Nucleic Acids: Structure & DNA Replication

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课程笔记

Nucleotide Structure

Both DNA and RNA are polymers made of repeating units called nucleotides.
Each nucleotide is formed from a pentose sugar, a nitrogen-containing organic base, and a phosphate group.
In a DNA nucleotide, the pentose sugar is deoxyribose (with hydrogen at the 2' position).
In an RNA nucleotide, the pentose sugar is ribose (with a hydroxyl group at the 2' position).
DNA contains the bases adenine (A), cytosine (C), guanine (G) and thymine (T).
RNA contains the bases adenine (A), cytosine (C), guanine (G) and uracil (U); uracil replaces thymine.
The 2' hydroxyl group in ribose makes RNA more susceptible to hydrolysis, so DNA is the storage molecule and RNA is the transport molecule with a shorter lifespan.

The Phosphodiester Bond

Nucleotides join together via condensation reactions between the phosphate group of one nucleotide and the pentose sugar of the next.
A condensation reaction between two nucleotides forms a phosphodiester bond.
A phosphodiester bond consists of a phosphate group and two ester bonds.
Many phosphodiester bonds create a chain of alternating phosphate groups and pentose sugars known as the sugar-phosphate backbone.
The phosphodiester bonds link the 5' carbon of one sugar to the phosphate group, which is linked to the 3' carbon of the next sugar.

DNA Structure

DNA is a polynucleotide made of two polynucleotide strands that run in opposite directions, an arrangement called antiparallel.
Each strand has a sugar-phosphate backbone formed by alternating deoxyribose sugars and phosphate groups joined by phosphodiester bonds.
One DNA strand runs from 5' to 3', and the other runs from 3' to 5'.
The nitrogenous bases project from the backbone towards the interior of the double-stranded molecule.
The two antiparallel strands are held together by hydrogen bonds between complementary bases.
Adenine always pairs with thymine (two hydrogen bonds) and guanine always pairs with cytosine (three hydrogen bonds).
DNA is described as a double helix, referring to its three-dimensional shape.

DNA Function

The function of DNA is to hold or store genetic information.
DNA contains the instructions for the growth and development of all organisms.
DNA is protected in the nucleus, which provides security for the genetic material.
DNA is easily copied and therefore conserved throughout generations of cells and inherited between generations.

RNA Structure and Function

RNA is a polynucleotide made up of many nucleotides linked together in a chain.
RNA molecules are single-stranded and relatively short compared to DNA.
Each RNA strand has a sugar-phosphate backbone of alternating ribose sugars and phosphate groups linked by phosphodiester bonds.
The nitrogenous bases project out sideways from the single-stranded RNA molecule.
Examples of RNA include messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA).
The function of RNA is to transfer the genetic code from DNA out of the nucleus and carry it to the ribosomes in the cytoplasm.
Ribosomes 'read' the RNA to make polypeptides (proteins) in a process called translation.

Ribosomes

Ribosomes are small organelles that are either free in the cytoplasm or attached to the rough endoplasmic reticulum (in eukaryotic cells).
Ribosomes are the site of protein synthesis and 'read' RNA to make polypeptides in translation.
Ribosomes are formed from RNA and proteins; the RNA is known as ribosomal RNA (rRNA).
The rRNA in ribosomes has enzymatic properties that catalyse the formation of peptide bonds between amino acids.
Each ribosome is composed of a small subunit and a large subunit.
Eukaryotic cells have 80S ribosomes (60S and 40S subunits); prokaryotic cells, mitochondria and chloroplasts have 70S ribosomes (50S and 30S subunits).
The large subunit is the site of translation; mRNA sits between the two subunits and the ribosome moves along it.
Ribosomes are not surrounded by a membrane.

