Ethidium Bromide Binds to DNA

 
Last Monday's Molecule was ethidium, better known by the name of its common salt, ethidium bromide [Monday's Molecule #35]. Ethidium is a large planer molecule that binds tightly to DNA. It is often used in biochemistry laboratories to visualize fragments of DNA that have been separated on gels. The ethidium molecule is fluorescent—when illuminated with ultraviolet light it shines in the visible range. Here's a picture (below right) of DNA fragments that are illuminated by ethidium binding. It's from an old paper of mine (Moran et al. 1979)—these days you usually can't publish simple experiments like this.

Ethidium binds by inserting itself bewteen the stacked bases in double-stranded DNA. Note that the ring structure of ethidium is hydrophobic and resembles the rings of the bases in DNA. Ethidium is capable of forming close van der Walls contacts with the base pairs and that's why it binds to the hydrophobic interior of the DNA molecule.

Molecules that bind in this manner are called intercalating agents because they intercalate into the compact array of stacked bases. In doing so, they distort the double helix and interfere with DNA replication, transcription, DNA repair, and recombination. This is why intercalating agents are often potent mutagens.

The cartoon below shows the distortion of the sugar-phosphate backbone when an intercalating agent bind and it also shows that the DNA is lengthened when intercalating agents bind. This changes the properties of DNA considerably. One of the tricks in separating closed circular molecules of DNA from linear fragments (such as genomic DNA) is to treat the DNA with ethidium bromide. The intercalating agent doesn't bind to closed circular molecules because they can't be lengthened enough to allow insertion of the chemical between the bases. The normal circular plasmid DNA can then be separated from linear DNA with bound ethidium because binding of ethidium changes the overall density of DNA.



The structure shown above (right) is from Reha et al. (2002). It shows a molecule of ethidium lying between two A/T base pairs.

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Moran,L., Mirault, M-E., Tissières, A., Lis, J., Schedl, P., Artavanis-Tsakonas, S., and Gehring, W. (1979) Physical Map of Two D. melanogaster DNA Segments Containing Sequences Coding for the 70,000 Dalton Heat Shock Protein. Cell 17:1-8.

Reha, D., Kabelác, M., Ryjácek, F., Sponer, J., Sponer, J.E., Elstner, M., Suhai, S., and Hobza, P. (2003) Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4',6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124:3366-76.

The Name of Buddy Holley's Airplane was "American Pie"

 
Friday's Urban Legend: FALSE

From americanpie.com.

Basic errors in American Pie interpretations have been carried forward and sometimes get reported as being fact. One of the most tedious theories of recent times is that the plane that crashed killing Buddy Holly, Ritchie Valens and the Big Bopper was called 'American Pie'. This is wholly untrue and Don McLean released a press statement in 1999 to confirm this:

"the growing urban legend that "American Pie" was the name of Buddy Holly’s plane the night it crashed, killing him, Ritchie Valens and the Big Boppper, is untrue. I created the term." - Don McLean, 1999

For those (one or two) of you who don't know what we're talking about, here's a video that interprets the song—one of the best pop songs of all time, especially for us old fogies who actually listened to Buddy Holly, Ritchie Valens and J. P. Richardson, Jr. (The Big Bopper).



[Hat Tip: Karmen at Chaotic Utopia]

200,000 Visits

 
According to Sitemeter I've had 200,000 visits to Sandwalk since I started last November. I have to admit that I'm fascinated by these numbers. It's exciting to see that some people are reading Sandwalk and it's fun to find out where they're coming from and which articles are the most popular.

When my six month trial period was up, I expressed some disappointment about the popularity of Sandwalk [My Six Months Are Up!]. Things look a little better now so I've decided to give it another six months and see how it works out. I don't know what I'll do when I run out of Nobel Laureates.

The Story of DNA (Part 1)

Where Rosalind Franklin Teaches Jim and Francis Something about Basic Chemistry

What we now know as DNA was first isolated in 1868 by Johann Friedrich Miescher, a student in the lab of Ernst Felix Hoppe-Seyler. A later student of Meischer’s named Richard Altmann called the material "nucleic acid."

Nucleic acids are composed of pentose sugars and four bases. Guanine was discovered in bird droppings in 1848 (guano means excrement of sea birds). Adenine was identified in beef pancreas in 1885. Thymine originally came from calf thymus, hence its name.

By the 1920’s it was clear that there were two kinds of nucleic acid, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), although they weren’t called by those names until much later. Chromosomes were known to contain DNA. It was thought that DNA was some sort of structural element that provided stiffening. The four bases were believed to be present in equimolar quantities and they repeated endlessly along the chain. This idea was referred to as the "Tetranucleotide Hypothesis." It was advanced by leading chemists and widely believed to be correct.

William (Bill) Astbury took the first X-ray diffraction pictures of DNA in the 1930’s. These first images, and Astbury’s interpretation of them, dominated thinking for fifteen years. Astbury noted that there were strong reflections at 0.34 nm and he interpreted this to mean that the phosphate groups were 0.34 nm apart. In other words, the repeating nucleotides were spaced at 0.34 nm intervals. Astbury concluded from his images that the bases were stacked on top of each other like a pile of coins. Both of these assumptions were correct. However, Astbury saw an important reflection at 2.7 nm suggesting the structure repeated every 2.7 nm. If DNA was helical then this would indicate that there were about eight bases in each turn of the helix and each turn was 2.7 nm. This turned out to be very misleading. Astbury also thought that the sugar moiety and the base were in the same plane and this is incorrect.

