Ie \J~\o\..e., J-:- S. -l. J<, R, H,-Ile,'-. {'if!: CHAPTER. Coding Capacity: Proteins Seem to Be the Best Bet. "Bj61#dJ~)i>("ve,:;~ v. C. ff<2a~ ~ ~.

Transcription

1 Ie \J~\o\..e., J-:- S. -l. J<, R, H,-Ile,'-. {'if!: "Bj61#dJ~)i>("ve,:;~ I.,re. 22 CHAPTER v. C. ff<2a~ ~ ~. THE CHEMICAL NATURE OF THE GENE We have seen how studies of inheritance led to the idea that the characteristics of an organism are controlled by individual elements called genes. We've also explored some aspects of biological chemistry and examined the kinds of molecules found in living cells. Is it possible that genes are made of ordinary molecules? Could a molecule actually carry genetic information? In this chapter we will try to answer these questions. Coding Capacity: Proteins Seem to Be the Best Bet Could protein be the genetic material? Eukaryotic chromosomes contain very little carbohydrate; they are about 30 percent nucleic acid and from 60 to 75 percent protein. Most scientists in the first half of the twentieth century agreed that proteins were most likely to be the molecules that carry information. To begin with, protein molecules are composed of 20 amino acids, but nucleic acids are composed of just 4 nucleotides. Therefore, proteins should be better coding molecules (an alphabet with 20 lette~s ~ould be far more exgressive than one with just.::jj. Proteins also differ a great deal more from one species to another than nucleic acids do. Finally, although scientists at the time were familiar with the ability of protein enzymes to control and regulate chemical reactions, there was no evidence that nucleic acids were able to do anything. The Transforming Principle As early as the 1920s, a few people were working on projects that were eventually to identify the molecules responsible for inheritance. Frederick Griffith was a British scientist studying the way in which certain types of 408

2 live smooth,~~ 8!P;8 injection lethal Figure 22.1 A diagrammatic summary of Griffith's experiments on transformation. Something present in a heat-killed extract of virulent pneumonia bacteria was able to transform a nonvirulent strain of bacteria so that they caused pneumonia. Griffith recovered live, virulent bacteria from the infected mice. live rough harmless injection heat-killed smooth harmless fif~8 (psi ~ injeliion!kft~z:~~ blood from dead mouse combined heat-killed smooth and live rough bacteria cause pneumonia, a serious and often fatal lung disease. In 1928 Griffith had in his laboratory two different types-strains-of pneumonia bacteria. Both strains grew very well in special culture plates in his lab, but only one of them actually caused pneumonia in mice. Griffith managed to grow both strains succ~ssfully in the lab and noticed that he could distinguish one strain from the other simply by its appearance on a culture dish. The virulent (disease-causing) bacteria produced a jelly-like coating around themselves that made their colonies look smooth. The nonvirulent (harmless) strains made no such coating and instead grew into colonies with rougho'jagged edges. Griffith first tested to determine whether the coating might contain a disease-causing poison. To do this, he killed a culture of virulent cells and injected the dead cells (coatings and all) into the mice. The mice were not harmed by the injections. Griffith concluded that his suspicions about a poison were incorrect. Next he injected both live nonvirulent and killed virulent cells into a mouse (neither one of these injections by itself made any of the mice sick). To Griffith's surprise, when the two types of cells were injected at the same time, the mice developed pneumonia (Figure 22.1). Today it is hard to appreciate just how startling this result was. The live nonvirulent bacteria never made the mice sick, and neither did the killed virulent ones. Why should the combination of these two have an effect that was different from what was seen when the two were administered separately? To confuse matters further, Griffith recovered live bacteria from the animals that had developed the disease. Were these bacteria the same nonvirulent ones he had injected? He grew the bacteria on plates to find out. Now they formed the smooth colonies that were characteristic of the virulent strain. Griffith:S extract had transformed one kind of bacterium into another! Griffith called the process he had discovered transformation. Griffith spe~ulated that when the live and killed bacteria were mixed together, a "factor" was transferred from the killed cells into the live ones. This factor changed the characteristics of the live cells in a permanent way, so that they henceforth acted like the virulent ones. What had actually happened? The molecule of inheritance had been transferred, and it had changed the characteristics of the cell that received it. The Transforming Principle is DNA In 1942 Oswald Avery, a scientist at the Rockefeller Institute in New York City, devised a simple research project: He would determine the chemical nature of the material CHAPTER 22 Molecules and Genes 409

