CHAPTER 2 Molecular Genetics DNA Structure
In the following chapter. We will explore the molecular structure and function of the genetic material. We will follow this plan: 1. Nucleic Acids: Review of the Basics 2. Nucleic Acids, the research behind the discovery of the DNA molecule structure and function. 3. DNA structure revisited, beyond the basics. 4. Chromosome formation and structure in prokaryotes and eukaryotes.
Nucleic Acids: Review of the Basics The composition and Structure of DNA and RNA • DNA and RNA are polymers of monomers called nucleotides. • A nucleotide consists of three distinct parts: ¾A pentose sugar (deoxyribose or ibose). ¾A phosphate group PO4-3 in the form of a triphosphate. ¾A nitrogen base
The pentose sugar
Pentose = C5 Each carbon is numbered clockwise beginning at 12:00. Only four carbons are used in the cycle which is enclosed with an ester bond ( -O-). The fifth carbon is part of the cycle. The difference between deoxyribose and ribose is that ribose has one hydroxyl group located on Carbon #2 (C5H10O5) whereas deoxyribose does not have a hydroxyl group having instead a H (C5H10O4)
The Phosphate Group • There are three phosphate groups in a free nucleotide. These three phosphates are bond together by two high energy bonds. • One of the terminal phosphates forms a bond with the pentose’s carbon # 3. The phosphate will be used for binding different nucleotides during DNA synthesis.
The nitrogen bases • The are two types of nitrogen bases • Purines: Double cycle nitrogen bases: Adenine and Guanine • Pyrimidines: Single cycle nitrogen bases: Cytosine, Thymine and Uracil.
• The dNTPs (deoxyribonucleotides tri phosphates) nucleotides are bond together by an endergonic dehydration synthesis reaction. Water
• dNTP + dNTP • The reaction leads to the formation of a Phosphodiester bond between C3 of one nucleotide and C5 of the other nucleotide.
dNTP-dNTP
• The addition of multiple nucleotides using deshydration synthesis lead to the formation of a large chain. • Notice that the first nucleotide (C5) has a phosphate and the last nucleotide (C3) does not. Thus this strand of DNA “runs” from a 5’ 3’ and it is known as the “sense” strand.
The second strand • The second strand of DNA follows the same principles that we have studied so far. It is positioned “upside down” and it is a mirror image of the first strand (a 180 degree vertical rotation). • However, if this was a “true” mirror image, the nitrogen bases should be the same and that is not the case: The nitrogen bases are complementary. • Complementary bases mean, that if in the first strand we find a purine base, in the second strand we will find a pyrimidine and viceversa.
Complementary bases First strand (sense)
Second strand (antisense)
The two strands are linked together by hydrogen bonds. There are two hydrogen bonds between thymine and adenine and three hydrogen bonds between cytosine and guanine. The C-G pair bond is slightly stronger than the A-T
Once the two strands are bond together, the entire structure twists to the right (right handed or clock wise) to form a helical structure known as a “double helix”.
Nucleic Acids, the research behind the discovery of the DNA’s structure and function. • 1868 Frederich Miescher Isolated “nuclein” from the nuclei of cells. Nuclein is now known as DNA. • 1911. T.H. Morgan’s group showed that genes are located on chromosomes. • 1928 Frederick Griffith discovered genetic transformation of a bacterium and called the agent responsible the “transforming principle.” • 1941 Avery. MacLeod and McCarty showed that Griffith transforming principle
• 1948 Edwin Chargaff produced Chargaff rule: The amount of A=amount of T and the amount of C=amount of G. • 1952 Alfred Hershey and Martha Chase demonstrated that DNA was the genetic material. • 1950’s Rosaling Franklin and Maurice Wilkins provided photographs of X-ray diffractions and provided physical information about DNA: A double helix and their measurents. • 1953 In a leap of imagination James Watson and Francis Crick produced the double helix model of the molecule of DNA. • 1956Gierer and Schramm, and 1957 Fraenkel-Conrat and Singer concluded that RNA was the genetic material of some viruses.
The search for genetic material • Once T.H. Morgan’s group showed that genes are located on chromosomes (1911), the two constituents of chromosomes - proteins and DNA - were the candidates for the genetic material. • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928. • He studied Streptococcus pneumoniae, a bacterium that causes pneumonia in mammals. He used two strains: The R strain was harmless. The S strain was pathogenic.
• The experiment was as follows. He injected: S bacteria
Mouse dies Type III S bacteria were isolated from the mouse
R bacteria
Heat killed S bacteria
Live R +Heat killed S bacteria
Mouse survives Mouse survives Mouse dies No living bacteria were isolated from the mouse
No living bacteria were isolated from the mouse
Type III S bacteria were isolated from the mouse
Live R +Heat killed S bacteria
The mouse died and he recovered the
pathogenic strain from the mouse’s blood. The heat had killed all the molecules but
not “the information to make capsules”. The R bacteria had assimilated this material from the dead S bacteria. Griffith called this phenomenon
Type III S bacteria were isolated from the mouse
transformation, a change in genotype and phenotype due to the assimilation of a foreign substance (now known to be DNA) by a cell. The substance, he called “transforming principle”.
