Lecture 12: Nucleosides, Nucleotides, DNA and RNA PDF

BioC 3021 Notes Robert Roon Lecture 12: Nucleosides, Nucleotides, DNA and RNA Slide 1. Nucleosides, Nucleotides, DNA and RNA In this lecture, we will first study the structure of nucleosides and nucleotides. Then, we will examine the structure of the rel...

BioC 3021 Notes Robert Roon Lecture 12: Nucleosides, Nucleotides, DNA and RNA Slide 1. Nucleosides, Nucleotides, DNA and RNA In this lecture, we will first study the structure of nucleosides and nucleotides. Then, we will examine the structure of the related biopolymers, DNA and RNA. Slide 2. Functions of Nucleic Acids Nucleic acids carry out a number of cellular functions. -They store genetic information (usually as DNA, but some viruses use RNA.) -They transcribe and translate genetic information (Messenger RNA, transfer RNA, and ribosomal RNA are involved in information transfer.) -They carry out enzymatic reactions. (For example, portions of ribosomal RNA catalyze protein synthesis.) -They serve in the storage and transfer of energy. (eg. ATP, GTP) -They serve as signaling molecules: (eg. cAMP, cGMP) -They act as redox coenzymes. (eg. NADH, NADPH, FADH2, FMN) Slide 3. Nucleic Acid Components. Nucleic acids are the fourth major class of biopolymer that we consider. For other biopolymers such as proteins and carbohydrates, each monomer unit consists of a single component—amino acids for proteins and monosaccharides for carbohydrates. In contrast, the monomer units that make up nucleic acids consist of three different components—the nitrogenous bases, sugar residues, and phosphates. The nucleic acids received their name because they are highly concentrated in the nuclei of eukaryotic cells, and because they 1 BioC 3021 Notes Robert Roon contain phosphate residues that can act as acids. At physiological pH (about pH 7), the phosphate residues are mostly in the deprotonated or Bronsted base form, giving them a negative charge. Thus, nucleic acids such as DNA and RNA are anionic compounds. Slide 4. Nitrogenous Bases Here, we see the purine and pyrimidine ring structures of the nitrogenous bases. These bases consist of five different heterocyclic nitrogen compounds that can conveniently be divided into two classes—the pyrimidines and the purines. The pyrimidines have one six-membered heterocyclic ring system. The purines have a six-membered ring fused to a five-membered ring. To distinguish these two classes, it helps to remember that the larger name goes with the smaller compound with only one ring— pyrimidines are smaller. The smaller name then corresponds to the larger compound with two rings—that would be the purines. Mayo Clinic 6/30/11 5:13 PM Comment: The way that I remember these is Cut The “Py” (Cytosine and Thymine are Pyrimidines. There are three pyrimidines—cytosine, which occurs in both DNA Pies are one circle, and Pyrimidines are one ring). Pur As Gold (Adenine and Guanine are Purines). and RNA, thymine, which is generally found in DNA, and uracil, which is normally a component of RNA. The rule that localizes thymine to DNA and uracil to RNA is broken only very very rarely. The two purines, adenine and guanine, are found in both DNA and RNA. Slide 5. Five Nitrogenous Bases. We can now examine the exact structure of the five nitrogenous bases. Note that one hydrogen atom on each base is shown in red. Those hydrogen atoms are lost when the bases combine with ribose or deoxyribose to form nucleosides. The red hydrogen atoms thus designate the site where covalent attachment occurs between base and sugar. That attachment site is from the six-membered ring for pyrimidines and from the five-membered ring for purines. The substituents on the nitrogenous rings are vitally important for 2 BioC 3021 Notes Robert Roon the function of nucleic acids. There are only two types of substituents—amino nitrogen groups and carbonyl oxygen groups. The location of these substituents is critical because they are involved in the hydrogen bonds that occur when nucleotides in DNA and RNA hybridize to each other. The purine, adenine, has an amino group at the number six position on the six-membered ring. The other purine, guanine, has an amino group at the two position and a carbonyl oxygen at the number six position. The pyrimidine, cytosine, has an amino group at the four position and a carbonyl oxygen at the number two position. Uracil, which occurs primarily in RNA, has carbonyl groups at positions four and two. The DNA component, Thymine, also has carbonyl groups at positions four and two, and a methyl group at position 5. Slide 6. Sugar Components of Nucleic Acids. Next, we will review the sugar structures. On the right, we have β- D-ribofuranose—the structure that occurs in RNA. On the left is 2-Deoxy- β -D-ribofuranose, which is a component of DNA. The two compounds differ only at position 2 on the furanose ring. That carbon atom carries a hydroxyl group in D-ribofuranose, and