeptide chains very close to each other.The 3 left handed coiled peptide chains form a right handed superhelix.The high content of hydroxyproline forms hydrogen bonds between chains.The formation of covalent cross linkages between adjacent polypeptide chains are responsible for tensile properties of collagen (stretched without being broken). The specific arrangement into fibril and fibers. The staggered array (overhanging) gives marked flexibility to collagen. Types of collagen: The collagen super family of proteins includes more than twenty collagen types, as well as additional proteins that have collagen-like domains. The most common types of collagen are type I and type II. Type I contains two chains of α1 and one chain of α2 (α12α2), whereas type II collagen contains three α1 chains (α13). Synthesis of collagen: Intracellular Processing Synthesis of preprocollagen -chain. It is formed by the ribosomes on the rough endoplasmic reticulum. Preprocollagen is formed of N-terminal signal peptide, two extension peptides (one at C-terminal and one at the N-terminal) and procollagen - chain. The signal peptide directs the polypeptide chain into the endoplasmic reticulum..Conversion of preprocollagen -chain to procollagen -Chain Removal of the signal peptide by signal peptidase.Hydroxylation of some prolyl and some lysyl residues by prolyl and lysyl hydroxylase respectively. Both enzymes require vitamin C as cofactor. Glycosylation of some hydroxylysyl residues. Addition of glucose and galactose is catalyzed by glucosyl transferase and galactosyl transferase respectively.Formation of procollage. Oxidation of cysteine residues and formation of disulfide bonds, which are present in the C-terminal extension peptides and are needed to initiate and form the triple helix. The procollagen molecules are packaged into transport vesicles for export from the cell. Extracellular Processing Formation of tropocollagen: This formed is by removal of the two extension peptides from the amino and carboxyl terminals. These reactions are extracellular and catalyzed by two enzymes called procollagen aminoproteinase and procollagen carboxyproteinase (procollagen peptidases). The triple helix is further stabilized by formation of inter-chain cross linkages catalyzed by lysyl oxidase (copper dependent enzyme). Collagen fibril and fiber formation: The newly formed tropocollagen molecules assemble in a parallel and staggered array into collagen fibrils. These fibrils are then packed together to form collagen fibers.Maturation of the collagen: Collagen fibers are further stabilized by the formation of covalent cross-links, both within and between the triple helical units. These cross-links form through the action of lysyl oxidase, that oxidatively deaminates the ε-amino groups of certain lysine and hydroxylysine residues, yielding reactive aldehydes. Such aldehydes react with the ε- amino groups of unoxidized lysines or hydroxylysines. These cross links increase by age and collagen becomes less flexible. 32  Synthesis of Collagen Fiber Preprocollagen - chain Signal peptide N-terminal extension peptide C-terminal extension peptide Removal of signal peptide OH Hydroxylation and glycosylation OH Procollagen - chain OH Formation of C-terminal extension peptide disulfide bonds and initiation of folding of the three polypeptide chains of procollagen Formation of triple helical chains of procollagen Intracellular Steps Procollagen Removal of extension peptides by procollagen peptidases Tropocollagen Extracellular Steps Collagen hole zone Association of tropocollagen molecules into fibril and formation of covalent cross-linkages 50 nm Collagen fibril (staggered array) Aggregation of fibril into fiber Collagen fiber 2- Elastin - Elastin is an insoluble protein polymer synthesized from a precursor, tropoelastin, which is a linear polypeptide composed of about 700 amino acids that are primarily small and nonpolar (for example, glycine, alanine, and valine). Elastin is also rich in proline and lysine, but contains only a little hydroxyproline and no hydroxylysine. - It is present in elastic CT of ligaments, lungs and walls of blood vessels. 33 - Tropoelastin is secreted by the cell into the extracellular space. There it interacts with specific glycoprotein microfibrils, such as fibrillin, which function as a scaffold onto which tropoelastin is deposited. Some of the lysyl side chains of the tropoelastin polypeptides are oxidatively deaminated by lysyl oxidase, forming allysine residues. Three of the allysyl side chains plus one unaltered lysyl side chain from the same and neighboring polypeptides form a desmosine cross-link. - Elastin is fibrous when extended, globular when relaxed. It is highly elastic and can be stretched in different directions due to its desmosine content and its special arrangement of its chains into α chains and random sequences. 3- Fibronectin: - Fibronectin is a glycoprotein of ECM and in plasma. It is one of the adhesive proteins. Its main function is adhesion of cells to ECM. It consists of 2 identical domains joined by disulfide S - S bridges. It has several binding sites, which can bind to heparin, collagen, and cell surface receptors (integrins). Integrins are transmembrane adhesion receptors. They interact with extra cellular proteins as fibronectin and collagen at cell surface. They react with actin microfilaments in the cytosol. Through these interactions, the interior and exterior can communicate. 