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DNA polymerase III A prokaryotic enzyme with 5’ to 3’ Polymerase activity. The Hydrolysis of pyrophosphate (PPi) drives the energy required to create the covalent phosphodiester bond.A prokaryotic enzyme with 5’ to 3’ Polymerase activity.DNA polymerase is a multisubunit enzyme where the subunits of the enzyme form a ring that encircles and moves along the template strand of the DNA, thus serving as a sliding DNA clamp. Therefore the enzyme cannot diffuse away from its task.DNA polymerase III has proofreading capabilities and therefore the error rate of RNA transcription is 1 in 10 million bases (1X107).To ensure DNA replication fidelity, DNA polymerase III can edit its mistakes. DNA polymerase III has 3’ to 5’ Exonuclease activity.The 5’ to 3’ polymerase activity then replaces it with the correct nucleotide.
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RNA Primer Excision and Replacement by DNA polymerase I DNA polymerase III continues to synthesize DNA on the lagging strand until it is blocked by an RNA primer. DNA polymerase I excises the RNA primer in a 5’ to 3’ Exonuclease direction, and then fills the gap with a 5’ to 3’ Polymerase direction. It also proofreads the new chain using a 3’ to 5’ Exonuclease activity for DNA.3’-exonuclease is always for proofreading functions.DNA Ligase The Okazaki fragments are joined by DNA ligase, which forms the phosphodiester bond in the sugar phosphate backbone between the two nucleotides.The creation of the phosphodiester bond requires 2 high energy bonds.DNA polymerase DNA polymerases read the parental nucleotide sequences in a 3’ to 5’ direction, and they synthesize the new DNA strand only in a 5’ to 3’ direction. Antiparallel synthesis 15 Mg2+ DNA polymerase is magnesium (Mg2+) dependent as the divalent magnesium ion neutralizes the 2 anionic charges on the incoming nucleotide.The two newly synthesized stretches of DNA must grow in opposite directions, one in the 5’ to 3’ direction towards the replication fork, and one in the 5’ to 3’ direction away from the replication fork.The RNA Primer. DNA polymerase cannot initiate synthesis of a complementary strand of DNA on a totally single stranded template. DNA polymerase requires an RNA primer, which is a short piece of RNA base-paired to the DNA template. The free hydroxyl group on the 3’-end of the RNA primer serves as the first acceptor of the first deoxynucleotide. The RNA Primer , Primase This is a specific RNA polymerase that synthesizes the short stretches of RNA (~10 bases long) that are complementary and antiparallel to the DNA template. Synthesized in a 5’ to 3’ direction. Prokaryotic and eukaryotic DNA polymerases elongate a new DNA strand by adding deoxynucleotides, one at a time, to the 3’-end of the growing chain .It Reads 3’ to 5’ and synthesizes 5’ to 3’ direction SB2. Obtain, evaluate, and communicate information to analyze how genetic information is expressed in cells. a. Construct an explanation of how the structures of DNA and RNA lead to the expression of information within the cell via the processes of replication, transcription, and translation. Learning Targets _______Identify the structural components of DNA and RNA Success Criteria _______Can accurately identify the key structural components of DNA (deoxyribose sugar, phosphate group, nitrogenous bases: adenine, thymine, cytosine, guanine). _______Can identify the key structural components of RNA (ribose sugar, phosphate group, nitrogenous bases: adenine, uracil, cytosine, guanine). _______Can describe the differences in the sugar backbone of DNA and RNA (deoxyribose vs. ribose). _______Can identify the double-stranded structure of DNA and the single-stranded structure of RNA. _______Identify the parts of protein synthesis and the location of each process Success Criteria _______Can identify and describe the two main processes of protein synthesis: transcription and translation. _______Can correctly explain that transcription occurs in the nucleus where DNA is transcribed into mRNA. _______Can explain that translation occurs in the cytoplasm at ribosomes where mRNA is translated into amino acid sequences to form proteins. _______Compare and Contrast DNA to RNA Success Criteria _______Can clearly identify similarities between DNA and RNA, such as both being nucleic acids and containing nucleotide structures. _______Can explain differences in DNA and RNA, including sugar types (deoxyribose vs. ribose), strand number (double-stranded DNA vs. single-stranded RNA), and nitrogenous base usage (thymine in DNA vs. uracil in RNA). _______Can describe the function of DNA as genetic storage and the function of RNA in protein synthesis (mRNA, tRNA, rRNA). _______Analyze