a hydrogen atom in 2-Deoxy- β -D-ribofuranose. The “R” in RNA comes from ribose, and the “D” in DNA comes from deoxyribose. Slide 7. Nucleoside Structure. When a heterocyclic base combines with a sugar, the elements of water are split out and a nucleoside is produced. Note that nucleosides contain only a base and a sugar. (When one or more phosphates are added to a nucleoside, we refer to the resulting compound as a nucleotide.) The covalent bond between the base and the sugar is referred to as a β -N-glycosidic linkage. The term “β” comes from the β orientation of the bond from the anomeric 3 BioC 3021 Notes Robert Roon carbon of the sugar. The “N” refers to the fact that this is a bond from an anomeric carbon to a nitrogen atom of the base. In the example used here, the base, adenine, has combined with β -D- ribofuranose to give the nucleoside, adenosine. Slide 8. Numbering in Nucleosides. In this diagram, all the atoms of the base and sugar of adenosine are numbered using the conventions adopted years ago by biochemists. If we focus on the ribofuranose ring, we see that the five carbon atoms are numbered 1-5 starting with the anomeric carbon and proceeding clockwise. Those numbers are followed by a prime (‘) designator. These “prime” numbers become important when the various phosphate derivatives of nucleosides are considered. For example, in the backbone of DNA, we will encounter 3’-5’ phosphodiester linkages. Slide 9. Naming Nucleosides. There are five bases and two sugars that can commonly occur in nucleosides. That gives ten possible combinations of one base and one sugar. The ribose containing nucleosides are referred to as adenosine, guanosine, cytidine, uridine, and thymidine. Thymidine, (a combination of thymine with ribose that is not generally found in RNA), is also sometimes designated ribothymidine. The deoxyribose containing nucleosides are called deoxyadenosine, deoxyguanosine, deoxycytidine, deoxyuridine, and deoxythymidine. Because the combination of thymine and deoxyribose is common and the combination of thymine and ribose is uncommon, the name deoxythymidine is sometimes shortened to thymidine (to confuse the unwary). Just be aware that the term thymidine is used in two different ways. Notice that purine containing nucleosides are referred to using the suffix –osine and the pyrimidine containing nucleosides are designated using the suffix –idine. 4 BioC 3021 Notes Robert Roon Slide 10. Nucleotide Structure. The term nucleotide refers to a compound containing a base, a sugar, and at least one phosphate group. The phosphate can be esterified to any hydroxyl group of the sugar residue. For ribonucleotides, that would include the 2’, 3’and 5’ hydroxyl groups of the ribose. With deoxyribonucleotides, the phosphates can be esterified to the 3’ and 5’ hydroxyl groups of the deoxyribose. The compound on the lower part of the slide is adenosine 5’- monophosphate (abbreviated as 5’-AMP). The designation 5’- monophosphate indicates that a single phosphate group is esterified to the 5’hydroxyl group of the ribose. If the ribose were replaced with a deoxyribose residue, we would have deoxyadenosine 5’- monophosphate (5’-dAMP). Slide 11. Structure of 3’-dGMP. There are numerous possible combinations of base, sugar, and phosphate. The compound shown includes guanine linked to a deoxyribose residue that has a single phosphate esterified to the 3’ hydroxyl group. It is called 2’-deoxyguanosine-3’-monophosphate (3’-dGMP). Such a phosphoester linkage to a 3’ hydroxyl group is found in the backbone of DNA. Slide 12. Restricted Rotation about N-Glycosidic Bond. There is restricted rotation about the N-Glycosidic Bond. The nitrogenous bases and the sugar residues can have two orientations with respect to each other. The anti orientation, in which the base and sugar are rotated away from each other, seems to be favored in most circumstances. The syn orientation, in which the two components are lined up on the same side, can occur, but that orientation is much less common. 5 BioC 3021 Notes Robert Roon Slide 13. The Structure of Adenosine 5’-triphosphate (ATP). Often, nucleotides contain more than one phosphate residue. The compound shown is Adenosine 5’-triphosphate (5’-ATP, which is often further abbreviated as ATP). The ATP molecule has three phosphate residues linked to each other by two phosphoanhydride bonds. One of these phosphates is in turn linked to a ribofuranose residue by phosphoester bonds. The phosphoanhydride bonds are synthesized when two phosphate groups are chemically joined in a reaction with the elimination of water. The anhydride bonds of ATP are used as reservoirs of chemical energy in biological systems. Hydrolysis of these bonds releases a high amount of energy. In the example shown, the hydrolysis of ATP to ADP and inorganic phosphate (Pi) releases 7.3 kcal/mol (In recent years, it has become the convention to measure energy in joules rather than