Molecular Biology Instructor: Prof. Dr. Fadel A. Sharif
Nucleic Acid Structure and Organization Lecture 1
Course Syllabus Grades Assignments: 15% One Midterm exam worth 35% Final exam: 50% Text: Molecular Biology Lecture Notes. Hansen B. and Jorde L.B., 2002. Kaplan Inc. (Required)
Topics • 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.
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.
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.
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 BasesPromotes 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 double-helical molecule known as Watson-Crick DNA or B-DNA. • 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.
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)
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.
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 spectrophotometer2) Use A260 to calculate concentration:Constant: A260 of 1.0 = 50 ug / ml dsDNA • For Example:A260 was determined to be 0.10.1 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)