4- Fibrillin: It is a large glycoprotein, which is secreted by fibroblasts into the extracellular matrix. It provides a scaffold for deposition of elastin. Fibrillin is found in the suspensory ligament of lens and associated with elastin in the aorta and in periosteum. Marfan's syndrome: It is due to abnormalities (mutations) in the gene encoding fibrillin. It affects the eyes (produces ectopia lentis or dislocation of the lens), cardiovascular system (weakness of aorta and dilatation of ascending aorta), skeletal system (patients are tall and have long digits) and joints (hyperextensibility). Cartilage Proteins Cartilage is an avascular CT (its nutrition is by diffusion and osmosis). Proteins of the Cartilage include: Collagen and noncollagenous proteins. A) Collagen: - Type II forms 90-98% of the total collagen present. It is formed of 3 α1 chains. It contains more hydroxylysine and more glycosylation. - Minor forms of collagen are also present e.g. type V, VI, IX, X and XI. B) Noncollagenous proteins: 1- Aggrecan: It is the major proteoglycan present in cartilage. It has a very complex structure containing several types of GAGs (hyaluronic acid, chondroitin sulfate and keratan 34 sulfate) attached to a protein core and a link protein. It has a bottle brush shape. It is formed of a long chain of hyaluronic acid attached noncovalently to link proteins, which are attached noncovalently to core proteins. Different types of GAGs are projecting from these core proteins. 2- Different types of proteoglycans 3- Chondronectin: It has a role in binding type II collagen to the chondrocytes. Molecular organization of cartilage matrix: GAGs side chains bind electrostatically to collagen fibrils. GAGs side chains are acidic and therefore negatively charged, they repel each other. Hence, they attract water in between causing the molecule to form a gel. It plays an important role in compressibility of cartilage. Structure of aggrecan changes with age: - The keratan sulfate and protein of the monomer are increased. The average monomer size is decreased.The chondroitin sulfate content is decreased. The chondroitin sulfate chains become shorter. Bone Proteins Bone is a mineralized CT. Proteins of the bone include, collagen and noncollagenous proteins. A) Collagen: - Type [I] forms 90% of bone proteins. It is formed of two α1 and one α2 chains. - Minor forms of collagen Type [V]. B) Noncollagenous proteins: 1- Osteocalcin: - It is bone specific protein, synthesized by osteoblasts. - It forms the major non-collagenous matrix protein. - It contains 3-5 γ-carboxy glutamate residues, that bind to hydroxyapatite (bone mineralization). Carboxylation of glutamate residue to γ-carboxy glutamate residue is mediated by vitamin K dependent enzyme. - Osteocalcin synthesis is induced by calcitriol (active form of vitamin D3). - Its serum level is used as a marker of bone formation. 2- Osteonectin: It is a glycoprotein, plays a role in bone miniralization as it binds collagen and has a high affinity to Ca2+ and hydroxyapatite. 3- Different types of proteoglycans: They are important in bone development and bone mineralization. 35  Nucleotides and nucleic acid Nucleotides are the building units of the major nucleic acids of the cell; RNA and DNA. Nucleotides are formed of: nitrogenous base, pentose sugar and phosphate.  The Nitrogenous Bases: These may be pyrimidine or purine bases. Purine Pyrimidine Pyrimidine bases include:  Uracil (present in RNA)  Thymine (present in DNA)  Cytosine (present in RNA and DNA) Purine bases include:  Adenine (present in RNA and DNA)  Guanine (present in RNA and DNA) NUCLEOSIDES Nucleosides are formed from nitrogenous bases and a pentose sugar (sugar + base).. The anomeric carbon D-ribose or 2'-deoxyribose is linked through a glycosidic bond to the N9 of a purine or N1 of a pyrimidine 36  NUCLEOTIDES -Nucleotides are formed when phosphate is esterified to a nucleoside (sugar + base + phosphate).The most common site of phosphorylation of nucleotides found in cells is the hydroxyl group attached to the 5'-carbon of the ribose. The carbon atoms of the ribose present in nucleotides are designated with a prime (') mark to distinguish them from the backbone numbering in the bases. Nucleotides can exist in the mono-, di-, or tri-phosphorylated forms. Apart from their role as building units of nucleic acids, they also function as: Energy stores in the cells (ATP).,Coenzymes (NAD+, NADP+, FAD)., Mediators of hormone action (cAMP, a cyclic derivative of AMP formed from ATP)., Regulation of enzymatic reactions through allosteric effects on enzyme activity., Serving as activated intermediates in numerous biosynthetic reactions (S-adenosylmethionine (SAM) involved in methyl transfer reactions). Synthetic Nucleotide Analogues Many nucleotide analogues are chemically synthesized and used for their therapeutic potential. The nucleotide analogues can be utilized to inhibit specific enzymatic activities. A large family of analogues are used as anti-tumor agents, for instance, because they interfere with the synthesis of DNA and thereby preferentially kill rapidly dividing cells such as tumor cells.Allopurinol (purine analogs) is used to treat gout. Allopurinol competitively inhibits the activity of xanthine oxidase, an enzyme involved in de novo purine biosynthesis. Polynucleotides Polynucleotides are formed by the condensation of two or more nucleotides. The condensation most commonly occurs between the alcohol of a 5'-phosphate of one nucleotide and the 3'-hydroxyl of a second, with the elimination of H2O, forming a phosphodiester bond. 37   Formation of polynucleotide strand: The formation of phosphodiester bonds in DNA and RNA exhibits directionality. The primary structure of DNA and RNA (the linear arrangement of the nucleotides) proceeds in the 5' ----> 3' direction. The common representation of the primary structure of DNA or RNA molecules is to write the nucleotide sequences from left to right synonymous with the 5' ----> 3' direction 38  NUCLEIC ACIDS There are 2 types of nucleic acids:  DNA (deoxy ribonucleic acid).  RNAs (ribonucleic acid). DEOXYRIBONUCLEIC ACID or DNA. - DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. Nearly every cell in a person’s body has the same DNA. Most DNA is located in the cell nucleus (nuclear DNA), but a small amount of DNA can also be found in the mitochondria (mitochondrial DNA). DNA is formed of two polynucleotide chains twisted around each other in a right- handed double helix. Human DNA consists of about 3 billion bases, and the sequence of these bases determines the information available for building and maintaining an organism. An important property of DNA is that it can replicate, or make copies of itself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. DNA Structure In analogy to protein structure, double stranded DNA (dsDNA) has a linear sequence (primary structure), secondary structure (right handed double helix), and tertiary structure (it is folded and packed in the cell). It is the sequence of nucleotides along the chain. 4 nucleotides are the "building blocks" of the polynucleotide chain of DNA (d-AMP, d-GMP, d-CMP & d- TMP).The 3' C of one nucleotide is linked to 5' C of next nucleotide by a phosphodiester bond. Polarity of sequence: each polynucleotide chain thus has two ends: a 5' P end and 3' (OH) end. The order of bases within each nucleotide is written in 5'→ 3' direction. Backbone: the sugar phosphate units (- P-S-P-S-P-) form the backbone of each strand, while nitrogenous bases (A,G,C,T) projecting inwards.The bases of the individual nucleotides are stacked on top of each other like the steps of a spiral staircase. DNA secondary structure: Two DNA strands form a helical spiral, winding around a helix axis in a right-handed spiral.The two strands run in opposite directions (antiparallel) i.e. one strand runs in 5'→ 3' direction and the other in 3'→ 5' direction, with hydrogen bonds between them..The sugar-phosphate backbones of the two DNA strands wind around the helix axis like the railing of a spiral staircase. 39 Base Pairs: Within the DNA double helix, A forms 2 hydrogen bonds with T on the opposite strand, and G forms 3 hyrdogen bonds with C on the opposite strand. The sequence of bases on one strand of DNA is complementary to that of the second strand, and the content of dAMP equals that of dTMP (A=T), while the content of dGMP equals that of dCMP (G=C). The rules of base pairing tell us that if we can "read" the sequence of nucleotides on one strand of DNA, we can immediately deduce the complementary sequence on the other strand. The 2 strands are called: Template strand: the strand which is copied during RNA synthesis. Coding strand: the opposite DNA strand. This strand has a base sequence which is identical to the base sequence of the formed mRNA (except that T is present instead of U). DNA tertiary structure: DNA is usually linear, but is sometimes circular as in cases of mitochondrial, bacterial or viral DNA. DNA may exist in a relaxed or supercoiled form. Supercoiling of DNA is important for its packing within the nucleus and for easier strand separation for replication and transcription. Supercoiling means a "coiled coil" of DNA, a level of coiling above the winding of the 2 helices around each other. Nucleosomes Nucleosomes form the fundamental repeating units of eukaryotic chromatin, which is used to pack the large eukaryotic genomes into the nucleus. Nucleosomes consist of a segment of DNA wound around a histone protein core.The nucleosome core particle consists of approximately 147 bp of DNA wrapped in left-handed superhelical turns around a histone octamer (8 histone proteins which are proteins rich in basic amino acids) Core particles are connected by stretches of "linker DNA", which can be up to about 80 bp long. Nucleosomes are folded through a series of successively higher order structures to eventually form a chromosome. Chromosomes Eukaryotic DNA is packaged into highly condensed structures called chromosomes. Chromosomes consist of one dsDNA molecule. Each somatic cell of your body has 23 pairs of chromosomes, one member of each pair contributed by your mother and the other by your father. (In germ cells - eggs and sperm - there are 23 individual chromosomes, not chromosome pairs.) One pair are the sex chromosomes, which can come in two forms, X and Y. A pair of X's gives a female, and an XY results in a male. 40 RIBONUCLEIC ACID (RNA) Ribonucleic acids are polynucleotides formed of mainly four nucleotides: AMP, GMP, CMP and UMP; these nucleotides are interconnected by phosphodiester bonds. RNA is made of a single polynucleotide strand. There are three main types of RNA: 1-Messenger RNA (mRNA)  mRNA.contains the information needed to build a protein. mRNA travels from the nucleus of a cell to ribosomes, the place where protein synthesis occurs, and is read by the ribosomes. The result is a protein. Hence the name , messenger RNA. The information that mRNA carries is written in genetic code - a sequence of bases. A sequence of three adjacent nucleotides that specifies one of twenty amino acids is called a codon. 