Download Gene Expression and DNA Replication

Molecular Biology
Instructor: Prof. Dr. Fadel A. Sharif
Lecture 1
Nucleic Acid Structure and
Organization
Course Syllabus
Text: Molecular Biology Lecture Notes. Hansen B.
and Jorde L.B., 2002. Kaplan Inc. (Required)
Grades
Assignments: 15%
One Midterm exam worth 35%
Final exam: 50%
Topics
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Nucleic Acid Structure and Organization
DNA Replication and Repair
Transcription and RNA Processing
The Genetic Code, Mutations, and Translation
Genetic Regulation
Recombinant DNA
Genetic Testing
What is Molecular Biology?
"Study of the synthesis, structure, and function of
macromolecules (DNA, RNA, and protein) and their
roles in cells and organisms"
OVERVIEW: THE CENTRAL DOGMA OF
MOLECULAR BIOLOGY
• An organism must be able to store and preserve its
genetic information (stored in the base sequence of DNA
molecules)
• pass that information along to future generations, and
• express that information as it carries out all the processes
of life.
• Classically, a gene is a unit of the DNA that encodes a
particular protein or RNA molecule.
The central dogma of Molecular Biology
What genes do?
-Genes replicate/
-genes direct RNA  protein synthesis/
-genes accumulate mutations
Gene Expression and DNA Replication
• Transcription, the first stage in gene expression, involves transfer
of information found in a double-stranded DNA molecule to the
base sequence of a single-stranded RNA molecule. If the RNA
molecule is a messenger RNA, then the process known as
translation converts the information in the RNA base sequence to
the amino acid sequence of a protein.
• When cells divide, each daughter cell must receive an accurate
copy of the genetic information. DNA replication is the process in
which each chromosome is duplicated before cell division.
Gene Expression
Comparison of Gene Expression and DNA Replication
Gene Expression
DNA Replication
- Produces all the proteins an
organism requires
- Transcription of DNA: RNA copy
of a small section of a
chromosome (average size of
human gene 104 - 105 nucleotide
pairs)
-Translation of RNA: protein
synthesis
- Occurs throughout interphase
- Transcription in nucleus
- Translation in cytoplasm
- Duplicates the chromosomes
before cell division
- DNA copy of entire chromosome
(average
size
of
human
chromosome,
108
nucleotide
pairs)
- Occurs during S phase
- Replication in nucleus
Lecture 2
The concept of the cell cycle
Can be used to describe the timing of some events
in a eukaryotic cell.
• The M phase (mitosis) is the time in which the
cell divides to form two daughter cells.
• Interphase is the time between two cell divisions
or mitoses. Gene expression occurs throughout all
stages of interphase.
The Eukaryotic cell cycle
Interphase is subdivided as follows:
• G1 phase is a period of cellular growth preceding DNA
synthesis. Cells that have stopped cycling, such as
muscle and nerve cells, are said to be in a special state
called Go.
• S phase is the period of time during which DNA
replication occurs. At the end of S phase, each
chromosome has doubled its DNA content and is
composed of two identical sister chromatids linked at the
centromere.
• G2 phase is a period of cellular growth after DNA
synthesis but preceding mitosis. Replicated DNA is
checked for any errors before cell division.
Reverse transcription
• Produces DNA copies of an RNA, is more commonly
associated with life cycles of retroviruses, which
replicate and express their genome through a DNA
intermediate (an integrated provirus).
• Also occurs to a limited extent in human cells, where it
plays a role in amplifying certain highly repetitive
sequences in the DNA
• Telomerase has reverse transcriptase activity.
Basic Structure of Nucleic Acids
• Repeating nucleotides linked by phosphodiester bonds
• DNA “backbone” = sugar (deoxyribose) + Phosphate
• RNA “backbone” = sugar (ribose) + Phosphate
Pentose Sugar
RNA
DNA
Negative (-) charge on Phosphate Units Give
DNA/RNA a Uniformly (-) negative charge !!!
Types of Nucleotides Based on Number of Phosphates
• Nucleoside = Base + Sugar
• Nucleotide = Base + Sugar + Phosphate (mono, di, tri)
What would the names be for ribose nucleotides?
Nitrogenous Bases Provide “Genetic Information”
• Order of bases in DNA is the “SEQUENCE”
• Two general types of nitrogenous bases
Purines (two rings)
Pyrimidines (one ring):
Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
Uracil (U) – only RNA
- Other purine metabolites, not usually found in nucleic acids,
include xanthine, hypoxanthine, and uric acid.