In 1944, Oswald Avery at the Rockefellar Institute in New York showed that bacteria could be transformed with pure DNA. This clearly indicated that DNA was the genetic material but the data was not widely accepted. The lack of acceptance was not due to Avery’s reputation since he was a recognized and highly respected scientist. It’s just that the concept of DNA as the genetic material didn’t fit with other data and couldn’t be reconciled with the supposed structure of DNA according to the Tetranucleotide Hypothesis. Nucleotides were known cofactors in metabolism and complex carbohydrates are usually structural as in cellulose and bacterial cell walls. Proteins, one the other hand, are special and unique.

Throughout the 1940’s everyone knew of Avery’s experiment but they set it aside as unexplainable. This is an excellent illustration of how science works. Usually it is a good idea to reserve judgment when a single experiment conflicts with the current paradigm.

Avery’s work stimulated Erwin Chargaff to take up the study of the chemistry of DNA. He carried out careful analyses of DNA from many sources and discovered that the base composition varied considerably. Some species had more guanosine and cytosine and less adenosine and thymidine while in others the relative compositions were quite different.

Chargaff also noted that the amount of adenosine was equal to the amount of thymidine and gaunosine equaled cytosine. It followed that the numbers of purines equaled the number of pyrimidines. For the most part, these molar ratios were not thought to be significant. What WAS important was the discovery that in DNA the four bases were not present in the same amounts. This destroyed the Tetranucleotide Hypothesis and paved the way to an important understanding: DNA could contain information now that the amounts of the bases could vary. It’s safe to say that only a small number of scientists appreciated this point. An even smaller number, not including Chargaff himself, appreciated the significance of A = T and G = C.

About this time James Dewey Watson was a graduate student at Indiana University in Bloomington, Indiana, He had gone there because of a famous geneticist, Hermann Muller, but Watson ended up in the lab of Salvador Luria working on bacteriophage. Luria, along with Max Delbruck, founded the ‘phage group—an elite group of scientists dedicated to discovering the secrets of life by working with the simplest organisms. They met every summer in Cold Spring Harbor and they visited each other often.

Watson’s Ph.D. thesis was unremarkable except for the fact that he completed it within four years and became Dr. Watson when he was only 21 years old. He set off to Europe on a post-doc. After a stint in Denmark with members of the phage group, he ended up in Cambridge.

After the war, an X-ray crystallography group was set up in Cambridge, England, to study the structure of proteins. The group was headed by Lawrence Bragg, a Nobel Laureate who developed the original theory of X-ray diffraction. The chief members of the group were Max Perutz, working on the structure of hemoglobin, and John Kendrew who worked with myoglobin. They later received Nobel Prizes for solving the first protein structures.

In the Fall of 1947 Francis Crick moved to Cambridge as an overage graduate student. He was 31 years old and had served in the Admiralty during the war. In the beginning he was associated with the biochemists and was in close touch with Fredrick Sanger. At the time Sanger was sequencing insulin. Although the results came in slowly over the next six years, Crick was aware of them instantly because he attended the monthly seminars. Sanger showed that every insulin molecule in beef pancreas had the same sequence of amino acids. This was the first direct evidence that proteins had a defined amino acid sequence and Crick realized right away that the sequence had to be encoded in the genes. Sanger got eh Nobel Prize for this work and later on received a second Nobel Prize for developing the technique of sequencing DNA.

1948: Linus Pauling was in Oxford as a visiting Professor on leave from the Californian Institute of Technology in Pasadena, USA. He discovers the α-helix and publishes the model with Richard Robert Cory on his return to California. This is the first clear indication of helices in macromolecules and soon after the model was published Max Perutz proves that hemoglobin contains α-helices. Pauling was the pre-eminent chemist at that time and his influence was enormous. He was in contact with the phage group but for the most part he was their major competitor. The fact that Pauling scooped the Cambridge group by coming up with a model for the &alpha:-helix will have an influence on later events..

1949: Crick moves to the Cavendish lab in Cambridge to study under Perutz. Crick had realized that it was important to learn X-ray diffraction in order to study structure. He had come to recognize the importance of information in the gene (whatever it was) and he hoped to discover how this information (sequence) gave rise to three dimensional structure. At this time Crick was interested in DNA but was not by any stretch convinced that it was the genetic material.

In the late 1940’s another structure group was established at King’s College in London, England. Maurice Wilkins joined the group and began to look at DNA fibers. In May of 1950, he had been given an excellent preparation of DNA containing large intact molecules (from Rudolph Signer in Bern, Switzerland). Wilkins’ first pictures were better than the fifteen-year-old images of Astbury. However, Wilkins’ interest in DNA was a sideline. The main focus of his work was proteins and he turned the DNA project over to a graduate student, Raymond Gosling.