2-Transfer RNA (tRNA) tRNA is a small RNA molecule (usually about 74-95 nucleotides) that transfers a specific active amino acids to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. The structure of tRNA is quite complex, but to simplify it, it looks like a 3-leafed clover, as it has 3 loops (the leafs) and one arm (stem). It has a 3' terminal site for amino acid attachment (the acceptor arm which has a characteristic CCA terminus). It also contains a three base region called the anticodon that can base pair to the corresponding three base codon region on mRNA. Each type of tRNA molecule can be attached to only one type of amino acid, but because the genetic code contains multiple codons that specify the same amino acid, tRNA molecules bearing different anticodons may also carry the same amino acid. The other two loops are the D arm that contains dihydrouridine and the TΨC loop where Ψ is a pseudouridine. 41 3-Ribosomal RNA (rRNA) rRNA, contributes significantly to the structure of the ribosomes in a cell. The remainder of the ribosomes is comprised of proteins made in the cytoplasm. Ribosomes have specific attachment sites that allow tRNA molecules and mRNA to be in the proper close contact that they require to synthesize proteins. 42  Replication, transcription & translation The human genome (full sets of genes) consists of 46 (23pairs) of chromosomes, half of which are inherited from the father and half from the mother. Genes: The gene is a DNA molecule formed of a sequence of deoxyribonucleotides. It is an element of heredity that is transmitted from parents to offspring. Human genes are discontinuous, i.e. formed of coding sequences (exons) and non-coding sequences (introns). Chromosomes: Genes are arranged in linear order, i.e. in the form of long strands of DNA wound around in tight coils, these DNA strands are called chromosomes. In human, there are normally 46 chromosomes (23 pairs) in each cell. Genome: It is the total genetic information carried on chromosomes in any cell. Gene Expression: The information for the synthesis of proteins is stored in the genes. For a protein to be synthesized the flow of genetic information goes as follows: from DNA to DNA (replication), DNA to RNA (transcription) then from RNA to protein (translation). REPLICATION Genetic information stored in DNA in the form of nucleotide sequence flows from DNA to DNA (replication), DNA to RNA (transcription) then from RNA to protein (translation). This genetic information flow is called as central dogma of molecular genetics. The major function of replication is to provide genetic information required by daughter cell from parent cell. When cell prepares for division, all the cell components must double. During S-phase of cell cycle, the concentration of deoxyribonucleotides increases to several folds and replication occurs. When cell divides, each daughter cell must contain the entire genetic information of the parent cell. Each parental strand of the DNA molecule acts as a template for the formation of a complementary new DNA strand. Therefore, each of the daughter DNA molecules is composed of one original (conserved) strand and one newly synthesized strand. This is called semi-conservative replication. 43 Enzymes of Eukaryotic Replication 1. DNA Polymerases DNA polymerases catalyze the formation of polynucleotide chains through the addition of successive nucleotides derived from deoxynucleoside triphosphates by removal of pyrophosphate. The polymerase reaction takes place only in the presence of an appropriate DNA template. Each incoming nucleoside triphosphate first forms an appropriate base pair with a base in this template. Thus, DNA polymerases are template-directed enzymes. They are only able to read the parental nucleotide sequence in the 3` to 5` direction. DNA polymerases add nucleotides to the 3`-end of a polynucleotide chain (synthesize the new strand in 5` to 3` direcrion). To initiate this reaction, DNA polymerases require a RNA primer with a free 3`- hydroxyl group already base-paired to the template. They cannot start from scratch by adding nucleotides to a free single stranded DNA template. DNA Ligas It joins ends of two segments of DNA by catalyzing the formation of phosphodiester bond. In prokaryotes, NAD+ is required whereas in eukaryotes ATP is required. DNA Helicas It catalyzes unwinding of DNA double helix. The separation of DNA strands requires energy which is supplied by hydrolyzing ATP. DNA Topoisomerases When DNA unwinds, supercoils are formed during DNA replication. They are removed by DNA topoisomerases. Topoisomerases I breaks a phosphodiester bond in one DNA strand (produces a cut or nick), allowing DNA to rotate freely around the other strand, then it reforms the phosphodiester bond. Topoisomerases II acts by making transient break in both DNA strands and reseals the break. DNA gyrase is a special type of topoisomerase II found in E-Coli, has unusual ability to introduce negative supercoils to relaxed circular DNA using energy from hydrolysis of ATP and is also required for separation of the interlocked molecules of circular DNA following chromosomal replication. 