3 Figure 22.2 The basic structure of a nucleic acid is illustrated in this dinucleotide. Both RNA and DNA consist of long chains in which the sugar and phosphate groups of adjacent nucleotides are joined by covalent bonds. responsible for the transformation effect in Griffith's experiments. Using Griffith's transformation system, Avery and his co-workers treated the transforming extract in ways that destroyed proteins, carbohydrates, lipids, and a kind of nucleic acid known as ribonucleic acid (RNA). Transformation still occurred. However, when they treated the transforming extract with an enzyme that destroyed another type of nucleic acid, deoxyribonucleic acid (DNA), transformation did not occur. Proteins did not carry genetic information. DNA did. Avery's results were treated with som,s!epticism)by his colleagues, if only because his answer seemed to make so little sense. Many in the scientific community had not suspected that nucleic acids were capable of playing such an important role. CLUES TO THE STRUCTURE OF DNA Base Composition We have already seen that nucleic acids are polymers of nucleotides. Long chains, polynucleotides, can be built by linking nucleotides together (Figure 22.2). By the late 1940s, scientists understood the general chemistry of the polynucleotide. Still there was nothing that might distinguish the nucleic acid from any other biological polymer. Some of the first clues to the structure of DNA came from experiments in which scientists determined the composition of DNA obtained from different organisms. DNA is a nucleic acid containing four nucleotide bases: adenine, cytosine, guanine, and thymine. Each of these bases is represented by a single letter: A, C, G, and T. Erwin Chargaff, a biochemist, was the first to notice a pattern in the relative percentages of the four bases. This pattern is apparent in Table Do you see the same p~ttern that Chargaff did? The percentages of the four bases are not static. They vary over a wide range, but the relative proportions of guanine (G) and cytosine (C) are always nearly equal; the same is true for the proportions of adenine (A) and thymine (T). In more symbolic form, we might express this observation as follows: [A] = [T] [C] = [G] This observation became known as Chargaff's rule. Chargaff himself had no explanation for the rule, but it did suggest something about the structure of DNA. Because there were always equal amounts of adenine and thymine, for example, the molecule had to be organized in a way that would account for the equivalence of the two bases. Therefore, to our basic knowledge of the chemical structure of a polynucleotide we can add a bit of information about the ratios of the various bases. The X-ray Pattern If a molecule can be crystallized, X-ray diffraction may produce a scattering pattern from the crystal that yields information about the molecule's internal structure. The repeating pattern of atomic bonds that exists within a crystal scatters a beam of X rays in a regular pattern. When researchers realized that DNA might be an interesting and important molecule, a number of X-ray crystallographers began to work on its structure. In most cases, however, the formation of good crystals from DNA turned out to be both a practical and a theoretical problem. Crystals were difficult to produce, and the patterns from successful crystals were difficult to interpret. Table 22.1 Base Composition (% of Total DNA Bases) SOURCE: E. Chargaff and J. Davidson, eds., 1955, The Nucleic Acids, New York: Academic Press. 410 PART 5 Molecules of Ufe

4 A few scientists, however, believed that DNA was such an interesting molecule that X-ray diffraction should be attempted even if perfect crystals couldn't be formed. One such person was Rosalind Franklin, a young scientist working with Maurice Wilkins, a crystallographer in London. Franklin drew a thick suspension of the fiber-like DNA molecules up into a glass capillary tube and used this DNA sample to scatter X rays. In the tube, she hoped, the thick suspension of DNA molecules would be forced to line up so that the molecules were parallel to the tube. Like spaghetti drawn through a straw, the molecules were all arranged in the same direction-not perfect enough to give a crystal-like pattern, but just good enough to yield a few clues about the structure of the DNA molecule. Interpreting the X-ray Pattern One of the X-ray patterns produced by Franklin's DNA samples is shown in Figure The pattern contains two critical clues to the structure of the DNA molecule (graphically summarized 'in the figure). Clue number 1: The two large dark patches at the top and bottom of the figure showed that some structure in the molecule was arranged at a right angle to the long axis of DNA and repeated at a distance of 3.4 A (see Appendix: The Metric System). In other words, something in the molecule was arranged l~ke the run){s of a ladder. Clue number 2: The X-like mark in the center of tlie pattern showed that something in the molecule was Figure 22.3 (left) DNA fibers were taken up in a thin tube so that most of them were oriented in the same direction. An X-ray diffraction pattern was then recorded on film. (bottom) X-ray diffraction pattern of DNA in the "8" form, as taken by Rosalind Franklin in Franklin's X-ray pattern contained two important clues to the structure of DNA. The large spots on the top and bottom of the pattern indicate that there is a regular spacing of 3.4 A along the length of the fiber. The "X"-shaped pattern in the center indicates that there is a zigzag feature in the molecule, which might be consistent with a helix. C HAP T E R 22 Molecules and Genes 411