calories. Standard conversion factors allow us to calculate that 7.3 kcal/mol is equal to 30.5 kJ/mol). The energy released by ATP hydrolysis can be coupled to energetically unfavorable biological reactions to promote product formation. The ability to couple favorable reactions with unfavorable reactions is one of the key elements that enable biological systems to survive and flourish. Both of the phosphoanhydride bonds of ATP release a high amount of energy upon hydrolysis, and both of these bonds can be utilized to promote unfavorable reactions. In contrast, the phosphoester bond that links the phosphates to the ribofuranose residue releases much less energy upon hydrolysis. That phosphoester bond is not generally used to promote unfavorable reactions. Slide 14. The ATP to ADP Cycle. The breakdown of ATP to ADP and Pi is only one half of a two phase process. There are a number of key biological reactions that are involved in ATP production (Energy Input). We will soon be discussing the glycolysis pathway and the TCA cycle. These 6 BioC 3021 Notes Robert Roon catabolic pathways provide the major mechanisms of ATP production in many living organisms. In these pathways, energy is released from the breakdown of organic compounds, such as glucose, pyruvate, and acetylCoA. That energy is coupled to the synthesis of ATP. The ATP hydrolysis reaction is then coupled to a variety of biological processes (Energy Output). ATP is used to promote the synthesis of biopolymers, to drive muscle contraction, and to energize the transport of ions and metabolites across biological membranes. Slide 15. UV Absorption Spectra of Nucleotides. The base components of nucleotides have conjugated double bond systems, which gives them the ability to absorb ultra violet light. They all absorb light in the range of 240 to 300 nm, but each base has a different spectra. Some of these spectra are sensitive to changes in pH. Protein molecules also absorb UV light in the range of 260-280 nm, but to a lesser degree. (The molar absortivity is lower for protein.) So, it is sometimes difficult to measure protein concentration in the presence of nucleic acid. Slide 16. Measuring UV Absorption. Many analytical procedures in biochemistry involve the measurement of the light absorbing characteristics of biological molecules. The absorbance of light by a compound in solution (A) is equal to the product of its molar extinction coefficient (ε) ), which is a characteristic unique to each different compound, times the length of the light path (b), times the concentration (C). That relationship is known euphemistically as Lambert-Beer Law (A = εbC). (Not to be confused with other laws which prohibit alcohol consumption by minors.) The analysis of light absorbance is performed using a spectrophotometer. Spectrophotometers have: 7 BioC 3021 Notes Robert Roon - a light source and a light filter that allows the machine to emit light at a specific wavelength (or a range of wavelengths). - a sample chamber containing a silica cuvette that holds a solution of the compound of interest - a detector that can measure the amount of light passing through the sample. Spectrophotometers are used in a number of ways in biochemical experiments. In the simplest experiments, the absorbance of a known compound can be used to determine the concentration of the compound in solution. Sometimes, chemical reactions can be assayed by measuring changes in absorbance (at a fixed wavelength) that correlates with the conversion of substrate to product. In more complex experiments, scanning spectrophotometers are used to monitor a range of wavelengths simultaneously in order to assay spectral changes that occur in electron-carrying proteins as they go from the reduced to the oxidized state. Slide 17. Phosphodiester Linkage in DNA and RNA In this slide, we see a short segment of DNA, that is, deoxyadenylyl-(3’-5’)-deoxyguanosine. The two nucleotide segments of this molecule are joined to one another by two phosphoester bonds—one bond to the 3-carbon of the upper deoxyribose ring, and the other bond to the 5-carbon of the lower deoxyribose ring. Collectively, these two phosphate linkages are referred to as a 3’-5’-phosphodiester bond. The upper portion of the deoxyadenylyl-(3’-5’)-deoxyguanosine molecule consists of a deoxyadenylyl residue that has a free hydroxyl group on the number 5 carbon of the deoxyribose ring. If this were the terminal residue of a long DNA chain, that deoxyadenylyl residue would be referred to as the 5’ hydroxyl end of the molecule. Below the deoxyadenylyl residue lies a 8 BioC 3021 Notes Robert Roon deoxyguanosine residue that has a free hydroxyl group on the number 3 carbon of the deoxyribose ring. In a DNA chain, this terminal residue would be called the 3’ hydroxyl end. There are three abbreviations that can be used for deoxyadenylyl- (3’-5’)-deoxyguanosine—those would be dApdG or (d)ApG or (d)AG. As we proceed from left