Nucleic Acids Flashcards
2 functions of nuclei acids
•Function primarily as informational molecules
•For the storage and retrieval of information regarding the primary sequence of polypeptide
2 types of nuclei acids
•Two types
(1) Deoxyribonucleic acid (DNA)
(2) Ribonucleic acid (RNA)
Describe nucleotide structure ( DNA and RNA are poynucleotides )
•Each nucleotide contains
1. A 5-carbon pentose sugar (ribose/ deoxyribose )
2. A nitrogenous base
3. One or more phosphate groups. ( gives negative charge to the nucleic acid. link the sugar on one nucleotide onto the phosphate of the next nucleotide to make a polynucleotide.)
4 bases in DNA
• Thymine • Adenine • Cytosine • Guanine
4 bases in RNA
• Uracil
• Adenine • Cytosine • Guanine
What are 2 types of bases and their examples
• DNA and RNA contain two types of bases
- Purine bases (two ring shaped molecules joined
together) - •Adenine (A) •Guanine (G) - Pyrimidine bases (a single ring) - •Cytosine •Uracil•Thymine
What is nucleoside and 2 examples
•Purine or pyrimidine base covalently attached to a sugar molecule
•RNA – Ribonucleoside
•DNA – Deoxyribonucleoside
What is nucleotide and 2 examples
(Base + sugar + phosphate)
•RNA – Ribonucleotide
•DNA – Deoxyribonucleotide
What is Dinucleotide
• Two nucleotides are joined by 3’5’phosphodiester bond
What is Polynucleotide
•Made up of many different nucleotides covalently joining with one another by 3’-5’ phosphodiester bonds.
•Each chain has polarity;
has 5’phosphate end 3’hydroxyl end.
DNA double helix details
In 1953
Watson and Crick
discovered the
3- dimensional model
of the DNA structure
Characteristic features of DNA double Helix
• Two DNA strands are wound around each other to form the double helix
• Run in opposite directions – Anti-parallel (one running from 5’ to 3’ and the other from 3’ to 5’)
- Two strands are twisted together around a common axis
- Right handed (spirals away, clockwise direction )
- 10 nucleotides in each strand per 360 turn of helix
•Hydrophilic sugar and phosphate groups form the backbone of the double helix.
•Hydrophobic bases are stacked inside the double helix.
•Bases form hydrogen bonds with one another.
•A purine forms a hydrogen bond with a pyrimidine
•A – T (2 H-bonds)
•G – C (3 H-bonds)
What is Chargaff’s Rule?
• Adenine and Thymine always join together
• Cytosine and Guanine always join together
COMPLEMENTARY BASE PAIRING
This is because there is exactly enough room for one purine and one pyramide base between the two polynucleotide strands of DNA.
What are 3 types of DNA structures
• B type (most common)
• A type (can form under certain in vitro conditions )
• Z type (can form under certain in vitro conditions )
Describe B type
• right-handed
•10 base pairs per turn. (1 bp is 0.34 nm)
•The intertwined strands make two grooves of different widths, referred to as the
major groove and the minor groove,
Helix width is 2 nm
What are major grooves & minor grooves
•Within the groove, the base pairs are exposed.
•which may facilitate binding with specific regulatory proteins/transcription factors.
•Site for binding of intercalating agents (e.g. Dactinomycin, a chemotherapeutic agent)
What is an intercalating agent and example
Distorts the helix and inhibit transcription
Ex. Dactinomycin, a chemotherapeutic agent)
G-C Content
Generally GC~50%, but extremely variable
nDistribution of GC is not uniform in genomes
CONSEQUENCES OF GC CONTENT
GC slightly denser:
Higher GC DNA moves further in a gradient
more stable DNA,
i.e. the strands do not separate easily.
5 Forces that help to form the DNA double helix
- Rigid phosphate backbone (phosphodiester bond )
- Base stacking interactions between bases (Van der Waals interactions)
- Hydrophobic interactions (highly negative phosphate backbone vs. non-polar bases facing interior)
- Hydrogen bonding (not the most energetically significant component) - comp paring
- Ionic interactions - salt stabilizes the duplex form
of DNA shielding of the phosphate backbone (e.g. Mg2+)
What is melting temperature (Tm)
T at which 1⁄2 the DNA sample is denatured
Denaturation & Renaturation
The two strands of the double helix separate reversibly at high temperatures
If the temperature is lowered, the strands renature (reanneal)
The rate of re-association is inversely proportional to the complexity of the DNA.
