Fundamentals of nucleic acids

Where is DNA contained

DNA is contained in chromosomes, which are found in the nucleus of most cells

what is genetics

the study of heredity and the variation of inherited characteristics/ the genetic properties or features of an organism

why are genetics important for pharmacists

helps choose medication

helps optimise drug dosage

what is gene therapy

Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patients cells

what is pharmacogenetics

pharmacogenetics is the application of genetic analysis to predict drug response, efficacy and toxicity

what are uses of pharmacogenetics

Drug discovery and development - identifying potential responders and non responders to a drug at an early stage using genetic variants that are markers of drug efficacy

Prescribing - Genetic test could function as an efficient factor in determining the best choice of drug for a given patient and condition based on their individual genotype

examples of genes go wrong

phenylketonuria - body cant break down phenylalanine which builds blood in brain, is treated with low protein die, can lead to learning difficulties, behaviour difficulties, epilepsy

neurofibromatosis - tumours grow along nerves, usually non cancerous

Epidermolysis bullosa - skin becomes fragile, friction can cause blisters

2 types of Nucleic acids

DNA - deoxyribonucleic acid , found in the nucleus with small amounts of mitochondria

RNA - Ribonucleic acid - found throughout the cell

Strcutures of Nucleic acids

DNA and RNA are nucleic acids

condensation polymers of nucleotides

nucleotides are composed of pentose, phosphate group, nitrogenous base

For DNA and RNA carbons run from 1- 5 prime

Adenine and guanine are what type of bases

thymine cytosine and uracil are what bases

double ring purines

single ring pyrimidines

nucleotides run in what direction

3 to 5 prime

what are the major and minor grooves of the DNA double helix

major groove= where the sugar phosphate backbones are further apart

minor groove - where they are closer together

this is important as many drugs interact with major or minor grooves

Explain DNA as a molecule of heredity

Frederick griffith 1928- found something from dead virulent cells could transform non virulent cells into virulent cells

Avery, MacLeod and MCCarty 1944- their experiments showed that DNA was the transforming factor

DNA vs RNA

DNA = double stranded, has deoxyribose, thymine

RNA= single stranded, ribose, uracil

DNA features

joined by hydrogen bonds

run antiparallel

A pairs with T

G pairs with C

mRNA function

generated during transcription

transcribed from DNA in nucleus

its sequence is complementary to DNA sequence

carries genetic info from nucleus to cytoplasm

tRNA function

helps decode mRNA sequence into a protein

binds with anticodon to codon on mRNA

carry specific amino acids at 3 end

rRna

part of ribosome

ribosomes composed of rRna and proteins

crucial for translation

3 proposed mechanisms for DNA replication

Conservative - original DNA is preserved and completely new molecule is synthesised

Semi - Conservative - Each new DNA Molecule consists of one strand from original molecule and one newly synthesised strand

Dispersive - The original DNA molecule is broken into fragments, and new DNA is synthesised through these fragments resulting in daughter molecules with interspersed old and new DNA

what was Meselon Stahl Experiment

DNA replication is semi - conservative experiment in 1958

grew bacteria containing light and heavy nitrogen (n14, n15)

Process of DNA Replication

Initiation - Proteins bind to the DNA at an origin of replication and begin to unwind double helix, creating a replication fork

elongation:

• Helicase unwinds the DNA double helix

• Single strand binding proteins - stabilise the unwound single strands

• Primase - synthesise a short RNA primer, providing a free 3 to hydroxl group for polymerase to start with

• DNA polymerase - reads template in 3 to 5 direction but synthesises the DNA strand in 5 to 3 direction by adding complementary nucleotides

• Due to anti parrel nature of DNA and 5 to 3 synthesis direction, replication occurs differently on two strands:

leading - strand - synthesise continuous 5 to 3 direction towards the replication fork

lagging strand - synthesised discontinuously in short fragments called okazaki fragments each requiring its own RNA primer

DNA polymerase - removes the RNA primers and replaces them with DNA nucleotides #

Ligase - seals okazaki fragments by forming phosphodiester bonds

3. Termination - Replication halts at specific termination sites

key concepts and terminology of transcription

template strand - one strand of the DNA molecule that is copied by RNA polymerase

RNA polymerase - the enzyme responsible for transcription. It reads the DNA template strand in the ‘3 to 5’ direction and d synthesise RNA in 5' to 3’ direction

Promoter - A DNA sequence that signals the start of a gene. Determines where transcription begins and which strand is used as a template

Terminator - A DNA sequence that signals the end of transcription

sense strand (coding strand) - The DNA strand has the same sequence as RNA transcript

Antisense strand: The DNA strand that is Complementary to the RNA transcript and serves as a template for transcription

stages of Transcription

initiation - RNA polymerase binds to promoter region on DNA unwinding small section of double helix

Elongation - RNA polymerase moves along the template strand synthesising the RNA molecule by adding complementary ribonucleotides. The DNA unwinds.

