Nucleotides and Nucleic Acids Notes

Genes and DNA: A Historical Account

Walter Sutton (1902): Argued that genes are located on chromosomes.
Homologous Recombination: During meiosis, it explains the inheritance patterns of alleles observed by Mendel.
The link between chromosomes and inheritance paved the way for discovering the molecular structure and function of genes.

Candidates for Hereditary Material

Chromosomes consist of both protein and DNA, assembled in the nucleus.
DNA is a major chemical component of the nucleus.
Early 1900s: Proteins were considered better candidates for hereditary material.
- Proteins have function, and the 20 amino acid “alphabet” seems more information-rich than DNA's 4-letter alphabet.

Transformation Experiment

Early 1920s: A harmless strain of bacteria can become infectious when mixed with a killed virulent strain.
The harmless strain is “transformed.”
The transforming material appears to be a gene.
1940s: Oswald Avery demonstrated that DNA is the transforming material.

Griffith's Experiment (1928)

Live R strain (rough, coatless) is non-infectious.
Live S strain (smooth, sugar coat) is infectious.
Heat-killed S strain is non-infectious.
Live R strain + heat-killed S strain becomes infectious, indicating a transforming principle.
Hypothesis: A transforming principle from the smooth strain turns the rough strain infectious.
Oswald Avery, Colin MacLeod, & Maclyn McCarty (1940s): Took up the challenge to identify the transforming principle.

Avery’s Experiment

S strain extract with sugar coat + protein + DNA, when mixed with Live R strain, can lead to transformation.

Bacteria and Viruses

Bacteria can exchange genetic material through mating (similar to plants and animals).
Bacterial viruses (phages) inject genetic material into bacteria, directing the host's enzymes to replicate new phage particles.
These serve as models for studying gene function.
1952: Alfred Hershey showed that DNA, alone, is responsible for producing new viruses within an infected cell.
Pilus is involved in bacterial DNA transfer.

Hershey-Chase Experiment

Protein coats of viruses labeled with $^{35}S$ . DNA labeled with $^{32}P$ .
After infection, viral particles are detached by shearing and separated by centrifugation.
$^{32}P$ (DNA) is found inside the infected cells, while $^{35}S$ (protein) remains outside, proving DNA carries the genetic information.

Structures of Nucleosides

Ribonucleoside: Example: Cytidine
Deoxyribonucleoside: Example: Deoxyguanosine

Structures of Common Bases

Pyrimidines:
- Cytosine (in DNA and RNA)
- Thymine (in DNA and some RNA)
- Uracil (in RNA)
Purines:
- Adenine (in DNA and RNA)
- Guanine (in DNA and RNA)

Nucleotides: Building Blocks of DNA

Composed of a sugar (deoxyribose), a phosphate group, and a nitrogenous base (e.g., cytosine).
Nucleoside: Base + Sugar
Nucleotide: Base + Sugar + Phosphate

Phosphodiester Bond

Alternating polymer of phosphate and sugar.
Hydrophobic bases (Cytosine, Thymine, Guanine, Adenine) and hydrophilic sugar-phosphate backbone.
Bonds link nucleotides together.

RNA Chain Fragment

Illustrates the structure of an RNA chain with adenine, cytosine, guanine, and uracil bases.
Shows the B-Glycosidic bond between ribose and each base.
Highlights the 5'-terminus and 3'-terminus of the RNA fragment.

Chargaff’s Rules

Base composition of DNA varies between species.
DNA from different tissues of the same species has the same base composition.
Base composition does not vary with age, nutrient state, or environment within a species.
For all cellular DNA: T = A and C = G.
Therefore, the sum of purine residues equals the sum of pyrimidine residues.

DNA Molecule

Right-handed coiled double helix (Watson & Crick model, 1953).
Major and minor grooves.
Chargaff’s rules: A:T = 1 and G:C = 1 (B-form DNA).
- $A=T$
- $G=C$

DNA Base Pairs

Cytosine pairs with Guanine (C-G) via three hydrogen bonds.
Thymine pairs with Adenine (T-A) via two hydrogen bonds.

Structure of DNA

Helical molecule with stacked bases separated by 3.4 angstroms.
Bases are on the inside, sugar-phosphates on the outside.
Complementary base pairing between A and T, and G and C.
- $[A] = [T]$ and $[G] = [C]$
- $[A + G] = [T + C]$ or $[purines] = [pyrimidines]$
Base pairs lie in the same plane, perpendicular to the helix axis, rotated 36° with 10 pairs per helical turn.
Double helix diameter is 20 angstroms, and the molecule is approximately 1 mm in length.
Two external helical grooves: major (deep and wide) and minor (shallow), allowing protein interaction with bases.
Two polynucleotide strands are antiparallel (3'-OH terminus of one strand is adjacent to the 5'-phosphate terminus of the other).