to right, the abbreviations leave out a bit more detail. However, they all refer to the same structure. Note that the convention is to always start the name at the 5’-end of the molecule. This convention is preserved for DNA and RNA and any other polynucleotide. Slide 18. Tetranucleotide Structure (DNA). This tetranucleotide sequence contains the four constituent bases of DNA—adenine, cytosine, guanine and thymine. The nucleotide units all contain deoxyribose, and they are all linked by 3’-5’- phosphodiester bonds. The way this structure is written implies that it is a segment of DNA that continues on in each direction. The phosphate residue at the upper left would lead on toward the 5’ end of the molecule, whereas the oxygen atom at the lower right would lead on to the 3’ end. Slide 19. Tetranucleotide Structure (DNA) with 3’ and 5’ Ends Just so there is no ambiguity, the 3’ and 5’ ends of the tetranucleotide are circled in this figure. Slide 20. Tetranucleotide Structure abbreviated (DNA). This slide shows two versions of the same sequence in abbreviated form. The vertical lines represent deoxyribose residues, with the numbers indicating the position of the five component carbon atoms. The diagonal lines with a central P represent the phosphodiester bonds that connect the 3’ and 5’ positions of the deoxyribose residues. The orientation of the DNA segment remains the same, 5’end to the left—3’end to the right. The upper 9 BioC 3021 Notes Robert Roon structure has free hydroxyl groups at the 5’ and 3’ends. The lower structure is identical except that the hydroxyl groups on each end are now phosphorylated. Slide 21. Tetranucleotide Structure (RNA). Here, we have a tetranucleotide sequence of RNA. The RNA sequence is very similar to that of DNA, but it does have two significant changes. There is a difference in base composition. The RNA sequence contains three of the same bases found in DNA—adenine, cytosine, and guanine. However, the fourth base is now uracil. A second difference is that the nucleotide units all contain ribose. The hydroxyl groups at the 2’ position are highlighted to show that this is ribose. The sugar residues continue to be linked by 3’-5’-phosphodiester bonds. Again, the implication is that the segment of RNA continues on in each direction with the phosphate residue at the upper left leading on toward the 5’ end of the molecule, and the oxygen atom at the lower right leading on to the 3’ end. Slide 22. Tetranucleotide Structure abbreviated (RNA). This slide shows two versions of the RNA sequence in abbreviated form. The vertical lines now represent ribose residues (note the 2’ hydroxyl groups). The numbers indicate the position of the five component carbon atoms of the ribose. The diagonal lines with a central P represent the phosphodiester bonds that connect the 3’ and 5’ positions of the ribose residues. The orientation of the RNA segments is again the same, 5’end to the left—3’end to the right. The upper structure has free hydroxyl groups at the 5’ and 3’ends, and in the lower structure, those hydroxyl groups are phosphorylated. Slide 23. Electron Micrograph of DNA. In this electron micrograph, we can see a segment of DNA containing thousands of base pairs. The preparation was spread out on a grid and then shadowed with an electron dense material 10 BioC 3021 Notes Robert Roon that sticks to DNA. Notice that in the region of the arrow, the two DNA strands have separated to form a loop. This may be a region where replication of the DNA was occurring. If you look closely, you can see that there are other places where the strands have separated. When we study DNA replication, we will observe that the process is semi-conservative with one strand going to each of the two progeny DNA molecules. Slide 24. Electron Micrograph of Circular DNA. The graphic shows two electron micrographs of circular mitochondrial DNA. On the left is a relaxed sample of mitochondrial DNA that has the double helical form, but is not very tightly twisted. On the right is a supercoiled sample in which extra twisting has forced the DNA into a very kinky morphology. This type of supercoiling occurs extensively during the process of DNA replication, and it also occurs to some extent during RNA transcription. If you want to see something similar to supercoiling, try twisting a rubber band between two pencils. At first the coiling is relatively smooth, but with more tightening the rubber band gets really squirrelly. Check it out. Slide 25. Double Helix Characteristics of DNA Most DNA exists as a double helix. The general form of a DNA double helix has the following characteristics. -The polynucleotide strands are antiparallel. One strand runs in the 5'->3' direction, and the other strand runs in the 3'->5' direction. -The bases in opposing strands are complementary. Adenine (A) is almost always paired with Thymine (T), and guanine (G) is paired with cytosine (C). -There are 10 base pairs per turn of helix. -The typology of the DNA helix exhibits major and minor grooves. Slide 26. Three Representations of the DNA Double Helix. 