44 General steps for eukaryotic DNA replication: Identification of the origin of replication by dna proteins. Unwinding of double stranded DNA to form single stranded DNA. Each strand will act as a template directing the synthesis of a daughter strand. This is done by DNA helicases. Single stranded DNA-binding proteins (SSBP) keep the two strands separated.Formation of multiple sites at which replication occurs (replication forks).Synthesis of RNA primers.Chain elongation: DNA polymerase elongates the primers according to the base pairing rule (A with T and C with G).Elongation occurs from the 5' to the 3' direction. DNA is a self correcting enzyme; it can remove an incorrect base and replace it.Excision of RNA primers and their replacement by DNA. Connection of the chain fragments by DNA ligase. Telomerase Eukaryotic cells face a special problem in replicating the 3`ends of their linear DNA molecules. Following removal of the RNA primer during copping of the end of lagging strand, there is no way to fill in the remaining gap with DNA. To solve this problem, and to protect the ends of the chromosomes from attack by nucleases, noncoding sequences of DNA complexed with proteins are found at these ends called telomeres. Telomere end consists of a repetitive sequence of T's and G's. In humans, telomeres consist of many (650 up to 2500) copies of 5`-TTAGGG-3` repeats. The TG strand is longer than its complement, leaving a region of single-stranded DNA at the 3'-end of the double helix that is a few hundred nucleotides long. The single-stranded region folds back on itself forming a structure that is stabilized by protein. This complex protects the ends of the chromosomes. Cells undergoing the aging process, the ends of their chromosomes get slightly shorter with each cell division until the telomeres are gone, and DNA essential for cell function is degraded, a phenomenon related to cellular aging and death. Cells that do not age (for example, germ-line cells and cancer cells) contain an enzyme called telomerase that is responsible for replacing these lost ends. Telomerase is a special kind of reverse transcriptase that carries its own RNA molecule of about 150 nucleotides long. (it uses its RNA strand as a template for synthesis of a complementary DNA strand). In that RNA are copies of the A/C sequence that is complimentary to the T/G repeat sequence. The RNA base-pairs with the terminal nucleotides at the single-stranded 3'-end of the DNA strand. Telomerase recognizes the single-strand 3` terminus and uses its RNA molecule as a template to elongate the parental strand by about 100 nucleotides (the process may be repeated). The elongated end is used as a template, for completing synthesis of the telomere of the lagging strand by DNA polymerase, RNA primer is removed and ends are joined by DNA ligase. 45 DNA Repair The replication process takes place at high accuracy but an error can occur for every 30,000 bases. Such errors in replication produce damaged DNA or DNA with altered base composition. Further, damage to DNA may result from the action of physical, chemical and environmental agents. The main mechanisms for DNA repair include mismatch repair, base excision repair and nucleotide excision repair. I- Mismatch Repair Mismatch repair corrects errors made when DNA is copied. For example, a C could be inserted opposite an A, T may replace C or the polymerase could insert two or more extra unpaired bases. Specific proteins scan the newly synthesized DNA, using adenine methylation as the point of reference. The template strand is methylated, and the newly synthesized strand is not. This difference allows the repair enzymes to identify the strand that contains the wrong nucleotide which requires replacement. An endonuclease produces a cut in the nonmethylated chain and an exonuclease removes the region containing the mutation, thus removing the faulty DNA. This defect is then filled in by DNA polymerase and then nicks are joined by DNA ligase. II- Base Excision-Repair (BER): Cytosine, adenine, and guanine bases in DNA are deaminated spontaneously to form uracil, hypoxanthine, or xanthine, respectively. DNA-glycosylases can recognize these abnormal bases and remove the base itself from the DNA. This removal marks the site of the defect (AP-site) and allows an apurinic or apyrimidinic endonuclease (AP-endonuclease) to excise the basic sugar and produce a cut in the DNA backbone. A deoxyribose-phosphate lyase removes the empty sugar-phosphate residue. Then a repair DNA polymerase, and a ligase returns the DNA to its original state. This mechanism is suitable for replacement of a single base but is not effective at replacing regions of damaged DNA. N.B. Depurination of DNA, which happens spontaneously owing to the thermal liability of the purine N-glycosidic bond, occurs at a rate of 5,000–10,000/cell/d at 37°C. Specific enzymes recognize a depurinated site and replace the appropriate purine directly. III- Nucleotide Excision Repair (NER) This mechanism is employed to correct larger defects in DNA (up to 30 bases in length). It is more complicated than BER. After defect recognition, unwinding of DNA 46 occurs, followed by excision of an area of DNA upstream and down- stream of the defective region. Then, the gap is filled by DNA polymerase and religated by a ligase. Hereditary DNA repair disorders Defects in the repair mechanism are responsible for several genetic disorders, including: Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging and death.Werner's syndrome: premature aging and retarded growth.Ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents. TRANSCRIPTION (RNA Synthesis) - RNA synthesis is catalyzed by RNA polymerase enzymes, which catalyze RNA synthesis from DNA molecule. Only one of the two DNA strands serves as a template for RNA synthesis and called template strand, the other strand is called the coding strand which have the same nucleotide sequence as RNA transcript with the exception of T base instead of U base. Types of RNA polymerases 1) Polymerase I : transcribes most of ribosomal RNA genes. 2) Polymerase II: transcribes mainly mRNA genes. 3) Polymerase III: transcribes mainly tRNA genes. General steps for mRNA synthesis: Initiation: It involves binding of RNA polymerase (RNAP) II to a specific region on the DNA known as the promoter region. In eukaryotic cells specific protein factors are needed to recognize the promoter. Elongation: After recognition of the promoter region, the RNA polymerase starts to synthesize a complementary transcript of the template DNA by using ribonucleoside triphosphates (ATP, GTP, UTP and CTP). Termination: The process of elongation continues until a terminal signal is reached, which is recognized leading to inactivation of RNAP and cleavage of RNA. The resulting RNA is called primary mRNA transcript. 47 Synthesis of mRNA I- Initiation The promoter region Two types of sequence elements are present in promoter (basal expression element). 1- TATA box: it defines where transcription is to commence along the DNA. This region has the sequence of TATAAA. The TATA box is usually located 20-30 bp upstream from the transcription start site in mammalian genes that contain it. The human TATA box is bound by TATA-binding protein (TBP) or TFIID. Binding of the TFIID to the TATA box sequence is thought to represent a first step in the formation of the transcription complex on the promoter. 2- CAAT box and GC box regions: these are present upstream (-40-200 bp) of the transcription start site and contribute to the mechanisms that control how frequently this event is to occur. N.B. The role of regulated expression elements is discussed later on (regulation of gene expression) Regulated expression elements Basal expression elements Distal Promoter regulatory proximal Promoter elements Coding region Hormone-response Enhancers elements CAAT & GC- TATA and And box Box rich region Tissue-specific +1 Elements Silencers III- Termination Termination signals exist far downstream of the coding sequence of eukaryotic genes. Transcription proceeds till the termination consensus sequence AAUAAA is reached. After RNA polymerase II has traversed the region of the transcription, RNA endonucleases cleave the primary mRNA transcript. Polymerase II-TFIIF complex is dissociated and is dephosphorylated by a phosphatase. A new cycle of transcription may start again. 48 Post-Transcriptional Processing of mRNA: - Primary mRNA transcript undergoes several modifications after transcription. - Processing of mRNA includes: Capping: At 5-end of the primary transcript a methylated guanine cap is added. Polyadenylation: At the 3-end of the primary transcript 10-30 nucleotides are removed and about 100-200 (A) residues are added.RNA splicing: Cutting out of introns (non-coding sequence) and joining the ends of neighbouring exons (coding sequence) to produce functional mRNA molecule. Synthesis of rRNAs The complete ribosome is formed of many fractions. The 45S genes for 18S, 5.8S and 28S rRNA are typically clustered together and tandemly repeated (one copy each of 18S, 5.8S and 28S occur, followed by untranscribed spacer DNA, then another set occurs and so on). RNA polymerase I transcribes these rRNA genes to produce a single 45 S precursor (pre-rRNA) molecule then undergoes RNA-processing in the nucleus which cleaves the precursor to release the mature 18S, 5.8S and 28S rRNA. 5S RNA gene is transcribed by RNA polymerase III. DNA RNA polymerase I 18 S 5.8 S 28 S 45 Pre-rRNA Cleavage by Ribonucleases (RNases) 18 S 5.8 S 28 S Ribosomal RNAs Synthesis of tRNAs The tRNA molecules in all organisms are base-paired internally to give a clover leaf appearance (explained before). Eukaryotic tRNA genes are all transcribed by RNA polymerase III. The primary transcript (pre-tRNA molecules) require up to four 49 different types of RNA processing steps as follows: Removal of the extra 5`sequence (leader sequence). Replacement of UU nucleotides with the CCA sequence at the 3`end. Modification of some bases e.g. methylation. Removal of short intron, which is present in