Later on the group in London decided that they needed to hire someone more senior to work on the structure of DNA. They found Rosalind Franklin, a chemist who had been working on the structure of complex chemicals in Paris, France. She was anxious to return to England and when she arrived in January 1951 she immediately took over the DNA project, presumably under the direction of Maurice Wilkins. Wilkins gave Rosalind Franklin the excellent DNA samples that he had obtained from Signer the previous summer.

In retrospect, it is clear that Wilkins never meant to assign complete control of the project to Franklin. He was looking for a collaborator even though he assigned Gosling to Franklin as her graduate student. This conflict between Franklin and Wilkins became more intense over the next two years until they were barely speaking to one another.

Meanwhile, the evidence that DNA was the genetic material was mounting to those who were paying attention. The famous Hershey-Chase experiment was being completed and word was spreading among the insiders. In this experiment, Hershey had labeled bacteriophage DNA with radioactive phosphorus and the protein were labeled with radioactive sulphur. After the phage adsorbed to the bacteria, the culture was put in a Waring blender and the resulting agitation knocked off the empty phage particles. The bacteria could then be separated from the radioactive sulphur labeled proteins. When the bacteria were concentrated by centrifugation the DNA was found to be in the bacteria.

After a short time the bacteria lysed producing a new burst of phage. The experiment clearly indicated that the injected DNA carried the information to produce new phage particles. The Waring blender experiment was much more sloppy than Avery’s earlier experiment but it was confirmation and it got quite a lot of people thinking about DNA as the genetic material, especially those who were associated with the phage group and understood the significance of a phage head stuffed with DNA. Watson was one of those who realized how important the experiment was.

During the winter of 1950-51 Watson was in Naples doing some experiments when he attended a seminar by Maurice Wilkins who was visiting from London. Wilkins showed his X-ray diffraction images of DNA fibers. This impressed Watson who then decided that he had to learn about diffraction techniques in order to solve the structure of DNA. He managed to obtain a fellowship, with the help of his phage buddies, to study under Bragg in Cambridge.

October 1951: Jim Watson arrives in Cambridge and meets Francis Crick. Watson was 23 years old. Crick was a 35 year old graduate student. Watson convinces Crick that genes are made of DNA and together they resolve to discover the structure of DNA and the secret of life. The two became fast friends and spent hours talking about biology. They are moved to a separate office of their own in order not to bother anyone else.

It’s important to note how crucial this meeting was. Watson had convinced himself that DNA was the stuff of life and he needed to solve the structure. Watson had the biological background from hanging out with the phage group and the bacterial geneticists. Crick had taught himself about structure and X-ray crystallography and was certain that structures would provide clues to the secret of life. Watson and Crick were thinkers and talkers rather than experimentalists, especially Crick. At the time, Watson and Crick were among the few people in the world who really “knew” that genes were made of DNA. They may have been the only scientists who desperately wanted to solve the structure of DNA and achieve fame and glory.

Crick was a personal friend of Maurice Wilkins, the man who had taken pictures of DNA fifteen months earlier. Crick knew about the Wilkins’ pictures and he knew that Rosalind Franklin was making slow progress on solving the structure. Within a few weeks of Watson’s arrival they had constructed a model based on Astbury’s data; what Crick and Watson remembered of the Wilkins data; and what Watson had learned from Rosalind Franklin. Here’s how it came together.

Alexander Todd in Cambridge had just worked out the chemical structure of DNA. The backbone consists of alternating sugar/phosphate groups joined through the 3′ carbon of one sugar and the 5′ carbon of the adjacent sugar residue. Crick was also aware of the unpublished results of Sven Furberg, a graduate student in London. Furberg had solved the three-dimensional structure of cytidylate, one of the nucleotides in DNA. He learned that the base and the sugar were at right angles to each other. Recall that Astbury’s conclusion was that they were in the same plane and that view had dominated thinking for fifteen years. Furberg had proposed that DNA formed a single-stranded helix with the bases sticking out and stacked on top of one another.

Watson went to a seminar by Franklin in London on Wednesday, November 21, 1951. While there, he learned that DNA contained several chains, that the chains were probably joined by hydrogen bonds between the phosphate groups, and that the structure was a helix. Watson also thought that Franklin had said that each unit of DNA (i.e., one turn of the helix) contained eight water molecules. In fact, Franklin had said that each nucleotide was associated with eight water molecules.

In a few short days of feverish activity Watson and Crick had built a model of DNA. It had three chains and the phosphate groups were on the inside with the bases projecting outward. Watson and Crick invited Wilkins, Franklin, and Gosling up from London to see their triumph. Unfortunately for them, the first model was destroyed in a few minutes as Franklin demonstrated that it was impossible. She showed them that their structure did not have enough water and that there was no way to form the phosphate-phosphate interactions that they had modeled. Watson and Crick admitted defeat.

Shortly after this fiasco, Lawrence Bragg (their boss) ordered Watson and Crick to stay away from DNA. The problem belonged to Wilkins and Franklin in London.