Nomenclature of the Ribonucleotide Series of
Compounds
Linkages to different carbon atoms in sugar:
• 1`–5` numbering is based on organic nomenclature
• This defines orientation of nucleic acids, 5` and 3` ends
• Two nucleotides are linked by a 5`, 3`-phosphodiester bond
5` Carbon linked to “Upstream”
Phosphate
3` Carbon linked to
“downstream” Phosphate
Nucleotides Base Pair By Hydrogen bonds
• Hydrogen bonds (H-bonds) form between purine and pyrimidine
bases in DNA and RNA
• Nitrogenous bases pair with complementary bases:
A pairs with T (A-T) = 2 H-bonds
A pairs with U (A-U) = 2 H-bonds (in RNA)
G pairs with C (G-C) = 3 H-bonds (stronger pairing)
H-bonds:
- H atom is shared between two atoms
-Typically between oxygen (O) and nitrogen (N) atoms
- Bond is strongest when three atoms are in a line (O-H-N)
- Strength ranges from ~ 2–6 kcal/mol (energy unit/bp)
Pairing Between Complementary Bases
Promotes Formation of Double-Stranded DNA
“Chargaff Rule” for Base Pairing
Using Chargaff's Rules
• In dsDNA (or dsRNA)
• % A = % T (% U)
• %G =%C
• % purines = % pyrimidines
A sample of DNA has 10% G; What is the %T?
Nucleic Acids
• Nucleotide is linked by 3',5' phosphodiester bonds
• Have distinct 3' and 5' ends, thus polarity
• Sequence is always specified as 5'3'
• Most DNA occurs in nature as a right-handed doublehelical molecule known as Watson-Crick DNA or BDNA.
• The hydrophilic sugar-phosphate backbone of each
strand is on the outside of the double helix. The
hydrogen-bonded base pairs are stacked in the center of
the molecule.
• There are about 10 base pairs per complete turn of the
helix.
• A rare left-handed double-helical form of DNA that
occurs in G-C-rich sequences is known as Z-DNA. The
biologic function of Z-DNA is unknown, but may be
related to gene regulation.
The B-DNA
Double Helix
A-form
B-form
Z-form
• B-form: sodium salt of DNA at very high relative humidity (92%)
• A-form: sodium salt of DNA in reduced humidity (75%). E.g.,
– DNA/RNA hybrid
– dsRNA
• Both A- & B-forms are right-handed
• Z-DNA: left-handed, assumed by dsDNA containing strands of
alternating purines & pyrimidines e.g., poly[dG-dC].[dG-dC]
Different ways to represent DNA sequence
5`-pApCpGpT-3`
5`-ACGT-3`
3`-TGCA-5`
5`-ACGT-3`
dsDNA Can be Denatured and Renatured
– Denaturing = “melting” = breaking H-bonds
– Renaturing = “annealing” = reforming H-bonds
Ways to Denature:
–
–
–
–
High heat: ~ 95°C will “melt” most DNA
High pH: Concentrated OH-will break H-bonds
Chemicals: Formamide, Urea, DMSO & Formaldehyde
Lowering salt conc. of DNA solution aids denaturation
Renature:
– Cool (room temperature) and given time (min-hr)
Melting and Renaturing DNA
The melting temperature (tm) for A given DNA is
when half of the DNA is single-stranded
Figure 1-1-10. DNA Melting Point
Tm Curve
% G + C Versus Tm
DNA and RNA Absorb Ultraviolet (UV) light:
• Peak absorbance is at 260 nm wavelength
• Damaging UV light (breaks DNA)
• DNA & RNA are quantified using this property
Hyperchromic effect: when two strands
separate the absorbance rises 30-40%.
Hypochromicity: caused by the fixing of the
bases in a hydrophobic environment by
stacking, which makes these bases less
accessible to UV absorption.
UV absorption of nucleotides
DNA & RNA have constant UV Absorbance:
Peak absorbance is at 260 nm wavelength
Absorbance at 260 nm (A260) is constant:
• Double-stranded DNA (dsDNA):
• A260 of 1.0 = 50 ug / ml
• Single-stranded DNA (ssDNA):
• A260 of 1.0 = 30 ug / ml
• Single-stranded RNA (ssRNA):
• A260 of 1.0 = 40 ug / ml
Determine dsDNA concentration with A260:
• For DNA:
1) Determine A260 with spectrophotometer
2) Use A260 to calculate concentration:
Constant: A260 of 1.0 = 50 ug / ml dsDNA
• For Example:
A260 was determined to be 0.1
0.1 x 50 ug / ml = 5 ug / ml dsDNA
Reuniting the Separated DNA Strands
Renaturation: when 2 separated strands, under proper
conditions, come back together again.
Annealing: base paring of short regions of
complementarity within or between DNA strands.
(example: annealing step in PCR reaction)
Hybridization: renaturation of complementary sequences
between different nucleic acid molecules. (examples:
Northern or Southern hybridization)