5 which dominated the picture could arise only ftom a helical structure." Watson and Crick had to account for several things. An ideal model for the structure of DNA would: I. Explain Chaigaff's rule. 2. Be able to carry coded information. 3. Be capable of replication. 4. Fit the chemistry known for polynucleotides. 5. Agree with the X-ray pattern's three predictions: (a) The molecule is helical. (b) One feature of the molecule is stacked at a spacing of 3.4 A. (c) The width of the molecule is about 20 A. Because the X-ray data were not detailed enough to determine a structure directly, Watson and Crick hoped to find an arrangement of nucleotide subunits that would be consistent with each piece of the puzzle, including the X-ray pattern. The Double Helix Model Figure 22.4 There were many false steps along the road to solving Franklin's X-ray pattern. For example, placing the nitrogenous bases inside the sugar-phosphate chains would give a fiber of the correct average width, but the different sizes of the bases, if there were no restrictions on how they were placed, would produce a "Iumpy" fiber. arranged in a zigzag fashion at a spacing of about 20 A. If DNA had a twisting, helical configuration, the X-ray pattern it produced from the side would indeed look like that "X." The X-ray pattern suggested that the molecule was a helix and that its diameter was about 20 A. The scientific challenge remaining was to put all of these clues together to determine the structure of DNA. In 1953 two young scientists working at Cambridge University in England learned about Franklin's remarkable X-ray pattern. The scientists, James Watson and Francis Crick, had been working with molecular models, twisting little bits of wood and paper into various shapes in an effort to determine how nucleotides could form a structure that could do all that DNA was able to do. According to their own accounts of that discovery, one look at Franklin's X-ray pattern was the last bit of evidence they required, and the problem was solved. In his book The Double Helix, James Watson wrote, "The instant I saw the picture my mouth fell open and my pulse began to race.... The black cross of reflections In early 1953 Watson and Crick believed that they had a sensible structure for the molecule: the double helix model. They published their ideas in a brief paper that appeared in April of that year. The details of their model were surprisingly simple. Watson and Crick realized they could account for the 3.4 A spacing in the X-ray pattern if they arranged the nitrogenous bases so that they were "stacked" on top of each other. The 20 A width of the molecule could be accounted for if they placed two antiparallel strands side by side in opposite directions and arranged the bases facing each other. The helical twist that was evident in the diffraction patterns could be accounted for as well. AU they had to do was twist the molecule so that the two strands twisted about each other. At first, however, there were two problems with the model. First, what kinds of forces might hold the two strands together? Second, how could one solve the problems posed by the sizes of the nitrogenous bases? Two of the bases, adenine and guanine, belong to a chemical group known as the purines. They have two carbon-nitrogen rings in their basic structures. The other two, thymine and cytosine, are pyrimidines: They have a single ring, meaning that they are quite a bit smaller than the purines. This would cause a problem in the model. If two pyrimidines were paired, the two strands would have to be much closer than when two purines were paired, making the model "lumpy" (Figure 22.4). Chargaff's rule showed how a "lumpy" helix could be avoided. If a purine was always paired with a pyrimidine, the helix wouldn't be lumpy. But was it possible for bonds 412 PART 5 Molecules of Life

6 to form between purines and pyrimidines to hold the two strands together? Watson and Crick remembered how hydrogen bonds (weak, noncovalent interactions) seemed to stabilize the structure of the a helix in proteins. To their delight, when James Watson drew a sketch of the bases, they could find perfect places for hydrogen bonds to form between A and T and between G and C (Figure 22.5). - The specific hydrogen bonding between the bases is known as base pairing. ~atso!!,by the way, had nev~r done very well in ch!!:!!!:-istry. and his sketch~owed~e ri~~onc~hird hxdrogen bond (highlighted in Figure 22.5) existed between G and C. Watson and Crick's insight solved one of the critical problems regarding the biological role of DNA. Prior to 1953, no one had been able to come up with a reasonable scheme for how a molecule might be replicated. But the structure of DNA itself contained an obvious answer to the riddle. Each strand is a complementary "copy" (although not an exact copy) of the other, which means that each contains the "information" required to reproduce the other strand. All that is required to copy the molecule is to separate the two strands and then to form a new strand for each original one by using the base-pairing rules suggested by Watson and Crick. James Watson and Francis Crick (Figure 22.6) had put together a three-dimensional model that accounts for the biological properties of DNA (Figure 22.7; see Theory in Action, The Prize, p. 414.) H / H '\ N\ H "H H Figure 22.5 The problem of placing nitrogenous bases was solved by James Watson, who drew sketches (top) showing how hydrogen bonds might pair thymine with adenine and guanine with cytosine. The modern representation of A- T and G-C base pairing (bottom) shows three hydrogen bonds in the G-C pair, something that was missing from Watson's first sketch. H H/' -- Figure 22.6 (left) James Watson and Francis Crick shortly after the publication of their double helix model for the structure of DNA. (right) The two strands of a DNA double helix are held together by hydrogen bonds between the nitrogenous bases. The strands run in antiparallel directions, forming a helix with a diameter of 20 A. C HAP T E R 22 Molecules and Genes 413