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Bibliography

Clayton, J. and Denis, C. eds. (2003) 50 Years of DNA. Nature/Pallgrave/Macmillan

Judson, H.F. (1996} The Eighth Day of Creation: Makers of the Revolution in Biology. expanded ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor N.Y. USA

Maddox, B. (2002) Rosalind Franklin: The Dark Lady of DNA. Perennial/HarperCollins

Watson, J.D. and Berry, A. (2003) DNA: The Secret of Life. Alfred A. Knope, New York, USA

Watson, J.D. (1168) The Double Helix. Atheneum, New York USA

The Three-Dimensional Structure of DNA

In order to understand the three-dimensional structure of DNA, it’s convenient to think of DNA as a ladder-like molecule with a very regular structure as shown below. The hydrogen bonding between base pairs is responsible for forming the regular structure with antiparallel strands.


The double-stranded helix forms as a result of interactions between adjacent base pairs. This interaction consists of van der Waals attractions and it causes the base pairs to come together so that they are in close contact. The result is a stack of base pairs, one on top of the other, with hardly any space between them. The usual term for these interactions are stacking interactions and they are the main force that holds the two strands together in a helical form.

The interior of double-stranded DNA is very hydrophobic which is just a fancy way of saying that water is excluded. Unlike proteins, the formation of double-stranded DNA is not an example of an entropy-driven hydrophobic effect, Instead it is enthalpic contributions in the form of stacking interactions that drive the reaction. However, the hydrophobic interior is essential because it stabilizes and protects the hydrogen bonds between the bases. These hydrogen bonds would not form if they were surrounded by water molecules since each of them could just as easily be replaced by hydrogen bonds with water.

The structure of the normal B form of DNA is shown on the right. B-DNA is a right-handed helix which means that if you think of it as a spiral staircase you will be turning to the right as you descend. Left-handed helices are found in some other rare forms of DNA. The width of B-DNA is 2.37 nm (nanometers) and it varies by only a small amount with base composition.

The distance between one base pair and the next is 0.33 nm, on average. This is called the rise as in the risers on a staircase. Some of the strongest reflections in the X-ray diffraction pattern of DNA are due to this repeat of 0.33 nm. The space between stacked bases is exaggerated in the cartoon (right).

The pitch of the helix is the distance to complete one turn of the helix. This value is usually given as 3.40 nm but it varies somewhat depending on base composition. On average, there are about 10.4 base pairs per turn of the helix in B-DNA and the angle of rotation between adjacent base pairs is about 34.6°.

Quoting from Horton et al. (2006)

The double helix has two grooves of unequal width because of the way the base pairs stack and the sugar–phosphate backbones twist. These grooves are called the major groove and the minor groove. Within each groove, functional groups on the edges of the base pairs are exposed to water. Each base pair has a distinctive pattern of chemical groups in the grooves. Because the base pairs are accessible in the grooves, molecules that interact with particular base pairs can identify them without disrupting the helix. This is particularly important for proteins that must bind to double-stranded DNA and “read” a specific sequence.

Two views of B-DNA are shown below. The ball-and-stick model (left) shows that the hydrogen bonds between base pairs are buried in the interior of the molecule where they are protected from competing interactions with water. The charged phosphate groups (purple and red atoms) are located on the outside surface. This arrangement is more evident in the space-filling model (right). The space-filling model also shows that functional groups of the base pairs are exposed in the grooves. These groups can be identified by the presence of blue nitrogen atoms and red oxygen atoms.


The length of double-stranded DNA molecules is often expressed in terms of base pairs (bp). For convenience, longer structures are measured in thousands of base pairs, or kilobase pairs, commonly abbreviated kb. Most bacterial genomes consist of a single DNA molecule of several thousand kb; for example, the Escherichia coli chromosome is 4600 kb. The largest DNA molecules in the chromosomes of mammals and flowering plants may be several hundred thousand kb long. The human genome contains 3 200 000 kb (3 x 10⁹ base pairs) of DNA.

©:Laurence A. Moran and Pearson/Prentice Hall 2007

The Chemical Structure of Double-Stranded DNA

 
Double-stranded DNA consists of two complementary polynucleotide chains where the bases on one strand form hydrogen-bonded associations with the bases on the other strand. There are only two pairs of bases that can form regular interactions where the edge of one base match the edge of another so that the two bases are joined by hydrogen bonds while lying in the same plane.

The A/T and G/C base pairs each have one purine (A or G) and one pyrimidine (T or C) which means that the size of each of the base pairs from one side to the other is almost the same. When two polynucleotide strands are laid side to side, as in the figure, the distance between the sugar residues on the two strands is the same for every base pair. What this means is that double-stranded DNA is a very regular structure in spite of the fact that the sequence of base pairs can be very different in different parts of the molecule.

The two strands are said to be complementary because all the bases in one strand are paired with the complementary bases on the other strand (A with T and G with C). This can only happen in a way that generates a regular structure if the two strands are antiparallel. If you look at the structure shown above you can determine the directionality of each strand by following the rules described in DNA Is a Polynucleotide. The left hand strand runs in the 5′→3′ direction from top to bottom while the right hand strand reads 3′→5′ from top to bottom.

The discovery of the structure of DNA by Watson & Crick was only made possible when they realized that the two strands of the helix had to be antiparallel.