the reasoning for enzymes usage in both DNA replication and protein synthesis. Success Criteria _______Can identify key enzymes involved in DNA replication (e.g., helicase, DNA polymerase, ligase) and explain their functions (e.g., helicase-unwinding DNA, DNA polymerase-synthesizing new DNA strands, ligase-sealing nicks in the DNA backbone). _______Can identify enzymes involved in protein synthesis (e.g., RNA polymerase) and explain their role in transcribing DNA into mRNA. _______Can analyze why enzymes are essential for speeding up chemical reactions…(ensuring accuracy, and catalyzing steps in replication and protein synthesis) _______Can provide specific examples of how enzyme malfunction can impact genetic replication or protein synthesis. _______Perform the steps of DNA replication and protein synthesis in order to demonstrate their understanding of how the structure of DNA supports the genetic expression in successive generations. Success Criteria _______Can demonstrate a step-by-step understanding of DNA replication, including unwinding, complementary base pairing, and proofreading. _______Can demonstrate the steps of transcription (formation of mRNA from DNA) and translation (conversion of mRNA into a polypeptide). _______Can show how DNA's structure (double helix, base pairing) ensures accurate replication for passing genetic information to offspring. _______Can illustrate how changes in DNA sequence can lead to changes in protein structure and function, thus affecting traits in successive generations.The Promoter Region is a region of DNA upstream where transcription is initiated for control of gene transcription Promoters are solely important for gene expression Pribnow Box in Prokaryotes. CAAT and Hogness box (TATA) in Eukaryotes They control the binding of RNA polymerases and Transcription factors (if Eukaryotic) Now RNA polymerase can transcribe DNA to RNA to further achieve the goal to mRNA to eventual protein formation Prokaryotic promoter are recognized by RNA polymerase and sigma factor (Sigma Subunit) Eukaryotic promoter is recognized and bounded by General Transcription Factors (GTF’s) and RNA Polymerase IIOrigin recognition ORC,MCM proteins helicase activity, ssDNA RPA, Primer synthesis polymers alpha/primase,Sliding clamp PCNA, primer removal RNase H, FEN1.Pol α/Primase synthesizes an RNA chain of 10 nucleotides followed by another 20 nucleotides of initiator DNA (iDNA). Because Pol α/Primase has no proofreading ability, this whole section of RNA/iDNA is removed. The exonucleases RNase H removes the RNA and Fen1 removed the iDNA. The gap is filled in by Pol δ and DNA ligase joins the 2 ends.Eukaryotic DNA Replication All have 5’ to 3’ Polymerase activity. What is a Plant Cell? Plant cells are eukaryotic cells that vary in several fundamental factors from other eukaryotic organisms. Both plant and animal cells contain a nucleus along with similar organelles. One of the distinctive aspects of a plant cell is the presence of a cell wall outside the cell membrane. Plant Cell Structure Just like different organs within the body, plant cell structure includes various components known as cell organelles that perform different functions to sustain itself. These organelles include: Cell Wall It is a rigid layer which is composed of polysaccharides cellulose, pectin and hemicellulose. It is located outside the cell membrane. It also comprises glycoproteins and polymers such as lignin, cutin, or suberin. The primary function of the cell wall is to protect and provide structural support to the cell. The plant cell wall is also involved in protecting the cell against mechanical stress and providing form and structure to the cell. It also filters the molecules passing in and out of it. The formation of the cell wall is guided by microtubules. It consists of three layers, namely, primary, secondary and the middle lamella. The primary cell wall is formed by cellulose laid down by enzymes. Cell membrane It is the semi-permeable membrane that is present within the cell wall. It is composed of a thin layer of protein and fat. The cell membrane plays an important role in regulating the entry and exit of specific substances within the cell. For instance, cell membrane keeps toxins from entering inside, while nutrients and essential minerals are transported across. Nucleus The nucleus is a membrane-bound structure that is present only in eukaryotic cells. The vital function of a nucleus is to store DNA or hereditary information required for cell division, metabolism and growth. 1. Nucleolus: It manufactures cells’ protein-producing structures and ribosomes. 