The Origins of Research on the Genetic Code

DNA was first observed in the 1800s by Friedrich Miescher, who named it 'nuclein'.
Many scientists initially doubted that DNA could carry the genetic code because of its relatively simple chemical composition.
DNA was thought to be too simple as it is made of only four repeating nucleotides, each composed of a phosphate group, deoxyribose and a nitrogen-containing base.
It wasn't until the 1940s that the role of DNA in genetic inheritance began to be more fully researched and understood.
By 1953, experiments confirmed that DNA carried the genetic code; the triplet code enables much variation (the code is universal and degenerate).
The location of DNA, protected in the nucleus, enabled the security of the genetic material rather than proteins that are found in the cytoplasm and susceptible to hydrolysis.
1953 was also the year in which Watson and Crick confirmed the double-helix structure of DNA using Rosalind Franklin's X-ray data.

Semi-Conservative Replication

Before a cell divides, it copies its DNA to ensure each daughter cell receives a full copy of the parental DNA.
DNA is copied by semi-conservative replication: each new DNA molecule has one original strand and one newly synthesised strand.
This process ensures genetic continuity between generations of cells and is essential for replacing body cells, growth and development.
DNA replication occurs during the S phase of interphase in the cell cycle.
Helicase unwinds the DNA double helix by breaking the hydrogen bonds between the base pairs, forming two single strands that act as templates.
Free nucleotides are attracted to the exposed bases by complementary base pairing.
DNA polymerase catalyses condensation reactions to join the new nucleotides together, forming the sugar-phosphate backbone.
The original strand and the new strand join together through hydrogen bonding between base pairs to form the new DNA molecule.
Free nucleotides exist as nucleoside triphosphates (activated nucleotides); the extra phosphate groups provide energy for replication.
DNA polymerase removes two phosphate groups from each nucleotide to release energy used to form phosphodiester bonds.

Calculating the Frequency of Nucleotide Bases

All DNA nucleotides contain the same phosphate group and deoxyribose sugar, but the nitrogenous base can vary.
The bases always pair up in the same way: Adenine pairs with Thymine (A-T) and Cytosine pairs with Guanine (C-G).
The bases in each pair are said to be complementary to one another.
Therefore, the frequency of adenine equals the frequency of thymine, and the frequency of cytosine equals the frequency of guanine.
The frequency of bases can be calculated if the number or percentage of one specific base is known.
For example, if 28% of bases are cytosine, then 28% are guanine (total 56%), leaving 44% for adenine and thymine, so adenine = 22% and thymine = 22%.

The Watson-Crick Model and Meselson & Stahl's Experiment

In 1953, Watson and Crick confirmed the double-helix structure of DNA and proposed the semi-conservative model of replication.
The conservative model suggested that the original DNA remains intact and the new DNA is made entirely of new strands.
Meselson and Stahl tested the theories using bacteria grown in heavy nitrogen (15N) followed by growth in light nitrogen (14N).
Bacteria grown in 15N had DNA containing only heavy nitrogen, which settled near the bottom of a centrifuge tube.
After one round of replication in 14N, if replication were conservative, original DNA would settle at the bottom and new DNA at the top.
If replication were semi-conservative, all DNA molecules would contain both heavy and light nitrogen and settle in the middle of the tube.
The DNA from the second centrifugation settled in the middle, confirming that each DNA molecule contained a mixture of heavy and light nitrogen isotopes.
If more rounds of replication occurred, the ratio of 15N:14N would go from 1:1 after the first round to 3:1 after the second and 7:1 after the third.
This experiment proved Watson and Crick's theory correct, confirming semi-conservative replication.
The experiment was well-controlled and repeatable, but only tested one organism (E. coli) and didn't show how enzymes like helicase and polymerase work.

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练习题

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1.Which of the following correctly lists the three components of a nucleotide?
Easy
- APentose sugar, phosphate group, nitrogen-containing organic base
- BPentose sugar, carboxyl group, nitrogen-containing organic base
- CHexose sugar, phosphate group, amino acid
- DPentose sugar, phosphate group, fatty acid
2.Which sugar is found in RNA nucleotides?
Easy
- ARibose
- BDeoxyribose
- CGlucose
- DFructose
3.Which base is found in RNA but not in DNA?
Easy
- AUracil
- BThymine
- CAdenine
- DGuanine
4.DNA is a polynucleotide made up of many nucleotides joined together.
Easy
True or false?
5.RNA molecules are double-stranded.
Easy
True or false?
6.How many hydrogen bonds form between guanine and cytosine in DNA?
Medium
- A3
- B2
- C1
- D4
7.Which enzyme unwinds the DNA double helix by breaking hydrogen bonds between base pairs?
Medium
- AHelicase
- BDNA polymerase
- CLigase
- DRNA polymerase
8.Which enzyme catalyses the formation of phosphodiester bonds between nucleotides during DNA replication?
Medium
- ADNA polymerase
- BHelicase
- CDNA ligase
- DRNA polymerase

