PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker CHAPTER 9 MOLECULAR STRUCTURE OF DNA AND RNA Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9.1 IDENTIFICATION OF DNA AS THE GENETIC MATERIAL To fulfill its role, the genetic material must meet several criteria 1. Information: It must contain the information necessary to make an entire organism 2. Transmission: It must be passed from parent to offspring 3. Replication: It must be copied In order to be passed from parent to offspring 4. Variation: It must be capable of changes To account for the known phenotypic variation in each species 9-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9.1 IDENTIFICATION OF DNA AS THE GENETIC MATERIAL The data of many geneticists, including Mendel, were consistent with these four properties However, the chemical nature of the genetic material cannot be identified solely by genetic crosses 9-4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Frederick Griffith Experiments with Streptococcus pneumoniae Griffith studied a bacterium (Diplococcus pneumoniae) now known as Streptococcus pneumoniae S. pneumoniae comes in two strains S  Smooth Secrete a polysaccharide capsule Protects bacterium from the immune system of animals Produce smooth colonies on solid media R  Rough Unable to secrete a capsule Produce colonies with a rough appearance 9-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

In addition, the capsules of two smooth strains can differ significantly in their chemical composition Figure 9.1 Rare mutations can convert a smooth strain into a rough strain, and vice versa However, mutations do not change the type of the strain 9-6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

In 1928, Griffith conducted experiments using two strains of S In 1928, Griffith conducted experiments using two strains of S. pneumoniae: type IIIS and type IIR 1. Inject mouse with live type IIIS bacteria Mouse died Type IIIS bacteria recovered from the mouse’s blood 2. Inject mouse with live type IIR bacteria Mouse survived No living bacteria isolated from the mouse’s blood 3. Inject mouse with heat-killed type IIIS bacteria 4. Inject mouse with live type IIR + heat-killed type IIIS cells 9-7 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.2 9-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Griffith concluded that something from the dead type IIIS was transforming type IIR into type IIIS He called this process transformation The substance that allowed this to happen was termed the transformation principle Griffith did not know what it was The nature of the transforming principle was determined using experimental approaches that incorporated various biochemical techniques 9-9 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The Experiments of Avery, MacLeod and McCarty Avery, MacLeod and McCarty realized that Griffith’s observations could be used to identify the genetic material They carried out their experiments in the 1940s At that time, it was known that DNA, RNA, proteins and carbohydrates are major constituents of living cells They prepared cell extracts from type IIIS cells containing each of these macromolecules Only the extract that contained purified DNA was able to convert type IIR into type IIIS 9-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-11 Avery et al also conducted the following experiments Figure 9.3 To further verify that DNA, and not a contaminant (RNA or protein), is the genetic material Figure 9.3 9-11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Hershey and Chase Experiment with Bacteriophage T2 In 1952, Alfred Hershey and Marsha Chase provided further evidence that DNA is the genetic material Figure 9.4 Inside the capsid They studied the bacteriophage T2 It is relatively simple since its composed of only two macromolecules DNA and protein Made up of protein 9-12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.5 Life cycle of the T2 bacteriophage Figure 10-5 Reproductive cycle of a T-even bacteriophage, as known in 1952. The electron micrograph shows an E. coli cell during infection by numerous phages.

The Hershey and Chase experiment can be summarized as such: Used radioisotopes to distinguish DNA from proteins 32P labels DNA specifically 35S labels protein specifically Radioactively-labeled phages were used to infect nonradioactive Escherichia coli cells After allowing sufficient time for infection to proceed, the residual phage particles were sheared off the cells => Phage ghosts and E. coli cells were separated Radioactivity was monitored using a scintillation counter 9-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the Hypothesis Only the genetic material of the phage is injected into the bacterium Isotope labeling will reveal if it is DNA or protein Testing the Hypothesis Refer to Figure 9.6 9-15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the Hypothesis Figure 9.6 9-16

Figure 9.6 9-17

The Data 9-18 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Interpreting the Data 9-19 Most of the 35S was found in the supernatant But only a small percentage of 32P These results suggest that DNA is injected into the bacterial cytoplasm during infection This is the expected result if DNA is the genetic material 9-19

Figure 10-6 Summary of the Hershey–Chase experiment demonstrating that DNA, and not protein, is responsible for directing the reproduction of phage T2 during the infection of E. coli.

RNA Functions as the Genetic Material in Some Viruses In 1956, A. Gierer and G. Schramm isolated RNA from the tobacco mosaic virus (TMV), a plant virus Purified RNA caused the same lesions as intact TMV viruses Therefore, the viral genome is composed of RNA Since that time, many RNA viruses have been found Refer to Table 9.1 9-20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 10-8 Reconstitution of hybrid tobacco mosaic viruses Figure 10-8 Reconstitution of hybrid tobacco mosaic viruses. In the hybrid, RNA is derived from the wild-type TMV virus, while the protein subunits are derived from the HR strain. Following infection, viruses are produced with protein subunits characteristic of the wild-type TMV strain and not those of the HR strain. The top photograph shows TMV lesions on a tobacco leaf compared with an uninfected leaf. At the bottom is an electron micrograph of mature viruses.