the anticodon loop. TRANSLATION (PROTEIN SYNTHESIS) The genetic information within the DNA is transcribed into mRNA in the nucleus; the latter is transported to the cytoplasm where it is translated into protein. Translation is the RNA directed synthesis of polypeptides. This process requires: The mRNA which is the template for the correct addition of individual amino acids. The synthesis of protein is directed by the mRNA template. The tRNAs carry activated amino acids (in the form of amino-acyl tRNA) into the ribosome.The ribosomes (composed of rRNAs and ribosomal proteins) are associated with the mRNA and containing the necessary enzymatic activities (peptidyl transferases) to catalyze peptide bond formation ensuring correct access of activated aminoacyl-tRNA complexes. Amino acids: activated and carried on tRNA. A source of energy (ATP and GTP). Different protein factors required for the process to be completed. THE GENETIC CODE - The sequence of nucleotides in DNA or RNA that determines the specific aminoacid sequence in the synthesis of proteins. It is the biochemical basis of heredity and nearly universal in all organisms.The correspondence between the sequence of bases in the mRNA nucleotides and the sequence of amino acids in the synthesized protein is called the genetic code. - Each three successive bases in the mature mRNA form a codon, which specifies an amino acid.In a codon each position of the 3 may be occupied by one of 4 bases (U,C,A,G), there are 43= 64 possible codons. - Three of them are non-coding and are used to terminate translation. The remaining 61 codons code for 20 amino acids. Therefore one amino acid can be specified by more than one codon (synonym codons). 50 Characteristics of genetic code: Specificity: It is triplet code. Each amino acid is coded by sequence of three nucleotides, known as codon (as indicated in the table). One codon function as initiation codon for protein synthesis i.e. AUG. Some codons do not code for any amino acid and they cause termination of polypeptide chain formation. They are called as nonsense codons or termination codons i.e. UAA, UAG or UGA. A given codon codes only one amino acid. But an amino acid may be indicated or coded by more than one codon known as synonym codons. Existence of more than one code word for an amino acid is known as degeneracy of genetic code. For example, arginine is coded by six synonym codons. The genetic code is mainly universal. The genetic codon is universal with the exception of few examples e.g. mitochondrial RNA reads four codons differently from the cytoplasmic RNA. Reading frame: Usually one reading frame will produce a functional protein (it is determined by the initiation codon). The correct reading frame is set in vivo by recognition of the initiation codon (AUG) by the ribosomes at the start of the coding sequence.For a given codon on mRNA, an anti-codon is present on tRNA. Codon and anti-codon always read from 5' → 3' direction. Further, codon and anticodon are anti-parallel and complementary in base composition. They interact with each other through base pairing. Synthesis of The Polypeptide Chain In Eukaryotes The process of translation is classified into three phases; initiation, elongation and termination. A- Initiation The process of initiation can be divided into four steps; ribosomal dissociation, formation of 43S preinitiation complex, formation of 48S initiation complex and formation of 80S initiation complex. 51 1- Ribosomal Dissociation: Two initiation factors (IF-1 and 3) bind to the 40S subunit of the 80S ribosome and produce its dissociation to 40S and 60S ribosomal subunits. 2- Formation of the 43S Preinitiation Complex: IF-2 combined with GTP bind with methionine-tRNA (Met-tRNA) to form a complex, which binds to the 40S ribosomal subunit (complexed with IF-1 and 3) to form the 43S preinitiation complex. 3- Formation of 48S Initiation Complex IF-4 (CAP binding protein) facilitates binding of mRNA to the 43S preinitiation complex to form 48S initiation complex. Then the complex scans the mRNA for the initiation codon (AUG) which is specific for Met-tRNA. This process is followed by hydrolysis of ATP and release of IF4. 4- Formation of the 80S Initiation Complex The binding of the 60S ribosomal subunit to the 48S initiation complex to form the 80S initiation complex involves hydrolysis of GTP. This step requires IF-5 and results in the release of the IF-1, 2 and 3. At this point, met-tRNA is on the P-site (peptidyl site) of the ribosome ready for elongation and the A-site is free ready to accept the next aminoacyl-tRNA. B- Elongation: Binding of an aminoacyl-tRNA: After the initiation complex is formed, addition of each amino acid to the growing polypeptide chain involves: binding of an aminoacyl-tRNA to the A site on the ribosome, formation of a peptide bond, and translocation of the peptidyl-tRNA to the P site. The peptidyl-tRNA contains the growing polypeptide chain. When Met- tRNAi (or a peptidyl-tRNA) is bound to the P site, the mRNA codon in the A site determines which aminoacyl-tRNA will bind to that site. The incoming aminoacyl-tRNA first combines with elongation factor EF1 bound GTP (hydrolyzed to GDP and Pi) before binding to A site of the mRNA-ribosome complex. This allows protein synthesis to continue. The ribosome