复习笔记

## Nucleotide Structure & the Phosphodiester Bond - Both DNA and RNA are **polynucleotides** – polymers made of many repeating **nucleotides**. - Each nucleotide is formed from: - A **pentose sugar** (carbons numbered 1′, 2′, 3′, 4′, 5′) - A **nitrogen-containing organic base** - A **phosphate group** - **DNA nucleotide**: deoxyribose sugar (H at 2′), phosphate, one of A, C, G, T. - **RNA nucleotide**: ribose sugar (OH at 2′), phosphate, one of A, C, G, U. - The 2′ hydroxyl group makes RNA more susceptible to hydrolysis → DNA is the storage molecule, RNA is the transport molecule with a shorter lifespan. - Nucleotides join via **condensation reactions** between the phosphate of one nucleotide and the pentose sugar of the next, forming a **phosphodiester bond**. - A phosphodiester bond consists of a phosphate group and two ester bonds. - The chain of alternating phosphate groups and pentose sugars is the **sugar-phosphate backbone**. ## DNA: Structure & Function - DNA consists of **two polynucleotide strands** that are **antiparallel** (one runs 5′→3′, the other 3′→5′). - Strands held by **hydrogen bonds** between complementary bases: - **A pairs with T** – two hydrogen bonds - **G pairs with C** – three hydrogen bonds - This is **complementary base pairing**; the pairs are **DNA base pairs**. - DNA is a **double helix** – a three-dimensional shape. - **Function**: to hold or store genetic information – the instructions for growth and development of all organisms. ## RNA: Structure & Function - RNA is a **single-stranded** polynucleotide; chains are relatively short compared to DNA. - Contains **ribose** instead of deoxyribose; contains **uracil (U)** instead of thymine (T). - Sugar-phosphate bonds are **phosphodiester bonds** (covalent). - Examples: **mRNA** (transcript copy of a gene), **tRNA**, **rRNA**. - **Function**: to transfer the genetic code from DNA out of the nucleus to ribosomes in the cytoplasm for **translation**. ## Ribosomes - Small organelles, free in cytoplasm or attached to rough ER (eukaryotes). - Site of **protein synthesis**; they ‘read’ RNA to make polypeptides in **translation**. - Formed from **RNA and proteins**; the RNA is **rRNA**. - rRNA has enzymatic properties that catalyse formation of **peptide bonds** between amino acids. - Composed of a **small subunit** and a **large subunit**; not surrounded by a membrane. - **80S** ribosomes (60S + 40S) in eukaryotes; **70S** (50S + 30S) in prokaryotes, mitochondria and chloroplasts. ## The Origins of Research on the Genetic Code - DNA first observed in the 1800s by Friedrich Miescher (named ‘nuclein’, 1869). - Many doubted DNA carried the genetic code because of its relatively simple chemical composition (only simple repeating nucleotides). - By 1953, experiments confirmed DNA carried the genetic code; the triplet code is universal and degenerate. - DNA’s location protected in the nucleus provides security; proteins in the cytoplasm are susceptible to hydrolysis. - 1953: Watson and Crick confirmed the double-helix structure using Rosalind Franklin’s X-ray data. ## The Process of Semi-Conservative Replication - Before a cell divides, DNA is copied so each daughter cell receives a full copy. - **Semi-conservative replication**: each new DNA molecule has one original strand and one newly synthesised strand. - Ensures genetic continuity; essential for replacing body cells and for growth and development. - Occurs during the **S phase of interphase**. - Steps: 1. **Helicase** unwinds the double helix by breaking hydrogen bonds between base pairs. 2. Two single polynucleotide strands act as templates. 3. Free nucleotides are attracted to exposed bases by base pairing. 4. **DNA polymerase** joins new nucleotides by catalysing condensation reactions. 5. Original and new strands join via hydrogen bonding between base pairs. - Free nucleotides exist as **nucleoside triphosphates** (activated nucleotides); extra phosphate groups provide energy. DNA polymerase removes two phosphate groups from each nucleotide to release energy used to form phosphodiester bonds. ## Calculating the Frequency of Nucleotide Bases - A pairs with T; C pairs with G. - Therefore frequency of adenine = frequency of thymine; frequency of cytosine = frequency of guanine. - Worked example: if 28% of bases are cytosine, then 28% are guanine (total 56%). This leaves 44% for A and T, so 22% adenine and 22% thymine. ## The Watson Crick Model - Watson and Crick proposed **semi-conservative replication**: each new DNA molecule contains one original and one newly synthesised strand, based on complementary base pairing. - The **conservative model** suggested the original DNA remains intact and the new DNA is made entirely of new strands. - **Meselson and Stahl** tested the theories using bacteria grown in heavy nitrogen (¹⁵N) then light nitrogen (¹⁴N). - After one round of replication, all DNA molecules contained both heavy and light nitrogen and settled in the middle of the centrifuge tube – supporting semi-conservative replication. - If conservative replication had occurred, original DNA would settle at the bottom and new DNA at the top. - Ratio of ¹⁵N:¹⁴N goes from 1:1 after first round, to 3:1 after second, to 7:1 after third. - The experiment proved Watson and Crick’s theory correct. - Evaluation: method could be replicated, well-controlled, produced observable bands, disproved conservative and dispersive models. However, only tested one organism (E. coli) and didn’t show how enzymes like helicase and polymerase work.