There’s a convention for writing DNA sequences. They all have to be written in the same direction and that’s 5′→3′. Thus, the sequence of the bases on the left hand strand is AGTC and the sequence of the bases on the right hand strand is GACT. This may seem a bit confusing if you don’t understand the convention.

One of the classic questions on undergraduate exams is to give the sequence of one strand of double-stranded DNA (e.g., TAACTGGCGGA) and ask students to write down the sequence of the other strand. You’d be surprised at how many students haven’t paid attention when we discuss antiparallel strands in DNA and naming conventions.

©:Laurence A. Moran and Pearson/Prentice Hall 2007

Dead Soldier's Mother Asks Us to Support Troops

 
Here's one of those difficult situations that can cause much confusion. The Toronto Star recently reported on the funeral of a soldier killed in Afghanistan [Support troops, dead soldier's mother asks Canadians]. Here's what was printed in the newspaper.

The mother of a soldier recently killed in Afghanistan beseeched Canadians on the day of her son’s funeral to support the country’s troops in Afghanistan.

Shortly before an honour guard piped the flag-draped coffin of Pte. Lane Watkins into an open field today, his mother Wanda read a family statement that suggested people should be extremely proud of the military’s efforts in a country that desperately needs Canada’s help.

"We don’t want any family to experience the terrible pain of losing their son or daughter, but if Canada and NATO abandon the Afghan people, the sacrifices Lane, our family and others have made will be for nothing," Watkins said.

"They deserve your respect. In supporting them, you’ll make our loss much easier to bear."

It's statements like this that make supporting our troops more difficult [What Does the "Support Our Troops" Ribbon mean to You?]. While we can all understand Wanda Watkins' grief, she conflates supporting our soldiers with supporting the mission. Since this is a common mistake, it means that any overt support for our soldiers—such as putting a yellow ribbon on your car—will usually be interpreted as support for Canada's role in Afghanistan.

I do not support the mission in Afghanistan and I urge the Canadian government to withdraw as soon as possible. Does this mean that I don't support individual soldiers who are carrying out the role assigned to them to the best of their ability? No it doesn't. They're are doing exactly what they are supposed to be doing and they deserve our respect and support because it's a dangerous job. As a matter of fact, they deserve even more support, and sympathy, because they're involved in a messy situation that they don't necessarily agree with.

Ms. Watkins wants us to stay in Afghanistan because otherwise her son will have died in vain. Unfortunately there's no way to avoid the obvious. If we eventually recognize that it is a mistake to be in Afghanistan then we will not have achieved "victory" and it will be difficult to justify the sacrifice of her son and the dozens of others who have died. To be blunt, they will have died in vain because we made a mistake by sending them into a dangerous situation where victory wasn't possible.

We cannot let such passionate appeals dissuade us from withdrawing if that's the best course of action. What would be the point of staying in Afghanistan if more lives will be lost for no gain? How many have to die in vain before we call it quits?

[The image is from The Royal Canadian Regiment Kit Shop]

Nobel Laureates: Francis Crick, James Watson, and Maurice Wilkins

 
 
The Nobel Prize in Physiology or Medicine 1962.

"for their discoveries concerning the molecular structure of nucleic acids and its significance for information transfer in living material"

Francis Harry Compton Crick (1916 - 2004), James Dewey Watson (1928 - ) and Maurice Hugh Frederick Wilkins (1916 - 2004) received the Nobel Prize in Physiology or Medicine for discovering the structure of DNA. This is one of the most notable and most deserving Nobel Prizes that has ever been awarded for a single discovery.

The presentation speech was delivered by Professor A. Engström of the Royal Caroline Institute.

Your Majesties, Your Royal Highnesses, Distinguished Audience.

An attempt to explain the significance of the discovery which has led to this year's Nobel Prize award in Physiology or Medicine could begin at a point which seems to be far from the precise world of biophysics and biochemistry. We could ask the question: «How do we define a fine portrait or a good caricature?»

A caricature is a drawing - or sometimes a sculpture, a piece of prose or poetry - in which the individual characteristics of the person being portrayed are emphasized. This something, strongly individual, could be a strange contour of the nose, a wild hair or a protruding chin. We all know, that we are very sensitive about the accuracy of the caricature. It must have qualities beyond those of a true picture. If the artist succeeds in producing the individual's specific variations of a common feature, the caricature becomes exciting and full of life, it is genuine. Thus, the artist must fuse the common general with the individual specific features.

When the scientist tries to disclose the physical and chemical characteristics of living matter in order to understand and to explain the great variety of living forms, he must always bear in mind this combination of generality and individuality. He can distinguish a number of general properties which are common to all living forms, for example the ability to extract nutrition from the environment and to multiply so that the offspring is given a life pattern similar to that of the parents. Thus he sees an extreme regularity. Further, when the scientist studies the physical and chemical characteristics of the organism or of its cells he discerns new signs of strict organization and internal order. But he cannot neglect noticing that each individual in one or more respects differs from other individuals of the same species. Within the framework of strong order there must be space for individual irregularities.

The discovery of the three-dimensional molecular structure of the deoxyribonucleic acid - DNA - is of great importance because it outlines the possibilities for an understanding in its finest details of the molecular configuration, which dictates the general and individual properties of living matter. DNA is the substance which is the carrier of heredity in higher organisms.