B-DNA and Z-DNA

Comparison of right-handed B-DNA and left-handed Z-DNA.
Z-DNA has a zig-zag sugar-phosphate backbone and a deep minor groove.
B-DNA has a smoother backbone and shallow grooves.

DNA Strands

Sense Strand: 5’ to 3’
Anti-sense Strand: 3’ to 5’
Obligatory base pairing suggests one DNA half serves as a template for the other during replication.

DNA Replication Evidences

1958: Two lines of evidence supported semiconservative replication:
- Meselson-Stahl Experiment: Used nitrogen isotopes to track new molecule construction across bacteria generations, showing that one strand of each molecule is passed unchanged to daughter cells.
- DNA Polymerase Discovery (Arthur Kornberg): Adds complementary nucleotides to a template provided by a half DNA molecule.

Meselson-Stahl Experiment Visualized

Demonstrates the distribution of heavy (15N) and light (14N) DNA after successive generations of replication, supporting the semiconservative replication model.

One Gene, One Protein Hypothesis

Archibald Garrod (1902): Described alkaptonuria as an “inborn error of metabolism.”
- Mutation in a gene impairs waste elimination phenotype.
- Dark urine reflects this error (catabolism of Phe and Tyr).
George Beadle and Edward Tatum (1941): Using Neurospora (bread mold), they demonstrated the “one gene, one protein” hypothesis.

Bread Mold Experiment

Illustrates the concept of how genes control metabolic pathways leading to growth on minimal medium.

Mutating a Gene for Vitamin A Synthesis

Shows how a mutation in a gene responsible for making vitamin A results in no growth on minimal medium.

Creating Mutants

Using X-rays to create mutants and then testing them on complete and minimal media.

Testing Mutants

Testing mutants on minimal medium supplemented with individual amino acids or vitamins to identify the specific deficiency.

Rescuing a Mutant with Vitamin B6

Demonstrates how supplementing a mutant with vitamin B6 restores growth on minimal medium.

RNA as an Intermediate

RNA acts as an intermediate between DNA and protein.
- DNA in the nucleus, proteins in the cytoplasm.
Watson & Crick proposed the “central dogma,” where information flows from DNA to proteins via a carrier (RNA).
How does DNA sequence function as a code? What is the role of RNA?

Ribose Nucleic Acid (RNA)

Contains ribose sugar, a phosphate group, and a nitrogenous base (uracil).

DNA vs. RNA

Feature	DNA	RNA
Nucleotide sugar	Deoxyribose	Ribose
Nucleotide bases	A, G, C, T	A, G, C, U
Number of chains	Two	One
Secondary structure	Double helix	Loops and stems
Purines	Adenine, Guanine	Adenine, Guanine
Pyrimidines	Cytosine, Thymine	Cytosine, Uracil

Types of RNA

Messenger RNA (mRNA)
Transfer RNA (tRNA)
Ribosomal RNA (rRNA)

Zamecnik's Experiment

Radioactive proteins were associated with ribosomes.
Low molecular weight RNA was associated with unincorporated radioactive amino acids.
Proposed that rRNA is the carrier molecule in protein synthesis.

Template for Protein Synthesis

Sydney Brenner proved that rRNA is not the template.
A third RNA, mRNA, carries information to the ribosomes.

Adaptor Molecules

Francis Crick proposed that there must be adaptor molecules, 20 of them, one for each amino acid.

tRNA as Adaptor Molecules

Mahlon Hoagland worked with aminoacyl tRNA synthetase.
Amino acids first attach to a low MW “soluble” RNA (tRNA) and are then transferred to proteins in ribosomes.
tRNAs pair with amino acids to ferry them to ribosomes for protein synthesis.

Cracking the Genetic Code

Marshall Nirenberg and Har Khorana deciphered how the nucleotide language of mRNA is translated into the amino acid language of a protein.
DNA words (codons) are three letters long.
- 1 nucleotide = 4
- 2 nucleotides = 4 x 4 = 16
- 3 nucleotides = 4 x 4 x 4 = 64

Identifying the Genetic Code

Johann Matthaei (postdoc of Marshall) tested the 3-nucleotide codon theory.
Using E. coli cell-free extract + DN’ase and synthetic mRNA (poly U), he found that UUU = Phe.