11 BioC 3021 Notes Robert Roon Here are three different models illustrating the DNA double helix. On the left is a ribbon model, which emphasizes the coiling of the two strands and shows the non-covalent base pairing that occurs between the strands. In the middle is a space filling model, which lets you see that the DNA molecule is really very densely packed with very little space in the interior of the helix. Other models suggest that there is open space between the atoms in DNA, but this realistic depiction of the actual space occupied by the atoms puts that illusion to rest. Finally, on the right is a wire frame model, which is particularly effective at showing the alternating deoxyribose and phosphate residues that serve as the covalent backbone for DNA. This model also gives us an excellent look at the base pairing. We can see that the bases radiate to the inside of the helix, that they are planar and perpendicular to the axis of helix, and also that the non-covalent interaction between these bases involves two (AT) or three (GC) hydrogen bonds per base pair. Slide 27. Features of the DNA Double Helix. The following list summarizes the standard features of the DNA double helix: -The two strands of the DNA double helix run in an antiparallel fashion, one 5’ to 3’ and the other 3’ to 5’. -The negatively charged phosphate residues are on the outside of the helix. -The bases, which radiate toward the interior of the opposing strands, are complementary: That is, A is paired with T (or T with A) and G is paired with C (or C with G). There are two hydrogen bonds between the AT pair and three hydrogen bonds between the GC pair. -In addition to hydrogen bonding, the helix is stabilized by aromatic stacking of successive bases. These bases lie parallel to each other in each strand of the helix. -It takes 10 base pairs to make one complete turn of the double helix. 12 BioC 3021 Notes Robert Roon -A close look at the model of the double helix reveals that there is a wide major groove and a narrow minor grove that spiral around the helix. Slide 28. Feature of Base Pairing In DNA and RNA -Base pairing refers to the interactions between two nucleotides on opposite strands of complementary DNA or RNA. -The two bases that are connected to each other by hydrogen bonds are called a base pair (often abbreviated bp). -In DNA base pairing, adenine (A) forms a base pair with thymine (T), and guanine (G) forms a base pair with cytosine (C). -In RNA base pairing, thymine is replaced by uracil (U)—adenine (A) forms a base pair with uracil (U), and guanine (G) forms a base pair with cytosine (C). -Hydrogen bonding underlies base-pairing. Appropriate geometrical correspondence of hydrogen bond donors and acceptors allows only the "right" pairs to form stable interactions. Slide 29. More Features of Base Pairing -The larger purine bases, adenine and guanine, are doubly-ringed structures. The smaller pyrimidine bases, cytosine and thymine (and uracil), are singly-ringed structures. -Purines are only complementary with pyrimidines. -Pyrimidine-pyrimidine pairings are energetically unfavorable because the molecules are too far apart for hydrogen bonding to be established. -Purine-purine pairings are energetically unfavorable because the molecules are too close, leading to overlap repulsion. -The only other possible pairings are GT and AC; these pairings are mismatches because the pattern of hydrogen donors and acceptors do not correspond. Slide 30. Three Base Pairs of DNA. The ball and stick model shows three base pairs of DNA. You can see in this diagram that the individual bases are planar, but the base 13 BioC 3021 Notes Robert Roon pairs are slightly tilted with respect to each other. Slide 31. Structure of the AT base pair. Base pairing occurs between complementary bases in DNA (and does not occur between non-complementary bases). The complementation between those base pairs represents the most important example of hydrogen bonding in living systems. Base pairing is at the heart of DNA replication, of transcription of DNA to RNA, and of translation of RNA to protein. The diagram of an AT base pair shows that the complementation between these two bases involves the formation of two hydrogen bonds. One hydrogen bond occurs between the hydrogen atom of the 6-amino group of adenine and a carbonyl oxygen atom of thymine. A second hydrogen bond occurs between one of the ring nitrogen atoms of adenine and a hydrogen atom attached to a ring nitrogen atom of thymine. For the AT base pair, the distance between the 1’ C of the deoxyribose of one chain to the 1’ C of the deoxyribose of the other chain is about 1.11 nm. Slide 32. Structure of the GC base pair. We have just seen that the pairing between A and T occurs at a distance of 1.11 nm. The distance for the GC