Stryer's Biochemistry, 4th Edition

Chapter 4. "DNA and RNA: Molecules of Heredity"

DNA is a polymer of deoxyribonucleotide units.

What is a nucleotide? A nucleotide consists of a nitrogenous base, a sugar, and one or more phosphate groups. The sugar is deoxyribose. Relative to ribose, deoxyribose lacks an oxygen in the two position. The nitrogenous base is a purine or a pyrimidine.

The purines are:

adenine (A) and guanine (G);

The pyrimidines are:

thymine (T) and cytosine (C).

By definintion, a nucleoside is a base attached to a sugar while a nucleotide is a base attached to a sugar which contains at least one phosphate group. Typically, the phosphates are attached to the sugar in the 5' position. The prime indicates that the substitution is on the sugar, i.e., not on the base.

The backbone of DNA is invariant throughout the long polymer, consisting of a long chain of deoxyriboses linked by phosphates. The 3' hydroxyl of the sugar of one dexoyribonucleotide is joined to the 5' hydroxyl of the adjacent sugar by a phosphodiester bridge. The variable part of DNA is the sequence of four kinds of bases. Each nucleotide unit of DNA is called deoxycytidylate, deoxythymidylate, deoxyguanylate, and deoxyadenylate. (p.77)

The book offers a simplified format for writing DNA sequences. We will stick with convention, i.e., sequences always are in 5' ==> 3' (5'hydroxy to 3'hydroxy). Thus, ACG and GCA refer to different compounds with different nucleotide sequences.

The Avery Experiment--read letter on pg 79 of your book, its great!

Transformation of pneumococci by DNA revealed that genes are made of DNA.

Until 1944, nuclear proteins were thought to be the carriers of genetic information. But this is when Avery conducted a seemingly simple experiment which proved unambiguously that DNA was the stuff of life.

Protocol: It was known that a bacterium termed, "Pneumococcus Type III" (also refered to as "the old man's friend") could be cultured in the laboratory. On agar plates the organism took on either of two morphologies: rough or smooth. The smooth forms were known to be lethal to mice while the rough forms were not. (It turns out that the R forms lack an enzyme needed to make a polysaccaride layer which coats and protects the bacterium).

Avery killed the pneumococci by heat and extracted the DNA. It was characterized with regard to optical properties, biochemical properties (protease and lipase resistant, nuclease sensitive), physical properties (electrophoretic profile), and by elemental analysis. In all respects, the material was DNA---not contaminated by protein or lipid etc.

After exhaustive purification and characterization, Avery and coworkers incubated R-type bacteria with DNA from S-forms resulting in the transformation of some R forms into S-forms. These transformed S forms were then found to be lethal to mice. This landmark experiment demonstrated that DNA was the genetic material of life-heredity.

Another pivitol experiment. Hershey and Chase 1952

It was then "known" that DNA does not contain sulfur and that protein does not contain phosphate (now known to be not completely true). The experiment employed the T2 bacteriophage virus which resembles a bizzare creature under electron microscopy. It was suspected that the virus might act like a little hypodermic needle, attaching to the host with its tail and injecting the contents (nucleic acid) into the bacterium.

Hershey and Chase cultured E. coli (we're always picking on E. coli!) with the T2 virus in medium containing ³⁵S amino acids and ³²P.

The phage liberated from the bacteria (the virus causes the lysis of it's host cell) were then purified and another batch of bacteria were infected with the radiolabelled phage. After a short period, the bacteria together with the attached phage were suspended in a Waring blender at 10,000 rpm.

The phage protein coat was thus sheared off the bacteria which were collected by centrifugation. The pellet of bacteria contained most of the ³²P and virtually none of the ³⁵S . . . meanwhile, the blender treatment had no effect on the ability of the phage to infect the bacteria.

The authors stated simply that "a physical separation of the phage T2 into genetic and non-genetic parts is possible."

The discovery of the DNA double helix by Watson and Crick in 1953

Based on an X-ray diffraction pattern of DNA produced by Rosalind Franklin and Maurice Wilkins, Watson and Crick were able to deduce the double helical structure of DNA and to render obvious its mechanism of replication. Important features of their model of DNA:

1. Two polynucleotide chains coiled around a common axis.
2. The purine and pyrimidine bases are on the inside of the helix --perpendicular to the axis of symetry. The sugar phosphate backbone is on the outside. The sugars are oriented nearly at right angles to the bases.
3. The diameter of the helix is 2OAngstroms. Adjacent nucleotides are separated by 3.4 Angstroms, the rotational relationship is 36 degrees. Thus, the overall structure of DNA repeats itself every 10 nucleotides, at intervals of 34 angstroms.
4. The two chains are held together by hydrogen bonds between base pairs: adenine with thymine; and guanine with cytosine. There is no room for a purine-purine pair.
5. Watson and Crick deduced that adenine must base pair with thymidine and that guanine with cytosine because of steric and hydrogen-bonding factors. This steric constraint does not restrict in any way the linear sequence of nucleotides.

If DNA is genetic material, then how is it replicated?

Watson and Crick first suggested semi-conservative replication. But it took an experiment by Messelson and Stahl to prove it.

Theoretic possibilities:

• Conservative replication: One daughter chromosome and one parental.
• Dispersive replication: Two chromosomes with randomly replicated units on both copies.
• Semi-conservative: Each strand of DNA in a chromosome makes a copy of itself.