moves along the mRNA in the 5' to 3' direction, translating the successive codons. Chain elongation occurs by sequential addition of amino acids to the C-terminal end of the ribosome bound polypeptide. Formation of peptide bond: In the first round of elongation, the amino acid on the tRNA in the A site forms a peptide bond with the methionine on the tRNA in the P site. In subsequent rounds of elongation, the amino acid on the tRNA in the A site forms a peptide bond with the peptide on the tRNA in the P site. The 60S ribosomal subunit contains peptidyl transferase which catalyzes the formation of peptide bond between the amino group of the new aminoacyl-tRNA in the A-site with the carboxylic group of the peptidyl-tRNA occupying the P-site. This results in attachment of the growing peptide to the tRNA in the A-site. Translocation involves elongation factor EF2 and GTP (hydrolyzed to GDP and Pi). After removal of the peptide chain from tRNA in the P-site, this 52 tRNA is quickly transferred to the E-site (as the ribosome moves) to be discharged out of the ribosome. Now the A-site is free to accept a new aminoacyl-tRNA. The process of peptide synthesis continues until a termination codon is reached. Eukaryotic ribosome can form 6 peptide bonds per second. 53  C- Termination The elongation steps are repeated until a termination (stop) codon moves into the A site on the ribosome. Since no tRNAs with anticodon that can pair with the stop codon, a release factor binds to the ribosome instead, causing peptidyl transferase to hydrolyze the bond between the peptide chain and tRNA. The newly synthesized polypeptide is released from the ribosome, which dissociates into its individual subunits, releasing the mRNA. This step requires energy (conversion of GTP to GDP and Pi) Polyribosomes (Polysomes) Many ribosoms can work on the same mRNA molecule and these aggregates are called polyribosomes or polysomes and are attached to the wall of the endoplasmic reticulum to form rough ER. In such cases each ribosome is about 80-100 nucleotide apart on mRNA. Chromatin Structure: - The physical structure of the DNA, as it exists compacted into chromatin, can affect the ability of transcriptional regulatory proteins (termed transcription factors)RNA polymerases to find access to specific genes and to activate transcription from them. The presence of the histones and methylation of cytosine bases in CG-rich regions (CG islands), most affect accessibility of the chromatin to RNA polymerases and transcription factors. Transcriptional Initiation: - This is the most important mode for control of eukaryotic gene expression. Specific factors that exert control include the strength of promoter elements within the DNA sequences of a given gene, the presence or absence of enhancer sequences (which enhance the activity of RNA polymerase at a given promoter by binding specific transcription factors). A promoter is a region of DNA that initiates transcription of a particular gene. Promoters are located near the transcription start sites of genes, on the same strand and upstream on the DNA (towards the 5'region of the sense strand). Promoters can be about 100–1000 base pairs long - Enhancers are special cis-acting elements on DNA that facilitate initiation of transcription; they act by binding certain proteins that facilitate the binding of transcription factors to the promotor. However, they can be close to or thousands of base pairs away from promoter and they can occur on either strand of DNA. - Silencers are special cis-acting elements in DNA that inhibit initiation of transcription. They act by binding certain proteins that produce inhibition of transcription. 54 Transcript Processing and Modification: - Eukaryotic mRNAs must be capped and polyadenylated, and the introns must be accurately removed. Several genes have been identified that undergo tissue-specific patterns of alternative splicing, which generate biologically different proteins from the same gene. RNA Transport: A fully processed mRNA must leave the nucleus in order to be translated into protein. Transcript Stability: Unlike prokaryotic mRNAs, whose half-lives are all in the range of 1--5 minutes, eukaryotic mRNAs can vary greatly in their stability. Posttranscriptional Processes Affecting mRNA may include the following: o RNA splicing: Tissue-specific protein products (protein isoforms) can be made from the same pre-mRNA through differential processing, particularly the use of alternate splice sites. For example, tropomyosin (TM) is an essential protein of the contractile apparatus in the different types of muscle, and its pre-mRNA undergoes differential splicing to yield seven tissue-specific TM isoforms. o Messenger RNA editing: Coding information of mRNA can be changed by RNA editing. An example in the liver, the single apolipoprotein gene (apoB gene) is transcribed into a mRNA that direct synthesis of apoB-100 protein (100-kDa). In the intestine, the same gene direct synthesis of a primary transcript, then by the action of a cytidine deaminase that coverts a CAA codon in mRNA to UAA at single specific site, which is a termination signal, and a apo B-48 protein (48-kDa) is the result of translating this edited form of mRNA. Both proteins have different functions. o Messenger RNA stability: The 5`-cap and th