9-21 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9.2 NUCLEIC ACID STRUCTURE DNA and RNA are large macromolecules with several levels of complexity 1. Nucleotides form the repeating units 2. Nucleotides are linked to form a strand 3. Two strands can interact to form a double helix 4. The double helix folds, bends and interacts with proteins resulting in 3-D structures in the form of chromosomes 9-22 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-23 Figure 9.7 Three-dimensional structure Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Nucleotides The nucleotide is the repeating structural unit of DNA and RNA It has three components A phosphate group A pentose sugar A nitrogenous base Refer to Figure 9.8 9-24 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.8 9-25 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-26 These atoms are found within individual nucleotides Figure 9.9 However, they are removed when nucleotides join together to make strands of DNA or RNA A, G, C or T A, G, C or U Figure 9.9 The structure of nucleotides found in (a) DNA and (b) RNA 9-26 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Base + sugar  nucleoside Example Adenine + ribose = Adenosine Adenine + deoxyribose = Deoxyadenosine Base + sugar + phosphate(s)  nucleotide Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) Adenosine triphosphate (ATP) Refer to Figure 9.10 9-27 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-28 Figure 9.10 Base always attached here Phosphates are attached there Figure 9.10 9-28 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Nucleotides are covalently linked together by phosphodiester bonds A phosphate connects the 5’ carbon of one nucleotide to the 3’ carbon of another Therefore the strand has directionality 5’ to 3’ The phosphates and sugar molecules form the backbone of the nucleic acid strand The bases project from the backbone 9-29 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

ribonucleic acid (RNA) Fig. 9.9 (TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. O O Base Base P O CH2 P O CH2 5¢ O 5¢ O O– O – 4¢ 1¢ 4¢ 1¢ H H H H H H H H 3¢ 2¢ Phosphate 3¢ 2¢ Phosphate O H O OH 2¢ Deoxyribose Ribose Repeating unit of deoxyribonucleic acid (DNA) Repeating unit of ribonucleic acid (RNA)

Figure 9.11 9-30 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 10-12 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 10-12 (a) Linkage of two nucleotides by the formation of a – phosphodiester bond, producing a dinucleotide. (b) Shorthand notation for a polynucleotide chain. Figure 10-12 Copyright © 2006 Pearson Prentice Hall, Inc.

A Few Key Events Led to the Discovery of the Structure of DNA In 1953, James Watson and Francis Crick discovered the double helical structure of DNA The scientific framework for their breakthrough was provided by other scientists including Linus Pauling Rosalind Franklin and Maurice Wilkins Erwin Chargaff 9-31 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Linus Pauling In the early 1950s, he proposed that regions of protein can fold into a secondary structure a-helix To elucidate this structure, he built ball-and-stick models Refer to Figure 9.12b Figure 9.12 9-32 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Fig. 9.13 Rosalind Franklin

Photo 51 x-ray diffraction of wet DNA showing the B form double helix taken by Rosalind Franklin and Raymond Gosling on Friday, 2nd May, 1952 by long exposure started the previous day

Rosalind Franklin She made marked advances in X-ray diffraction techniques with DNA The diffraction pattern she obtained suggested several structural features of DNA Helical More than one strand 10 base pairs per complete turn 9-34 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Erwin Chargaff’s Experiment Chargaff pioneered many of the biochemical techniques for the isolation, purification and measurement of nucleic acids from living cells It was already known then that DNA contained the four bases: A, G, C and T 9-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Testing the Hypothesis An analysis of the base composition of DNA in different species may reveal important features about the structure of DNA Testing the Hypothesis Refer to Figure 9.14 9-36 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.14 9-37

Figure 9.14 9-38

The Data 9-39 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Table 10-3 Copyright © 2006 Pearson Prentice Hall, Inc. Table 10.3 DNA Base Composition Data Table 10-3 Copyright © 2006 Pearson Prentice Hall, Inc.