Procedure: The investigators grew E. coli in medium containing ¹⁵NH₄Cl as the only nitrogen source. After many generations to ensure complete incorporation of the heavy isotope, the bacteria were switched to medium that contained ¹⁴N, the ordinary isotope of nitrogen. The question: What was the distribution of the ¹⁴N and ¹⁵N in the DNA molecules after successive rounds of replication? The separation/distribution was carried out by density-gradient equilibrium sedimentation. Here a small amount of DNA is dissolved in concentrated cesium chloride having a density close to that of the DNA (ca. 1.7 g/cm³). Centrifugal force sets up the gradient between 1.66 and 1.76 g/cm³. This allowed separation between heavy ¹⁵N-DNA from lighter ¹⁴N-DNA.

Found: DNA was harvested from the bacteria at various times following the switch from heavy to light nitrogen. All DNA corresponded to the heavier nitrogen at the beginning. Within one generation, all of the heavy form had been transformed to a single lighter form. And after two rounds of replication there were two species of DNA based on density corresponding to the density of the first generation daughter molecules and a new lighter band. All consistent with the semi-conservative mode of replication.

DNA Molecules are very long.

DNA must be very long in order to encode the proteins necessary to support life. The chromosome of E. coli is a single circular molecule of double helical DNA that is 4 million base pairs long. The molecular weight of this would be 2.6 ~ 10⁹Daltons! If severed and layed out flat, the bacteria's DNA would stretch to 1.4mm -- having a width of 20 Angstroms. The largest chromosome of Drosophila melanogaster contains a DNA molecule of 62 million base pairs-stretching out to a distance of 2.1 cm. A human genome consists of about 3 billion base pairs. A good question would be in regard to how it is all stuffed into a microscopic cell. Two additional distinguishing characteristics of DNA from different organisms are linear vs. circular and relaxed vs. supercoiled. Many bacteria possess circular chromosomes while their viruses are circular or interconvert between circular and linear. DNA can also undergo supercoiling to form a "superhelix". This is important for storage and packing of DNA but also for regulation of gene activity (subject of later chapter).

DNA is replicated by specific polymerases

In 1958 Arthur Kornberg discovered the enzyme DNA polymerase (now called DNA polymerase 1 as others have been discovered). It is too simplistic to think that it is singly required for DNA replication--- instead over 20 proteins are involved. However, considered in isolation, this is how it works:

1. The enzyme catalyzes the stepwise addiditon of deoxyribonucleotides to a DNA chain as shown in the picture. There are several required components of the reaction: all four of the deoxyribonucleotide triphosphates must be present as well as Mg⁺⁺.
2. DNA polymerase adds nucleotides to the 3'-hydroxy terminus of a pre-existing DNA. A primer chain with a free 3'-hydroxy group is needed.
3. Of course, a DNA template is required otherwise the DNA polymer would consist of nonsense code. The template may be either single or double stranded.
4. How does the reaction proceed? DNA polymerase catalyzes the nucleophilic attack of the 3'OH terminus of the primer on the innermost phosphorous atom (alpha-position) of a deoxyribonucleotide triphosphate.
5. Although the reaction occurs at the free 3'hydroxyl group, the overall reaction catalyzes the addition of nucleotides in the 5' to 3' direction.
6. How does the enzyme know which nucleotide to add next? The incoming nucleotide must be complimentary to the opposing base on the template strand. H-bonds play a crucial role.
7. Another important feature of DNA polymerase 1 is that it also corrects its mistakes---it has an editing function. As a result it is highly accurate---with roughly 1 mismatch per 100 million base pairs. What does this number mean? Evolutionary Diversity vs. Mother Nature's mistakes.

Some viruses have only single stranded DNA (+) strand (i.e., Phi-X174).

Note that in this and similar cases, the virus infects a host cell and therein produces a complimentary strand (-) which is used to replicate (+) strand for further virus production.

The genes of some viruses are composed of RNA.

The structure of RNA differs from that of DNA in two important respects: First, all of the sugar residues are ribose, and second, uracil is used instead of thymdine. RNA cannot form a normal double helix because of steric reasons mostly relating to the 2' hydroxy feature.

Tobacco mosaic virus is an RNA virus which replicates in cells by first producing a complementary RNA strand (- strand) which subsequently serves as a template for synthesis of a large number of + RNA particles for packaging into a virus particle. All of these processes are carried out by RNA dependent RNA polymerases. By convention, the + strand of viral RNA is the one which serves as a template for synthesis of proteins (mRNA).

RNA Tumor viruses and other Retroviruses.

These viruses are remarkable in that they replicate through DNA intermediates within the host cell. The best studied of the tumor viruses is the Rous Sarcoma virus which contains a single strand of RNA. HIV-1 also operates on this same mechanism. Both viruses are termed "retroviruses" because they do everything backwards, i.e., genetic information flows from RNA to DNA rather than the conventional route of DNA to RNA.

Infection begins with delivery of the + strand of viral RNA into the host cell. It serves as a template for a complementary - strand of DNA by reverse transcriptase (also packaged with the virus particle). This enzyme is an RNA directed DNA polymerase. The - strand of DNA then serves as the template for synthesis of the corresponding + strand, thereby completing synthesis of a double helical DNA version of the viral genome. This DNA copy is incorporated into the chromosomal DNA of the infected cell and replicated along with normal cellular DNA where it may reside forever.

Point: AZT subverts reverse transcriptase leading to unproductive viral particles but as yet nothing can be done to selectively rid the cell of the incorporated viral genome.