Avery. MacLeod and McCarty showed that Griffith transforming principle was DNA.
Allow sufficient time for the DNA to be taken up by the type R bacteria. Add an antibody that aggregates R bacteria that has not been transformed and remove by centrifugation. Plate the remaining (the transformed bacteria) on petri dishes.
The Hershey and Chase Experiment • In 1952, Alfred Hershey and Martha Chase showed that DNA was the genetic material of the phage T2. • The T2 phage, consisting almost entirely of DNA and protein, attacks Escherichia coli (E. coli), a common intestinal bacteria of mammals. • This phage can quickly turn an E. coli cell into a T2-producing factory that releases phages when the cell ruptures.
Bacteriophage T2 lytic life cycle
• To determine the source of genetic material in the phage, Hershey and Chase designed an experiment where they could label protein or DNA and then track which entered the E. coli cell during infection. – They grew one batch of T2 phage in the presence of radioactive sulfur, marking the proteins but not DNA. – They grew another batch in the presence of radioactive phosphorus, marking the DNA but not proteins. – They allowed each batch to infect separate E. coli cultures. – Shortly after the onset of infection, they spun the cultured infected cells in a blender, shaking loose any parts of the phage that remained outside the bacteria.
– The mixtures were spun in a centrifuge which separated the heavier bacterial cells in the pellet from lighter free phages and parts of phage in the liquid supernatant. – They then tested the pellet and supernatant of the separate treatments for the presence of radioactivity.
• Hershey and Chase found that when the bacteria had been infected with T2 phages that contained radio-labeled proteins, most of the radioactivity was in the supernatant, not in the pellet. • When they examined the bacterial cultures with T2 phage that had radio-labeled DNA, most of the radioactivity was in the pellet with the bacteria. • Hershey and Chase concluded that the injected DNA of the phage provides the genetic information that makes the infected cells produce new viral DNA and proteins, which assemble into new viruses.
The Chargaff Rule • 1948 Edwin Chargaff hydrolized DNA from different organisms and demonstrated that the composition of double stranded DNA was 50% purine and 50% pyrimidine. A+T =1 C+G • Furthermore, he demonstrated that the amount of Adenine was very similar to the amount of Thymine and the amount of Guanine was similar to the amount of Cytosine. These equivalencies are known as Chargaff rules.
Watson and Crick discovered the double helix by building models to conform to X-ray data • By the beginnings of the 1950’s, the race was on to move from the structure of a single DNA strand to the three-dimensional structure of DNA. • Among the scientists working on the problem were Linus Pauling, in California, and Maurice Wilkins and Rosalind Franklin, in London.
• Maurice Wilkins and Rosalind Franklin used X-ray crystallography to study the structure of DNA. – In this technique, X-rays are diffracted as they passed through aligned fibers of purified DNA. – The diffraction pattern can be used to deduce the three-dimensional shape of molecules. Franklin concluded that DNA was helical structure with two distinctive regularities (grooves) of 0.34 nm and a 3.4 nm along its axis.
The Watson and Crick Model Watson and Crick used the data produced by Wilkins and Franklin to produce a three dimensional model of the structure of DNA.
The key breakthrough came when Watson put the sugar-phosphate chain on the outside and the nitrogen bases on the inside of the double helix. The sugar-phosphate chains of each strand are like the side ropes of a rope ladder, pairs of nitrogen bases, one from each strand, form rungs and the ladder forms a twist every ten bases.
And the rest is History!
One more time, let’s revise the chemical structure and physical characteristics of the molecule of DNA.
Double Stranded DNA Can Occur in Three Conformations
A-DNA is the dehydrated form.. It is not usually found in cells. It is a right-handed helix with 10.9bp/turn, with the bases inclined 13° from the helix axis. ADNA has a deep and narrow major groove, and a wide and shallow minor groove. A-DNA
B-DNA
Z-DNA
Double Stranded DNA Can Occur in Three Conformations
A-DNA
B-DNA
B-DNA is the hydrated form of DNA, the kind normally found in cells. It is also a right-handed helix, with only 10.0bp/turn, and the bases inclined only 2° from the helix axis. B-DNA has a wide major groove and a narrow minor groove, and its major and minor Z-DNA grooves are of about the same depth.
Double Stranded DNA Can Occur in Three Conformations
A-DNA
B-DNA
Z-DNA is a left-handed helix with a zigzag sugar-phosphate backbone that gives it its name. It has 12.0bp/turn, with the bases inclined 8.8° from the helix axis. Z-DNA has a deep minor groove, and a very shallow major groove. Its existence in living cells has not Z-DNA been proven.