4 Important applications of PCR
-Critical importance in any technique that relies on complementary base pairing
-use temperature or denaturants.
nPCR
nSouthern blots
nNorthern blots
nDNA-DNA
hybridization
EXPLAIN PCR
I. DENATURATION ~95°C
DNA STRANDS SEPARATE
- ANNEALING
PRIMERS ADHERE T O DNA STRANDS
~55°C - EXTENSION ~72°
TAQ POLYMERASE .g,COMPLEMENTARY STRAND
6 Factors Affecting Tm
G-C content of the sample
Presence of intercalating agents (anything that disrupts H-bonds or base stacking)
Salt concentration
pH (extreme pH disrupts H-bonds) nLength
Denaturants
Chromatin in DNA packaging in chromosomes
Chromatin
• Chromatin is the chromosomal material in the nuclei of eukaryotic cells.
• Chromosomes become visible only during the cell division.
Consists of
• dsDNA
• Histones; most abundant chromatin proteins • Non-histone proteins ( most are acidic)
• RNA (small quantity)
Histones in DNA packaging in chromosomes
• A set of small basic proteins
• Basic amino acids (positively charged) abundant in
histone proteins are Histidine, lysine, and Arginine.
• Form ionic bonds with negatively charged
phosphate groups.
• They bind with DNA and stabilize DNA structure.
• Also, participate in gene regulation.
4 levels of packaging of genome ?
•Level I - the B form of the double helix ( 2 nm short region of DNA double helix)
•Level II - The nucleosome ( 11 nm beads on a string form of chromatin , like thread loosely around a spool )
(beads-on-a-string structure)
•Level III – Chromatin fiber ( 30nm chromatin fibre of packed nucleosome like bundled thread )
•Level IV - Radial loop domain (300 nm protein scaffold that together make 700 nm chromatin )
3 brief facts on dna packaging
• The genomic DNA of a eukaryotic cell is in the nucleus.
• The human genome consists of about 3 x 109 DNA base pairs (about 2 m in length).
• Eukaryotic nuclear DNA is tightly packaged in chromosomes.
Describe Level II - The nucleosome
• A complex of DNA and histone proteins
• Eight core histone proteins aggregate to form the
histone core (octamer complex) of the nucleosome.
• Nucleosome - DNA wrapped two times around the
octamer core/octamer complex.
• The DNA fragment is negatively supercoiled in the left-handed direction.
• Nucleosomes are linked together by “linker ‘ DNA.
• Produces the “beads–on–a–string” appearances seen in electron micrographs.
Describe Level III - chromatin fiber and 2 types
• It involves the coiling of nucleosomes into
chromatin fiber.
• This level of condensation involves the interaction of another histone protein
Solenoid or zigzag
Explain Level IV –Radial loop domain
• Involves a new structure called the chromosome scaffold.
• Scaffold - made up of several protein (non histone protein ) components including lamins and topoisomerases.
• 30 nm chromatin fiber is anchored to the chromosome scaffold and is organized into Radial Loop Domains.
7 Biomedical importance of DNA
• Storage of genetic information in humans.
To understand the molecular basis of diseases. E.g. genetic disorders.
• For the diagnosis of diseases; Molecular biology techniques like Southern blotting, PCR, gene sequencing
• Molecular targets of therapy; e.g. antibiotics, anti- cancer drugs, gene therapy.
• Genetic engineering; manufacturing of drugs/vaccines
• To predict how someone responds to a drug; pharmacogenomics.
• Medico-legal applications; DNA fingerprinting.
How many levels of organisation in RNA structure
3
Describe 3 levels of organisation
Primary:
Covalent bonds
Secondary/Tertiary Non-covalent bonds
• H-bonds (base-pairing)
• Base stacking
Where are RNA structures found
in both nucleus and cytosol.
2 functions of RNA
•Needed to convert DNA information into
polypeptide sequences.
•In some viruses, RNA serves as the primary database with no DNA involvement.
4 classes of RNA
•Messenger RNA (mRNA)
•Transfer RNA (tRNA)
•Ribosomal RNA (rRNA)
•Small RNA
Describe mRNA. Structure . Where synthesised. Function
• Single-stranded.
•Serves as a template for protein synthesis. (•carries genetic code to the site of protein
synthesis(ribosome))
•Synthesized in the nucleus and passes to the cytoplasm.
•Higher molecular weight compared to other types of RNA.
•Short half-life.
Describe tRNA structure. 2 functions .
•Smallest RNA.
•Contains intra-strand/intra-chain base
pairing.
•serves as an “adaptor” molecule that carries its specific amino acid
•At least 20 tRNA molecules in every cell, at least one corresponding to each of the 20 amino acids.
•Involved in the translation process.
•contain a high percentage of unusual bases (eg. dihydrouracil)
3 functions of rRNA
•Contributes to structure and function of ribosomes.
•Necessary for ribosomal assembly and binding of mRNA to ribosomes.