7 ~ iii\);1;z l:~ ~ ~ ~ ~.".. ;1 THEORY N ACT 0 N The Prize TheX.ray pattern that provided the critical c1uesjorwatson and Crick was made by Rosalind Franklin, Her pape iw hichcqn tainedthepa ttern. was actually published in the very same issueofnatufethat..<;ontained the double helix paper, Needless to s~y.she was norexactlyovedqycd that a de tai ledinte rp tetationo(hetown data was, publishedsimul c c c tarieously c c wit\) theda~itself, C C C c; "c kc CCCC WatsonandCnc hav_enev~rc CCC C CCC denied that Fra~kIin'spatterncohtairiedtheim~ttant C.. '. newinfqrmation.,; C that made the.1rcmodel possible, But their acuons, Ihterpreu~gthedata aftetanadvan~c look.wet~~thical,c 0 ~ f F kl" b' h h C ne 0 ran. ins )ogr~p ers as argued that Watson and Crick deprived Franklin of the credit she deserved for making one of the fundamental scientific discoveries of the twentieth century. Unfortunately, Rosalind Franklin lived long enough to see only the first hint of how important her work had been. She died of cancer in 1957, just as DNA was beginning to become a central focus in biological research. A few years later, the Nobel Prize committee decided that the time was right to award the Prize to the group that had developed the double helix model. The Nobe.lPrize is never givepposthumously. It was awarded to Watson and Crick, and Franklin's as socia te, Maurice Wilkins. 414 PAR T Molecule~ of Life

8 ~ DNA REPLICATION 3 5' The DNA molecule, as we see, suggests a method for its own replication. Watson and Crick pointed this out in a paper published shortly after the announcement of the double helix structure. Figure 22.8 illustrates how the double helix is unwound to enable each strand to serve as the template for the synthesis of a new strand. The rules of complementary base pairing help to control the proeess and ensure that each newly synthesized strand has the appropriate base sequence. The replication process is said to be semiconservarive, which means that the two original strands of the helix are separated and that at the end of replication, each strand is paired with one of the newly synthesized strands. In this way, the replication process produces two identical DNA molecules, each composed of one "old" strand and one "new" strand. DNA Polymerase It was soon discovered that DNA does not replicate in isolation but rather requires a number of special enzymes to unwind the double helix and synthesize new DNA strands. The most important enzyme in this process is known as DNA polymerase. The action of DNA polymerase is illustrated in Figure DNA polymerase contains a binding site for attachment to the DNA strand and also a binding site for nucleotides. The nucleotides that attach to the enzyme are triphosphates: Like ATP, they have three phosphate groups attached to them. DNA polymerase binds the correct nucleotide to the growing DNA strand and uses the energy from splitting off two of the three phosphates to form a covalent bond linking the nucleotide to the growing chain. As DNA polymerase moves along the chain to attach the next molecule, part of the enzyme "proofreads" the work it has just done by checking the nucleotide pair to ensure that the proper base pairing has taken place. If an incorrect nucleotide has been inserted, this portion of the enzyme swiftly removes the nucleotide from the chain, and the molecule starts work over again. This intricate process of base pairing, covalent bond formation, and proofreading is at the heart of DNA replication in all organisms. In tiny bacteria, a few DNA polymerase molecules may work to replicate the single DNA molecule that contains all of the cell's genetic information. In larger organisms, thousands of DNA polymerase molecules may work at scattered sites throughout many chromosomes to complete DNA replication in time for cell division to begin. DNA polymerase complex 3' y ---8 Parent molecule "'\ 3 5'. synthesis 5' DNA polymerase -~ ;8 Original strands are unwound e Complementary '.,. new strands-- synthesized using base sequence of original strands complex " - ~ Figure 2~.8 Becauseach strand of the DNA molecule is complementary to the other strand, each may serve as a template against which a new strand may be constructed. DNA replicates in semi-conservative fashion. Each strand of the helix serves as a template against which a new strand is assembled, following base-pairing rules. 3' '/ C HAP T E R 22 Molecules and Genes 415