2. Nucleopore: Nuclear membrane is perforated with holes called nucleopore that allow proteins and nucleic acids to pass through. Plastids They are membrane-bound organelles that have their own DNA. They are necessary to store starch and to carry out the process of photosynthesis. It is also used in the synthesis of many molecules, which form the building blocks of the cell. Some of the vital types of plastids and their functions are stated below: Leucoplasts They are found in the non-photosynthetic tissue of plants. They are used for the storage of protein, lipid and starch. Chromoplasts They are heterogeneous, colored plastid which is responsible for pigment synthesis and for storage in photosynthetic eukaryotic organisms. Chromoplasts have red-, orange- and yellow-colored pigments which provide color to all ripe fruits and flowers. Central Vacuole It occupies around 30% of the cell’s volume in a mature plant cell. Tonoplast is a membrane that surrounds the central vacuole. The vital function of the central vacuole apart from storage is to sustain turgor pressure against the cell wall. The central vacuole consists of cell sap. It is a mixture of salts, enzymes and other substances. Golgi Apparatus They are found in all eukaryotic cells, which are involved in distributing synthesized macromolecules to various parts of the cell. Ribosomes They are the smallest membrane-bound organelles which comprise RNA and protein. They are the sites for protein synthesis, hence, also referred to as the protein factories of the cell. Mitochondria They are the double-membraned organelles found in the cytoplasm of all eukaryotic cells. They provide energy by breaking down carbohydrate and sugar molecules, hence they are also referred to as the “Powerhouse of the cell.” Lysosome Lysosomes are called suicidal bags as they hold digestive enzymes in an enclosed membrane. They perform the function of cellular waste disposal by digesting worn-out organelles, food particles and foreign bodies in the cell. In plants, the role of lysosomes is undertaken by the vacuoles. Chloroplasts It is an elongated organelle enclosed by phospholipid membrane. The chloroplast is shaped like a disc and the stroma is the fluid within the chloroplast that comprises a circular DNA. Each chloroplast contains a green colored pigment called chlorophyll required for the process of photosynthesis. The chlorophyll absorbs light energy from the sun and uses it to transform carbon dioxide and water into glucose. Structure of Chloroplast Chloroplasts are found in all higher plants. It is oval or biconvex, found within the mesophyll of the plant cell. The size of the chloroplast usually varies between 4-6 µm in diameter and 1-3 µm in thickness. They are double-membrane organelle with the presence of outer, inner and intermembrane space. There are two distinct regions present inside a chloroplast known as the grana and stroma. • Grana are made up of stacks of disc-shaped structures known as thylakoids or lamellae. The granum of the chloroplast consists of chlorophyll pigments and are the functional units of chloroplasts. • Stroma is the homogenous matrix which contains grana and is similar to the cytoplasm in cells in which all the organelles are embedded. Stroma also contains various enzymes, DNA, ribosomes, and other substances. Stroma lamellae function by connecting the stacks of thylakoid sacs or grana. The chloroplast structure consists of the following parts: Membrane Envelope It comprises inner and outer lipid bilayer membranes. The inner membrane separates the stroma from the intermembrane space. Intermembrane Space The space between inner and outer membranes. Thylakoid System (Lamellae) The system is suspended in the stroma. It is a collection of membranous sacs called thylakoids or lamellae. The green colored pigments called chlorophyll are found in the thylakoid membranes. It is the sight for the process of light-dependent reactions of the photosynthesis process. The thylakoids are arranged in stacks known as grana and each granum contains around 10-20 thylakoids. Stroma It is a colorless, alkaline, aqueous, protein-rich fluid present within the inner membrane of the chloroplast present surrounding the grana. Grana Stack of lamellae in plastids is known as grana. These are the sites of conversion of light energy into chemical energy. Chlorophyll It is a green photosynthetic pigment that helps in the process of photosynthesis. Functions of Chloroplast Following are the important chloroplast functions: • The most important function of the chloroplast is to synthesize food by the process of photosynthesis. • Absorbs light energy and converts it into chemical energy. • Chloroplast has a structure called chlorophyll which functions by trapping the solar energy and is used for the synthesis of food in all green plants. • Produces