Nucleotide: The repeating unit of DNA and RNA, formed from a pentose sugar, a nitrogen-containing organic base and a phosphate group.
Phosphodiester bond: The bond formed by a condensation reaction between the phosphate group of one nucleotide and the pentose sugar of the next; it consists of a phosphate group and two ester bonds.
Sugar-phosphate backbone: The chain of alternating phosphate groups and pentose sugars produced by many phosphodiester bonds in DNA or RNA.
Antiparallel: The arrangement of the two DNA polynucleotide strands running in opposite directions (one 5′→3′, the other 3′→5′).
Complementary base pairing: The specific pairing of bases: adenine with thymine (two hydrogen bonds) and guanine with cytosine (three hydrogen bonds).
Double helix: The three-dimensional shape formed by DNA molecules, consisting of two antiparallel polynucleotide strands held together by hydrogen bonds.
Semi-conservative replication: The process of DNA replication where each new DNA molecule contains one original strand and one newly synthesised strand.
Helicase: The enzyme that unwinds the DNA double helix by breaking the hydrogen bonds between the base pairs on the two antiparallel polynucleotide strands.
DNA polymerase: The enzyme that synthesises new DNA strands from the two template strands by catalysing condensation reactions to form the sugar-phosphate backbone and removing two phosphate groups from each nucleotide to release energy.
Ribosome: A small organelle that is the site of protein synthesis; formed from RNA and proteins, composed of a small and a large subunit, and not surrounded by a membrane.
rRNA: Ribosomal RNA; a specific type of RNA that forms part of the structure of ribosomes and has enzymatic properties that catalyse the formation of peptide bonds between amino acids.
Pentose sugar: A sugar with five carbon atoms; deoxyribose and ribose are both pentose sugars.
Polynucleotide: A polymer made up of many nucleotides joined together in a long chain; DNA and RNA are polynucleotides.
Nucleoside triphosphates: Activated nucleotides that exist as free nucleotides in the nucleus; the extra phosphate groups activate the nucleotides and provide energy for DNA replication.
Meselson and Stahl: Two scientists who tested theories of DNA replication using bacteria grown in heavy nitrogen (¹⁵N) followed by growth in light nitrogen (¹⁴N); their results supported the semi-conservative model.

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