DNA is organized in Chromosomes • 1. Cellular DNA is organized into chromosomes. A genome is the chromosome or set of chromosomes that contains all the DNA of an organism. • 2. In prokaryotes the genome is usually a single circular chromosome. In eukaryotes, the genome is one complete haploid set of nuclear chromosomes; mitochondrial and chloroplast DNA are not included.
Viral Chromosomes • A virus is nucleic acid surrounded by a protein coat. The nucleic acid may be dsDNA, ssDNA, dsRNA, or ssRNA, and it may be linear or circular, a single molecule or several segments. For example: • The T-even phages (T2, T4, and T6) have dsDNA in one linear DNA molecule. • φFX174 is a small, simple virus with one short ssDNA chromosome • Bacteriophage l chromosome’s is a linear molecule of dsDNA, but after the virus infects its host, the chromosome becomes circular.
Prokaryotic Chromosomes • The typical prokaryotic genome is one circular dsDNA chromosome, but some prokaryotes have a main chromosome and one or more smaller ones. The smaller ones are called plasmids. Both Eubacteria and Archaebacteria lack a membranebounded nucleus, and their DNA is densely arranged in a cytoplasmic region called the nucleoid.
• If E. coli was gently lysed, it would releases one 4.6-Mb circular chromosome, highly supercoiled. A 4.6-Mb double helix is about 1mm in length, about 103 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell. • Supercoiling is “super twisting” the DNA. It is
DNA length is about 1000 x cell length
Prokaryotes also organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently. The compaction factor for looped domains is about 10fold. In E. coli there are about 100 domains of about 40kb each.
Eukaryotic chromosomes • Most eukaryotes have a diploid number of chromosomes. • Eukaryotic chromosomes are made of chromatin, a combination of DNA and proteins, and its levels of condensation varies during the different stages of the cell life cycle. When looked with an electron microscope, chromatin shows a structure of “beads-on-a-string” structure. These “beads” are called” nucleosomes. • These nucleosomes are the units of chromatin formed by the proteins and the DNA.
• Histones are abundant, small proteins with a net (+) charge. The five main types are H1, H2A, H2B, H3, and H4. • Two molecules of each histone H2, H2A, H3 and H4 group together to form the “histone core” and the DNA wraps around 1.65 times (about 147 bp fragment). This is called a nucleosome. Nucleosome cores are about 11 nm in diameter.
The function of the histones is to condense the DNA. The nucleosome core provides a 7-fold condensation factor. The DNA between nucleosomes is called Linker DNA. The size of the DNA linker varies within and among organisms. H1 further condenses the DNA by connecting nucleosomes to create chromatin with a diameter of 30nm, for an additional 6-fold condensation.
This is called the 30 nm chromatin fiber.
The formation of this 30 nm chromatin fiber is explained by the solenoid model. This model proposes that the nucleosomes form a spiral with 6 nucleosomes per turn.
Further packing of the chromatin fibers is less understood. It is believed that loops of DNA attached themselves to non-histone proteinaceous “scafolds”. In cross section it seems like the loops are arranged like the petals of a flower around the “scafold”. An average human chromosome has about 2000 domains (loops).
Summary of the condensation and coiling of chromatin in Eukaryotic chromosomes. A methaphasic chromosome is 400 times thicker than the naked DNA.
• Eukaryotic chromosomes levels of condensation varies during the different stages of the cell life cycle. The most disperse is when the DNA is going to duplicate (S phase) and the most condensed is during metaphase of Mitosis and Meiosis. • There are two types of chromatin: Euchromatin and Heterochromatin. • Euchromatin condenses and decondenses with the cell cycle. It is actively transcribed, and lacks repetitive sequences. Euchromatin accounts for most of the genome in active cells.
• Heterochromatin remains condensed throughout the cell cycle. It replicates later than euchromatin, and is transcriptionally inactive. There are two types based on activity: • Constituitive heterochromatin: occurs at the same sites in both homologous chromosomes of a pair, and consists mostly of repetitive DNA (e.g., centromeres). • Facultative heterochromatin varies between cell types or developmental stages, or even between homologous chromosomes. It contains condensed, and thus inactive, euchromatin (e.g., inactivated Xchromosomes or Barr bodies).
• Centromeres and telomeres are eukaryotic chromosomal regions with special functions. • Centromeres are the site of the kinetochore, where spindle fibers attach during mitosis and meiosis. They are required for accurate segregation of chromatids.
• The telomeres are usually the “ends” of the chromosome (the fragment of DNA at the ends). • Telomeres are needed for chromosomal replication and stability. Generally composed of heterochromatin, they interact with both the nuclear envelope and each other. All telomeres in a species have the same sequence.
• The total amount of DNA in the haploid genome of a species is its C value . • A genome is the information in one complete haploid chromosome set.