NADPH and molecular oxygen (O 2 ) by photolysis of water. • Produces ATP – Adenosine triphosphate by the process of photosynthesis. • The carbon dioxide (CO2) obtained from the air is used to generate carbon and sugar during the Calvin Cycle or dark reaction of photosynthesis. Mitochondria “Mitochondria are membrane-bound organelles present in the cytoplasm of all eukaryotic cells, that produce adenosine triphosphate (ATP), the main energy molecule used by the cell.” What are Mitochondria? Popularly known as the “Powerhouse of the cell,” mitochondria (singular: mitochondrion) are a double membrane-bound organelle found in most eukaryotic organisms. They are found inside the cytoplasm and essentially function as the cell’s “digestive system.” They play a major role in breaking down nutrients and generating energy-rich molecules for the cell. Many of the biochemical reactions involved in cellular respiration take place within the mitochondria. The term ‘mitochondrion’ is derived from the Greek words “mitos” and “chondrion” which means “thread” and “granules-like”, respectively. It was first described by a German pathologist named Richard Altmann in the year 1890. Structure of Mitochondria • The mitochondrion is a double-membraned, rod-shaped structure found in both plant and animal cell. • Its size ranges from 0.5 to 1.0 micrometers in diameter. • The structure comprises an outer membrane, an inner membrane, and a gel-like material called the matrix. • The outer membrane and the inner membrane are made of proteins and phospholipid layers separated by the intermembrane space. • The outer membrane covers the surface of the mitochondrion and has a large number of special proteins known as porins. Cristae The inner membrane of mitochondria is rather complex in structure. It has many folds that form a layered structure called cristae, and this helps in increasing the surface area inside the organelle. The cristae and the proteins of the inner membrane aid in the production of ATP molecules. The inner mitochondrial membrane is strictly permeable only to oxygen and ATP molecules. A number of chemical reactions take place within the inner membrane of mitochondria. Mitochondrial Matrix The mitochondrial matrix is a viscous fluid that contains a mixture of enzymes and proteins. It also comprises ribosomes, inorganic ions, mitochondrial DNA, nucleotide cofactors, and organic molecules. The enzymes present in the matrix play an important role in the synthesis of ATP molecules. Functions of Mitochondria The most important function of mitochondria is to produce energy through the process of oxidative phosphorylation. It is also involved in the following process: 1. Regulates the metabolic activity of the cell 2. Promotes the growth of new cells and cell multiplication 3. Helps in detoxifying ammonia in the liver cells 4. Plays an important role in apoptosis or programmed cell death 5. Responsible for building certain parts of the blood and various hormones like testosterone and estrogen 6. Helps in maintaining an adequate concentration of calcium ions within the compartments of the cell 7. It is also involved in various cellular activities like cellular differentiation, cell signaling, cell senescence, controlling the cell cycle and in cell growth. Disorders Associated with Mitochondria Any irregularity in the way mitochondria function can directly affect human health, but often, it is difficult to identify because symptoms differ from person to person. Disorders of the mitochondria can be quite severe; in some cases, they can even cause an organ to fail.Lipids are large, nonpolar organic molecules. They do not dissolve in water. Lipids include triglycerides (trie-GLIS-uhr-IEDZ), phospho- lipids, steroids, waxes, and pigments. Lipid molecules have a higher ratio of carbon and hydrogen atoms to oxygen atoms than carbohydrates have. Because lipid molecules have larger numbers of carbon-hydrogen bonds per gram than other organic com- pounds do, they store more energy per gram. Fatty Acids Fatty acids are unbranched carbon chains that make up most lipids. Figure 3-10 shows that a fatty acid contains a long carbon chain (from 12 to 28 carbons) with a carboxyl group, —COOH, attached at one end. The two ends of the fatty-acid molecule have different properties. The carboxyl end is polar and is thus hydrophilic or attracted to water molecules. In contrast, the hydro- carbon end of the fatty-acid molecule is nonpolar. This end tends not to interact with water molecules and is said to be hydrophobic (HIE-droh-FOH-bik), or “water fearing.” In saturated fatty acids, such as palmitic acid, which is shown in Figure 3-10, each carbon atom is covalently bonded to four atoms. The carbon atoms are in