•Performs peptidyl transferase activity (ribozymes)
4 species of small RNA and functions
Species of Small RNA
• Small nuclear RNA (snRNA) > mRNA processing
• Small nucleolar RNA (snoRNA) > rRNA processing
• Micro-RNA (miRNA) > regulatory RNA
• Small Interfering RNA (siRNA) > Regulatory RNA
4 groups of RNA functions
• Storage/transfer of genetic information
• Structural
• Catalytic
• Regulatory
RNA in viruses and examples
• many viruses have RNA genomes
single-stranded (ssRNA)
e.g., retroviruses (HIV), SARS-CoV-2
double-stranded (dsRNA) e.g. Rota virus
Structural role of RNA
rRNA, which is major structural component of ribosomes.
Catalytic role of RNA
•rRNA has peptidyl transferase activity
(Enzymatic activity responsible for peptide bond formation between amino acids in growing peptide chain)
• Also, many small RNAs are enzymes. “ribozymes”
Regulatory role of RNA
• miRNA forms imperfect RNA-RNA duplexes (multiple mRNA targets)
• siRNA usually forms perfect RNA-RNA hybrids (specific mRNA targets)
• both inhibit gene expression (gene- silencing effects)
•Potential therapeutic agents for cancers and infections.
- Biomedical importance of RNA
•Storage of genetic information in
pathogens.
• to understand the pathophysiology and the genetic basis of diseases. E.g. gene expression studies
•RNA therapeutics; anti-cancer therapy
•Prevention of disease; mRNA vaccines
(Pfizer/Moderna)
•For the diagnosis of diseases; reverse- transcriptase(RT)-PCR, Northern blotting
What is genome
• total genetic information carried by a cell/organism.
What is gene
• A unit of heredity.
• It is a region of DNA that is responsible for a
particular characteristic.
• Only about 10% human DNA contain genes.
• Every gene in humans/animals has a specific sequence.
What is gene sequence
The order of the nucleotide arrangement. Ex: ACCGTATC
3 types of genetic variants
Nuclear sequence-level and structural variants
Mitochondrial variants
3 types of Nuclear sequence-level variants
• Single nucleotide variants
• Small insertions
• Small deletions
5 Nuclear structural variants
• large insertions,
• Large deletions
• copy-number variants
• Inversions
• Translocations
What are epigenetic modifications abd their importance (5 diseases )
•Do not involve altereation of DNA coding sequence.
• Reversible
•Inhereted/transgenerational (but may be
gradualy lost over several generations)
• Involve
Enzymes; DNA methyltransferase
Smal-RNAs; microRNA, siRNA
•Important in tumorigeneisis/carconogenesis, neurodegenrative diseases, autism spectrum disorder, autoimmune diseases
3 types of epibenthic modification ( with further)
•DNA methylation
•Histone modifications • Methylation
• Acetylation
• Phosphorylation
•RNA-mediated modifications (e.g. gene silencing effects of miRNAs and siRNAs)
MCQ 1
Regarding human dsDNA,
A. Amount of adenine equals the amount of thymine.
B. Total amount of purines equals total amount of pyrimidines.
C. is a right-handed helix
D. Thermostability is determined by the phosphodiester bonds.
E. Packaginginvolvenon-histoneproteins.
TTTFT
MCQ 2
Regarding histone proteins;
A. are negatively charged at physiological pH.
B. Modulate gene expression.
C. Facilitate packing of nucleosomes into a solenoid.
D. forms the chromosome scafold.
E. Are subject to covalent modifications.
FTTFT
MCQ 3
Ribonucleic acids
A. are polymers
B. cannot be separated by electrophoresis
C. Has regulatory functions
D. is not found in chromatin
E. Ribozymes are examples
TFTFT
MCQ 4
The following are epigenetic changes
A. single nucleotide variant in a nuclear gene
B. A deletion in a mitochondrial gene
C. Chromosomal translocation
D. DNA demethylation
E. Histone acetylation
FFFTT
Regarding small RNAs;
A. are non-coding RNAs
B. tRNA is an example
C. Have gene silencing effects
D. Form hybrids with other RNAs
E. InvolvedinRNAprocessing
TFTTT
MCQ 6
The following factors affect melting temperature of DNA
A. pH
B. Presence of denaturants
C. Nucleotide composition of DNA
D. Length of DNA
E. Presence of magnesium ions
TTTTT
MCQ 7
Denaturation of DNA
A. is irreversible
B. A step in polymerase chain reaction
C. Involves breaking of phosphodiester bonds
D. Occurs in-vitro
E. Induced by chemicals
FTFTT
MCQ 8
Regarding DNA double helix
A. B-type is predominant in vivo
B. Has purine-purine hydrogen bonds
C. Is a polycation
D. Stabilized by bas-stacking interactions
E. Bases are exposed at the major groves
TFFTT
MCQ 9
The components of nucleosome
A. histones
B. rRNA
C. Linker DNA
D. Chromosome scafold
E. Radial looped domains
TFFFF
MCQ 10
Regarding epigenetic modifications
A. irreversible
B. inherited
C. Alters coding DNA sequence
D. Involve covalent modifications by enzymes
E. Do not alter gene expression
FTFTF.