Interpreting the Data The compelling observation was that Percent of adenine = percent of thymine Percent of cytosine = percent of guanine the percentage of C + G does not necessarily equal the percentage of A + T This observation became known as Chargaff’s rule It was crucial evidence that Watson and Crick used to elucidate the structure of DNA 9-40 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Watson and Crick Familiar with all of these key observations, Watson and Crick set out to solve the structure of DNA They tried to build ball-and-stick models that incorporated all known experimental observations A critical question was how the two (or more strands) would interact An early hypothesis proposed that the strands interact through phosphate-Mg++ crosslinks Refer to Figure 9.15 9-41 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

This hypothesis was, of course, incorrect! Figure 9.15 This hypothesis was, of course, incorrect! 9-42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Watson and Crick They went back to the ball-and-stick units They then built models with the Sugar-phosphate backbone on the outside Bases projecting toward each other They first considered a structure in which bases form H bonds with identical bases in the opposite strand ie., A to A, T to T, C to C, and G to G Model building revealed that this also was incorrect 9-43 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Watson and Crick They then realized that the hydrogen bonding of A and T resembled that between C and G So they built ball-and-stick models with AT and CG interactions These were consistent with all known data about DNA structure Refer to Figure 9.16 Watson, Crick and Maurice Wilkins were awarded the Nobel Prize in 1962 Rosalind Franklin died in 1958, and Nobel prizes are not awarded posthumously 9-44 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Fig. 9.16

The DNA Double Helix General structural features (Figures 9.17 & 9.18) Two strands are twisted together around a common axis There are 10 bases per complete twist The two strands are antiparallel One runs in the 5’ to 3’ direction and the other 3’ to 5’ The helix is right-handed As it spirals away from you, the helix turns in a clockwise direction 9-45 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Key Features •Two strands of DNA form a •The bases in opposite strands Fig. 9.17 (TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Key Features 2 nm 5¢ end 3¢ end •Two strands of DNA form a right-handed double helix. H HO H H H H H H P O– O •The bases in opposite strands hydrogen bond according to the AT/GC rule. A S H2N N O P O P S O N CH2 O O S G C P N H P N S O– H S G C P 5¢phosphate O P O CH2 N O N NH NH2 2 H •The 2 strands are antiparallel with regard to their 5¢to 3¢ directionality. P S O– H H H H H H H H O– C S O S P H H2N N N CH2 O P O P S O •There are ~10.0 nucleotides in each strand per complete 360° turn of the helix. P N H H N S P O O N S O H A T S O P O CH2 O N NH P 2 O– H H H H H H H H G C S H H O– S P O H H N N CH2 O P O P C G S N H O– S N P O O H T A N H2N S P O P O CH2 O N O P S O– P H H H H H CH3 P S 3¢ hydroxyl OH H G One complete turn 3.4 nm P S 3¢ end 5¢ end C G S P P T A S S P G C P S A T One nucleotide 0.34 nm S P S S P P S C G S P S C G P

Ball-and-stick model of DNA Space-filling model of DNA Fig. 9.18 (TE Art) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Minor groove Minor groove Major groove Major groove Ball-and-stick model of DNA Space-filling model of DNA

The DNA Double Helix General structural features (Figures 9.17 & 9.18) The double-bonded structure is stabilized by 1. Hydrogen bonding between complementary bases A bonded to T by two hydrogen bonds C bonded to G by three hydrogen bonds 2. Base stacking Within the DNA, the bases are oriented so that the flattened regions are facing each other 9-46 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The DNA Double Helix General structural features (Figures 9.17 & 9.18) There are two asymmetrical grooves on the outside of the helix 1. Major groove 2. Minor groove Certain proteins can bind within these grooves They can thus interact with a particular sequence of bases 9-47 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.17 9-48 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.18 9-49 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The Three-Dimensional Structure of DNA To fit within a living cell, the DNA double helix must be extensively compacted into a 3-D conformation This is aided by DNA-binding proteins Refer to 9.21 This topic will be discussed in detail in Chapter 10 9-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

DNA wound around histone proteins Figure 9.21 9-56

RNA Structure The primary structure of an RNA strand is much like that of a DNA strand Refer to Figure 9.22 vs. 9.11 RNA strands are typically several hundred to several thousand nucleotides in length In RNA synthesis, only one of the two strands of DNA is used as a template 9-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 9.22 9-58

Table 10-4 Copyright © 2006 Pearson Prentice Hall, Inc. Table 10.4 RNA Characterization Table 10-4 Copyright © 2006 Pearson Prentice Hall, Inc.

Different types of RNA secondary structures are possible Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary base-pairing A to U and C to G This allows short regions to form a double helix Different types of RNA secondary structures are possible Refer to Figure 9.23 9-59 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Complementary regions Held together by hydrogen bonds Figure 9.23 Noncomplementary regions Also called hair-pin Have bases projecting away from double stranded regions 9-60 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Molecule contains single- and double-stranded regions Many factors contribute to the tertiary structure of RNA For example Base-pairing and base stacking within the RNA itself Interactions with ions, small molecules and large proteins Molecule contains single- and double-stranded regions These spontaneously interact to produce this 3-D structure Figure 9.24 Figure 9.24 depicts the tertiary structure of tRNAphe The transfer RNA that carries phenylalanine 9-61 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 10-1 Copyright © 2006 Pearson Prentice Hall, Inc. Figure 10-1 Simplified view of the central dogma describing information flow involving DNA, RNA, and proteins within cells. Figure 10-1 Copyright © 2006 Pearson Prentice Hall, Inc.