effect full, or saturated. In contrast, linoleic acid, also shown in Figure 3-10, has carbon atoms that are not bonded to the maximum number of atoms to which they can bond. Instead, they have formed double bonds within the carbon chain. This type of fatty acid is said to be unsaturated. Triglycerides Three classes of lipids important to living things contain fatty acids: triglycerides (fats), phospholipids, and waxes. A triglyceride is composed of three molecules of fatty acid joined to one molecule of the alcohol glycerol. Saturated triglycerides are composed of saturated fatty acids. They typically have high melting points and tend to be hard at room temperature. Common dietary saturated triglycerides include butter and fats in red meat. In contrast, unsaturated triglycerides are composed of unsaturated fatty acids and are usually soft or liquid at room temperature. Unsaturated triglycerides are found primarily in plant seeds where they serve as an energy and carbon source for germinating plants. Phospholipids Phospholipids have two, rather than three, fatty acids attached to a molecule of glycerol. They have a phosphate group attached to the third carbon of the glycerol. As shown in Figure 3-11, the cell membrane is made of two layers of phospholipids, called the lipid bilayer. The inability of lipids to dissolve in water allows the mem- brane to form a barrier between the inside and outside of the cell. Hydrophilic “head” Phospholipids Hydrophobic “tail” Phospholipids Water Water The lipid bilayer of a cell membrane is a double row of phospholipids.The “tails” face each other.The “head” of a phospholipid, which contains a phosphate group, is polar and hydrophilic.The two tails are two fatty acids and are nonpolar and hydrophobic. FIGURE 3-11 H C H C O OH H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H H C H C H H H H C H H C H H C H H C H H C H C O OH H C H H C H H C H C H C H C H H C H H C H C H H C H H C H C H H H H C H Fatty acids have a polar carboxyl head, highlighted in purple, and a nonpolar hydrocarbon tail, highlighted in green. FIGURE 3-10 Palmitic acid Linoleic acid mb06se_bchs02.qxd 5/18/07 10:49 AM Page 59 60 CHAPTER 3 1. Compare the structure of monosaccharides, dis- accharides, and polysaccharides. 2. How are proteins constructed from amino acids? 3. How do amino acids differ from one another? 4. Describe a model of enzyme action. 5. Why do phospholipids orient in a bilayer when in a watery environment, such as a cell? 6. Describe how the three major types of lipids differ in structure from one another. 7. What are the functions of the two types of nucleic acids? CRITICAL THINKING 8. Applying Information Before a long race, run- ners often “carbo load.” This means that they eat substantial quantities of carbohydrates. How might this help their performance? 9. Recognizing Relationships High temperatures can weaken bonds within a protein molecule. How might this explain the effects of using a hot curling iron or rollers in one’s hair? 10. Applying Information You want to eat more unsaturated than saturated fats. Name examples of foods you would eat more of and less of. SECTION 2 REVIEW Waxes A wax is a type of structural lipid consisting of a long fatty-acid chain joined to a long alcohol chain. Waxes are waterproof, and in plants, form a protective coating on the outer surfaces. Waxes also form protective layers in animals. For example, earwax helps pre- vent microorganisms from entering the ear canal. Steroids Unlike most other lipids, which are composed of fatty acids, steroid molecules are composed of four fused carbon rings with various functional groups attached to them. Many animal hor- mones, such as the male hormone testosterone, are steroid com- pounds. One of the most familiar steroids in humans is cholesterol. Cholesterol is needed by the body for nerve and other cells to func- tion normally. It is also a component of the cell membrane. NUCLEIC ACIDS Nucleic acids are very large and complex organic molecules that store and transfer important information in the cell. There are two major types of nucleic acids: deoxyribonucleic acid and ribonucleic acid. Deoxyribonucleic acid, or DNA, contains information that deter- mines the characteristics of an organism and directs its cell activi- ties. Ribonucleic (RIE-boh-noo-KLEE-ik) acid, or RNA, stores and transfers information from DNA that is essential for the manufactur- ing of proteins. Some RNA molecules can also act as enzymes. Both DNA and RNA are polymers, composed of thousands of linked monomers called nucleotides (NOO-klee-uh-TIEDS). As shown in Figure 3- 12, each nucleotide is made of three main components: a phosphate group, a five-carbon sugar, and a ring-shaped nitrogenous base.