Deoxyribonucleic acid is a high polymer composed of a few types of building blocks, which occur in large numbers. These building blocks are a sugar, a phosphate, and nitrogen-containing chemical bases. The same sugar and the same phosphate are repeated throughout the giant molecule, but with minor exceptions there are four types of nitrogenous bases. It is for the discovery of how these building blocks are coupled together in three dimensions that this year's Nobel Prize in Physiology or Medicine has been awarded to James Dewey Watson, Maurice Hugh Frederick Wilkins, and Francis Harry Compton Crick.

Wilkins investigated deoxyribonucleic acid of various biological origins by X-ray crystallographic techniques. Such techniques are the most powerful tools which can be used to investigate the molecular structure of matter. Wilkins' X-ray crystallographic recordings indicated that the very long molecular chains of deoxyribonucleic acid were arranged in the form of a double helix. Watson and Crick showed that the organic bases were paired in a specific manner in the two intertwined helices and showed the importance of this arrangement.

The deoxyribonucleic acid molecule can also be looked upon as two interwoven spiral staircases, forming one staircase. The outside of this staircase consists of the phosphate and sugar molecules. The steps are formed by the paired bases. If it were possible to stain each base separately, that is each half-step, and if it were also possible for a person to climb this staircase, this person would get an impression of a tremendous variety. Soon he would discover, however, that red always was coupled to blue, and black to white. Also, he would notice that the steps sometimes had black to the right, and white to the left, or the reverse, and that the same variation was true also for the red-blue steps. The climber, who in molecules of human deoxyribonucleic acid had to ascend millions of steps, would see an endless variation in the sequence of red-blue, blue-red, black-white, and white-black steps. He would ask, what is the meaning of this, and he would realize that the staircase contained a kind of message, the genetic code.

Deoxyribonucleic acid is no staircase in which one can climb; it is a very active biological substance. It has been shown that a number of the steps - most likely three - via another nucleic acid, ribonucleic acid, regulates which amino acids shall be coupled into a protein chain during its synthesis. Thus the order of amino acids in a protein is fundamentally determined by a certain sequence of bases in the nucleic acid. Thus the nucleic acid controls the production of the highly specific proteins, which are the specialized workers of the organism. All the various types of proteins produced take part in a team-work which is subordinated to the needs of the whole organism. Certain characteristics of this team-work, certain specific features in some of the proteins, make the individual unique.

The code contained in the deoxyribonucleic acid is transferred in cell division, that is in the normal growth of the organism, and also in the fusion of the sexual cells. In this way the code of the deoxyribonucleic acid can start and control the development of a new individual which has striking similarities with its parents.

Today no one can really ascertain the consequences of this new exact knowledge of the mechanisms of heredity. We can foresee new possibilities to conquer disease and to gain better knowledge of the interaction of heredity and environment and a greater understanding for the mechanisms of the origin of life. In whatever direction we look we see new vistas. We can, through the discovery by Crick, Watson and Wilkins, to quote John Kendrew, see «the first glimpses of a new world».

Dr. Francis Crick, Dr. James Watson, and Dr. Maurice Wilkins. Your discovery of the molecular structure of the deoxyribonucleic acid, the substance carrying the heredity, is of utmost importance for our understanding of one of the most vital biological processes. Practically all the scientific disciplines in the life sciences have felt the great impact of your discovery. The formulation of double helical structure of the deoxyribonucleic acid with the specific pairing of the organic bases, opens the most spectacular possibilities for the unravelling of the details of the control and transfer of genetic information.

It is my humble duty to convey to you the warm congratulations of the Royal Caroline Institute and to ask you to receive this year's Nobel Prize for Physiology or Medicine from the hands of His Majesty the King.

Nucleotides Can Adopt Many Different Conformations

 
Individual nucleotides in solution can adopt many different conformations. For example, the deoxyribose sugars can bend and twist in many different ways. Two of the most stable conformations are shown on the left. They are the conformations found in some DNA structures.

The C-2′ endo conformation (top) allows the maximum separation between the oxygen atoms at the 5′ and 3′ positions. What this means is that any polynucleotide strand with sugars in this conformation will be extended. On the other hand, strands and helices with sugars in the C-3′ endo conformation will be much more compact with tighter helices.

If you look at all of the bonds that make up the sugar-phosphate backbone of a polynucleotide strand you will see that there are a lot of possible conformations. You can have free rotations around many of these bonds. Now, as it turns out, the structure of double-stranded DNA has a well-defined conformation where many of these bond angles are fixed but if you were handed two strands of DNA you would be hard pressed to bend them into the proper conformation of real DNA.

This is one of the reasons why it was so hard to predict the structure of DNA even though the chemical structure of the polynucleotides was known.

In addition to sugar puckers and bond rotations around the backbone, there are also many conformations of the base relative to the sugar. The two extreme conformations involve rotation around the β-N- glycosidic bond shown in green in the figures below. In the anti- conformation (left) the base is rotated so that it is as far from the sugar as possible. This is the most stable conformation and it's found in most DNAs. In the syn conformation (right) the base is rotated so that it's over the sugar leading to a much more compact structure.