base pair is essentially the same (1.08 nm). Both the AT and the GC interactions involve a purine interacting with a pyrimidine. If a purine bonded to a purine, the bond distance would be greater because purines are larger than pyrimidines. If two pyrimidines were bonding, the distance would be correspondingly smaller. Because base pairing always involves a purine bonding to a pyrimidine, the strands of the DNA double helix remain at a constant distance from one another. There are three hydrogen bonds in the GC pair: -A carbonyl oxygen of guanine bonds to a cytosine amino group 14 BioC 3021 Notes Robert Roon -A hydrogen attached to a nitrogen atom on the guanine ring bonds to a nitrogen atom of the cytosine ring -An amino group of guanine bonds to a carbonyl oxygen of cytosine Collectively, the energy of these three GC hydrogen bonds is greater than the total energy of the two AT bonds. Traditionally, this was thought to be due to the greater number of bonds in the GC pair. However, recent data suggest that other factors may play a role in the collective affinity of these base pairs. In any case, it would take more energy to separate the two strands of DNA from a polymer with GC bonds than it would to separate the strands from a similar AT polymer. Slide 33. Watson and Crick. The photograph shows Drs. Watson and Crick sometime in the mid 1950’s, soon after they published their seminal paper outlining the features of the DNA double helix. That publication marks the beginning of modern molecular biology. Slide 34. E. coli DNA Spew. This is a famous electron micrograph (your classic spew or hurl) of an E. coli cell that was ruptured to release its DNA and then treated with a chemical agent to shadow the released DNA. What is amazing is the length of the DNA molecule in comparison to the size of the cell. The total length of that circular DNA molecule is crammed into a small region of the cell. Not only that, but the DNA is replicated and transcribed while remaining in a very compact form. That makes the high speed with which the E. coli DNA is replicated even more amazing. Slide 35. Meselson Stahl Experimental Details. When Watson and Crick proposed their model for the DNA double helix, they also suggested a mechanism for DNA replication. That mechanism involved the separation of the two parental strands, 15 BioC 3021 Notes Robert Roon each of which was then copied to create two new double strands. The two progeny DNA molecules would each have one parental and one new strand of DNA. That mechanism is referred to as semiconservative replication. Drs Meselson and Stahl set out to test this hypothesis. They grew E. coli for many generations in a media that was enriched in two “heavy” isotopes 2H and 15N. These isotopes were not radioactive, but they would make the product DNA extra dense or heavy. This added density of the heavy isotopes would increase the rate at which DNA would sediment in CsCl density gradient centrifugation. Normal DNA would sediment more slowly than the isotopically labeled material, and a mixed DNA sample containing one chain of normal DNA and one chain of heavy DNA would sediment at an intermediate rate. In the experiment, E. coli cells were initially grown for many generations on the heavy isotopes. The result was that the DNA was highly enriched with the heavy isotopes. They then shifted the cells to a normal growth media and took DNA samples after 1, 2, 3 and 4 generations of growth. The samples were then subjected to CsCl density gradient centrifugation, and the positions of the DNA bands were noted. Slide 36. Meselson Stahl Experimental Results. This photograph shows the experimental results of the Meselson Stahl Experiment. The banding patterns from the CsCl density gradient centrifugation change with time. At the initiation of the experiment, at time zero, only one heavy band is present. After one generation, there is a somewhat lighter band at an intermediate density. After two generations, there is about 50% intermediate and 50% light density material. After four generations, most of the DNA has shifted to the light density. These results are entirely consistent with the semiconservative mechanism of DNA replication. 16 BioC 3021 Notes Robert Roon Slide 37. Pattern of Semiconservative Replication. Here, we have an artist’s rendition of the expected (and observed) pattern of semiconservative replication. Initially, there is one DNA molecule containing two parental strands. After one generation, there are two progeny DNA molecules. Both of these molecules have one parental strand and one new strand. After two generations, there are four DNA molecules—two of the molecules have one parental strand and one new strand. The other two molecules have two new strands. If further generations were generated, there would always be two DNA molecules with one parental strand and one new strand. All the rest of the DNA molecules would contain only new strands. 17