Codon Examples

UUU = Phenylalanine (Phe)
AAA = Lysine (Lys)
GGG = Glycine (Gly)
CCC = Proline (Pro)

The Genetic Code Table

Illustrates the relationship between mRNA codons and amino acids.
Highlights the degeneracy of the genetic code.

Challenges in Deciphering the Code

Some codes were hard to decipher because the order (e.g., GGC, GCG, or CGG) could not be established biochemically.
Zamecnik’s group approached this problem using the activation of tRNA.

Structure of Transfer RNA

Structure solved by Robert Holley in 1962.
tRNA is single-stranded, but nucleotides form hydrogen bonds to create short double helix regions.
All tRNAs have a similar cloverleaf shape.
Each mRNA codon has a different tRNA with a specific anti-codon.

Completing the Genetic Code

Zamecnik’s group used a cell-free translation system, discovering that tRNA becomes activated when the amino acid binds to the tRNA region (ATP dependent).
Adding short RNA chains (3 or 6 nucleotides) to cell-free translation and identifying the amino acid of the bound tRNA.
Making specific nucleotide sequences to confirm the code.

The Complete Genetic Code

Shows all 64 codons and their corresponding amino acids.
Includes start and stop codons.

Start and Stop Codons

AUG is the start codon.
UAA, UAG, and UGA are stop codons.
A gene is a discrete DNA sequence encoding a protein, starting with a start codon and ending with a stop codon.

Colinearity in Bacteria

In bacteria, DNA and mRNA are colinear.
mRNA code is a faithful representation of the DNA from which it is transcribed.

Eukaryotic mRNA Editing

Transcription produces pre-mRNA, which undergoes splicing to remove introns, resulting in mature mRNA.

Flow of Genetic Information

DNA -> Pre-mRNA -> mRNA -> Proteins
Transcription, splicing, and translation processes.
Central dogma assumed informative flow is one way.

Reverse Transcription

Tumor viruses (like Rous Sarcoma Virus, RSV) integrate their DNA into the host cell DNA.
Retroviruses (like HIV) use reverse transcriptase to convert RNA to DNA.
- RNA -> DNA -> RNA -> Proteins

RNA's Multifaceted Roles

1960s: mRNA stores genetic material, tRNA and rRNA translate genetic information into proteins.
1980s: Some RNA acts as enzymes (ribozymes) to self-edit their own genetic codes.

MicroRNA (miRNA)

Small non-coding RNAs (~18-24 nucleotides).
Negatively regulate gene expression.
Involved in developmental timing, differentiation, cell death, and potentially cancer development.
At least 4,000 species known.

The RNA World Hypothesis

Why does RNA play so many roles in the flow of genetic information?
Why bother storing genetic information in DNA if RNA alone can do the job?
RNA has a greater capacity as a genetic material.
Single-stranded RNA is unstable and easily damaged by enzymes.
DNA evolved as a more stable molecule to pass information with accuracy.

Other Major Points

A genome is an entire set of genes.
Genes can be turned on and off.
Different genes are active in different cells.
Many non-coding RNAs are involved in protein synthesis.
Some DNA can jump (transposons).
Living things share common genes.

Some Key Notes

Base: Purines and pyrimidines
Nucleoside: Base + Sugar
Nucleotide: Base + Sugar + Phosphate
Phosphodiester bond
DNA:
- Double helix
- Antiparallel
- Base pairing
RNA:
- Single strand
- Secondary structures
- Performs many tasks
- Messenger RNA
- Transfer-RNA
- Ribosomal RNA

Absorption Properties of Nucleic Acids

Estimate nucleic acid concentration and purity using absorbance at different wavelengths.
Nucleic acid absorbs at A260, proteins at A280.
A260/A280: Pure DNA = 1.8, Pure RNA = 2.0
Extinction coefficient depends on the environment of the bases: Isolated nucleotides > ssDNA > dsDNA

Denaturation and Renaturation of Nucleic Acids

DNA: 40% gain in absorbance on denaturation.
$T_m$ (melting temperature): 80-100°C; depends on G+C content and DNA length.
Renaturation: important in vitro hybridization. Fast cooling vs. slow cooling affect renaturation efficiency.

Other Functions of Nucleotides

Storage and transfer of energy (ATP)
As cofactors (e.g., Coenzyme A)
As second messengers (cAMP)