When the structure of double-stranded DNA was being worked out, the conformations of the nucleotides were not known. It wasn't even obvious that the more stable anti conformation of free nucleotides would necessarily be the conformation in DNA. You can see that attempts to build a model of DNA without lots of additional information were doomed to failure. Several people tried and failed, including the greatest chemist of the day Linus Pauling.

©Laurence A. Moran 2007
[Figures are from Moran/Scrimgeour et al. Biochemistry 2nd ed. (1994) ©Neil Patterson Publishers/Prentice Hall.]

The Calvin Cycle: Regeneration

 
Nobody took me up on the offer to become an intelligent designer. The goal was to figure out a way of converting the five products of the Rubisco reaction into three new substrate molecules [The Calvin Cycle]. The five products are three carbon (3C) compounds and the three new substrate molecules are five carbon (5C) compounds. Here's how it's done ...

Two 3C molecules are joined to make one six carbon (6C) compound (fructose). Then two of the carbon atoms from fructose are transferred to another 3C molecule to make the first of the five carbon products (red, ribulose). This leaves a 4C molecule that is joined to another one of the 3C molecules to produce a seven carbon (7C) sugar called sedoheptulose. Two carbon are transferred from sedoheptulose to the last 3C molecule to produce a second 5C molecule. This leaves the third and last 5C molecule.

Of course there's a lot of fiddling in the pathway to get the molecules into the right form for these reactions. Here's the complete Calvin Cycle in all its glory. Click on it to see a bigger picture.



You can simplify the pathway a great deal by writing it like this ...

This shows you that, in spite of the complexity, the overall pathway takes three molecules of carbon dioxide and converts it to one molecule of glyceraldehyde 3-phosphate. That's the purpose of the Calvin Cycle, it fixes carbon.

The pathway is expensive. It uses two types of energy currency, ATP and NADPH, but these are produced in abundance by photosynthesis. It's a fair bet that this particular reaction is the ultimate source of 99% of the carbon atoms in your food.

There's a neat trick we can do with this reaction. We can use it to estimate the cost of synthesizing acetyl CoA—the substrate for the citric acid cycle and the product of the pyruvate dehydrogenase reaction. The pathway from glyceraldehyde 3- phosphate to acetyl CoA is coupled to the synthesis of two molecules of NADH and two molecules of ATP. If we subtract these from the cost of making glyceraldehyde 3-phosphate then the total cost of synthesizing acetyl CoA from CO₂ is 7 ATP + 4 NAD(P)H. This can be expressed as 17 ATP equivalents since each NADH is equivalent to 2.5 ATP.

Since the net gain from complete oxidation of acetyl CoA by the citric acid cycle is 10 ATP equivalents, the biosynthesis pathway is more expensive than the energy gained from catabolism. In this case, the "efficiency" of acetyl CoA oxidation is only about 60% (10/17 = 59%) but this value is misleading since it is actually the biosynthesis pathway (costing 17 ATP equivalents) that is complex and inefficient.

Tautomers of Adenine, Cytosine, Guanine, and Thymine

 
The four bases of DNA can exist in at least two tautomeric forms as shown below. Adenine and cytosine (which are cyclic amidines) can exist in either
amino or imino forms, and guanine, thymine, and uracil (which are cyclic amides) can exist in either lactam (keto) or lactim (enol) forms. The tautomeric forms of each base exist in equilibrium but the amino and lactam tautomers are more stable and therefore predominate under the conditions found inside most cells. The rings remain unsaturated and planar in each tautomer.


Fifty years ago it wasn't clear whether the amino or imino forms of the purines were stable under physiological conditions. (Or the lactam lactim forms.) As we will see, this uncertainty played a significant role in events leading up to the discovery of the structure of DNA.

We now know that all of the bases in the common nucleotides can participate in hydrogen bonding. The amino groups of adenine and cytosine are hydrogen donors, and the ring nitrogen atoms (N-1 in adenine and N-3 in cytosine) are hydrogen acceptors (see below). Cytosine also has a hydrogen acceptor group at C-2. Guanine, cytosine, and thymine can form three hydrogen bonds. In guanine, the group at C-6 is a hydrogen acceptor, and N-1 and the amino group at C-2 are hydrogen donors. In thymine, the groups at C-4 and C-2 are hydrogen acceptors, and N-3 is a hydrogen donor. (Only two of these sites, C-4 and N–3, are used to form base pairs in DNA.) The hydrogen-bonding patterns of bases have important consequences for the three-dimensional structure of nucleic acids.



©Laurence A. Moran and Pearson/Prentice Hall 2007

DNA Is a Polynucleotide

 
DNA is composed of nucleotides strung together to make a long chain called a polynucleotide. There are four basic nucleotides in DNA. They are; deoxyadenylate (A), deoxyguanylate (G), deoxycytidylate (C), and deoxythymidylate (thymidylate) (T).


There are a few things you need to know about the nucleotides in order to properly understand the structure of double-stranded DNA.

First, a nucleotide is composed of a base (adenine, guanine, cytosine, thymine) attached to a sugar (deoxyribose) to form a nucleoside. The nucleoside has an attached phosphate group and that makes it a nucleotide. The name of the nucleoside containing the base adenine is deoxyadenosine and if the phosphate group is attached at the carbon numbered 5′ (five prime) then the formal name of the nucleotide is 2′deoxyadenosine 5′-monophosphate (dAMP).

Those little numbers are important. The phosphate group can also be attached to the 3′ carbon to make another kind of nucleotide called 2′-deoxyadenosine 3′-monophosphate.

Normally the carbon atoms of the sugar are numbered 1, 2, 3 etc. but in a nucleoside the numbering of the nitrogen and carbon atoms of the base takes precedence. Thus, the sugar carbon atoms are numbered 1′, 2′ 3′ etc as shown on the left.

If you want to follow the discussion about DNA you have to take a bit of time right now and get familiar with the numbering of the sugar carbon atoms. Note that there's no attached hydroxyl group on the 2′ carbon atom. That's why this is 2′-deoxyribose.

You can string together a bunch of nucleotides to make single-stranded DNA. Inside the cell it's the job of DNA polymerase to make polynucletides from nucleotides. The structure of a typical polynucleotide (right) shows that the individual units are attached through their phosphate groups. The phosphate group on one 5′ carbon atom is attached to the 3′ carbon atom of the nucleotide above it.

This gives rise to the characteristic sugar-phosphate backbone of DNA. This linkage is called a 3′-5&prime (three prime, five prime) phosphodiester linkage. The bases are not involved in the covalent linkages between nucleotides.

This single-stranded polynucleotide chain has a free 5′ end at the top and a free 3′ end at the bottom and that's going to be true for all single-stranded chains. We're often interested in describing the directionality of this chain because it's important in synthesis and in degradation by nucleases. For example, the chain is synthesized in the 5′→3′ (five prime to 3 prime) direction. Which means that incoming nucleotides are added to the bottom of the chain during elongation.

By convention, the directionality is determined by reading across an individual nucleotide residue. In practice this means reading across a single deoxyribose sugar. Thus, reading from the top to the bottom of the strand shown above you cross the sugar carbons in the order 5′, 4′ and 3′. The direction is 5′→3′ (five prime to three prime). If you're talking about the direction from bottom to top then you read across the sugar in the order 3′, 4′, and 5&prime and the direction is 3′→5′ (three prime to five prime).

Are You a Non-Conformist?

 

You Are 79% Non Conformist

You are a pretty serious non conformist. You live a life hardly anyone understands. And while some may call you a freak, you're happy with who you are.

Are You a Nonconformist?

[Hat Tip: Mike, of course. It's scary to note that I'm slightly more of a non-conformist than he is.]

The Oldest Organisms on Earth

 
Today's Botany Photo of the Day is Pinus longaeva, bristlecone pine. Trees of this species are generally considered to be "the longest-lived of all sexually reproducing, nonclonal species." Many of them are over 4000 years old including this one, from Wheeler Peak in Nevada.

It is located in the same area as the oldest known tree, the 4,862 year old tree formerly known as "Prometheus" before it was cut down [The Martyred One].

If the world was created in 4004 B.C. then the deluge can be reliably dated to about 2450 B.C., which means that Prometheus was living for 400 years before the flood and must have survived it. Isn't that amazing?

The Calvin Cycle

Are You an Intelligent Designer?

The Rubisco reaction results in fixation of carbon dioxide and the production of two molecules of glyceraldehyde 3-phosphate each of which contains three caron atoms [Fixing Carbon: the Rubisco Reaction]. The starting substrate is ribulose 1,5- bisphosphate, a 5-carbon sugar derivative [Monday's Molecule #34]. Here's a schematic diagram of the reaction showing the carbon skeletons with the newly incorporated carbon atom in blue.
In order for this to become a cycle you have to regenerate the original substrate—a five-carbon compound. And in order for it to be a biosynthesis pathway you have to have net synthesis of one of the products. It was the working out of this stoichiometry that got Melvin Calvin the Nobel Prize in 1961 [Nobel Laureate: Melvin Calvin]. Do you think you can figure out the strategy for regenerating the five-carbon substrate? Here are the rules.

1. You start with three cycles of the Rubisco reaction, using up three 5C molecules and producing six 3C molecules. One of the 3C molecules enters the normal metabolic pathways and the other five are used to regenerate three 5C molecules (5 x 3C = 3 x 5C).
2. You can fuse molecules to create larger ones (e.g., 3C + 3C = 6C).
3. You can cleave large molecules to create smaller ones (e.g. 6C = 3C + 3C) as long as there are no intermediates with only one carbon (1C) or two carbons (2C).
4. You can swap 2C units between molecules provided that no products are 1C or 2C (e.g, 7C + 3C = 5C + 5C is allowed).
5. You can swap 3C units between molecules provided that no products are 1C or 2C (e.g, 7C + 3C = 4C + 6C is allowed).

Here's an outline of the Calvin Cycle. Your task, should you choose to accept it, is to design the regeneration pathway beginning with five molecules of glyceraldehyde 3-phosphate (5 x 3C) and ending with three molecules of ribulose 1,5- bisphosphate (3 x 5C). You just need to account for the carbon shuffling reactions.