Termination - RNA polymerase reaches terminator sequence, it detaches from DNA, newly synthesised RNA and RNA polymerase is released, dna double helix forms

mRNA processing in Eukaryotes

Exons - coding regions that are eventually translated into protein

Introns - non coding regions that are removed during processing

1.) 5’ Capping - Modified Guanine is added to 5’ end of primary transcript (cap protects mRNA from degradation)

2.) 3’ Polyadenylation - a tail of multiple adenine nucleotides is added to 3’ end of pre - MRNA (enhances stability)

3) Splicing - Introns are removed from primary transcript and exons are joins together (ensures coding sequences are present in mature mRNA)

Central Dogma of molecular biology

what is the genetic code

set of rules by which info encoded in DNA or RNA translated into proteins

4 bases and 20 amino acids
• Code coded by 1 base = 4 combinations
• Code coded by 2 bases = 16 combinations
• Code coded by 3 bases = 64 combinations

Characteristics of Genetic Code

Specificity - Each codon specifies only one amino acid eg; AUG always codes for methionine

Universality - The genetic code is nearly universal, The same codons specify the same amino acids in most organisms, from bacteria to humans

Degeneracy - The genetic code is degenerate meaning most amino acids are specified by more than one codon.eg; arginine has 6 different codons

key players in translation

mRNA - carries DNA to ribosome

tRNA - recognises codon with anticodon

A Site - where tRNA binds

P - where tRNA holding the growing polypeptide

E site - where tRNA leave the ribosome

Stages of Translation

initiation - small ribosomal subunit binds to mRNA at binding site, tRNA recognises and binds to start codon on mRNA

Elongation - tRNA with anticodon complementary enters A site, peptide bond is formed between trna in A site and polypeptide chain attached to trna in p site, Ribosome moves one codon down, tRNA in A site moveds to p site and p site moves to e site and cycle repeats

Termination - when stop codon is encountered in A site translation terminates

4 protein structures

primary - sequence of amino acids in a polypeptide chain

secondary structure - folding of polypoeptide chain into alpha helices and beta sheets

tiertiary -three dimensional shape of single polypeptide chain, resulting from interactions between amino acids and side chains

Quaternary structure - arrangement of multiple polypeptide chains

misfolding of proteins can lead to what

abnormal protein aggregates eg: diseases

DNA Packaging: Histones and nucleosome

primary level of DNA packaging in eukaryotes involves wrapping DNA double helix around proteins called histones

Histones:

core histones:`H2A, H2B,H3,H4

Linker histone ; H1

histones are rich in basic amino acids

Nucleosome -eight histone molecules form a histone octamer

approximately 146 base pairs of DNA wrap around histone octamer forming nucleosome bead. This compacts the DNA

explain the terns, nucleosome, solenoid and chromatin

• Nucleosome: DNA wrapped around histones.

• Solenoid: coiled arrangement of nucleosomes.

• Chromatin: DNA and proteins that make up chromosomes.

orders of chromatin structure

1.) Naked DNA (dna double helix)

2.) Nucleosomes (10mm) - beads on a string

3.) Solenoid (30mm)

4.) Chromatin fibre

5,) metaphase Chromosome

euchromatin and heterochromatin

euchromatin: ]

uncoiled,decondensed

loose dna histone interaction

regions with high transcriptional activity

Histones are not methylated

eg:active brain stem cells

Heterochromatin:

condensed, tightly packed, transcriptionally inactive

regions with low or no transcriptional activity

histones are methylated

eg: :resting cells

histone modifications

• acetylation: Generally associated with gene activation (euchromatin). Acetylated chromatin is open and transcriptionally active.

• Deacetylation: Generally associated with gene repression (heterochromatin). Deacetylated chromatin is compact and transcriptionally repressed.

• Methylation: Can be associated with either activation or repression, depending on the specific amino acid residue and context. Heterochromatic regions often exhibit histone methylation.

chromosome structure

chromosome - single long DNA molecule containing a array of many genes

karyotype - full set of chromosomes digitally arranged and matched with homologous partners

FISH - a technique using flouorescent probes to identify specific DNA

Chromatids - duplicated chromosome consists of two identical sister chromatids joined at the centromere

Centromere - Region of chromosome that separates into two arms: short(p arm) long (q arm)

Telomeres - Protective caps at ends of chromosome

Telomeres and Ageing

• Telomere Shortening: With each round of DNA replication, telomeres shorten due to the "end replication problem" (DNA polymerase cannot fully replicate the lagging strand).

• Senescence: Eventually, telomere shortening reaches a critical length, leading to cellular senescence (a state of irreversible cell cycle arrest).

• Telomerase: Stem cells and tumour cells often express telomerase, an enzyme that can extend telomeres, allowing for potentially unlimited cell division

Key terms in gene regulation

Promoter - signals where transcription by RNA polymerase should start

Transcription factor - proteins that bind to specific DNA to regulate gene expression

in Transcription in bacteria what is a promoter sequence

promoter sequence - define the direction of transcription and indicate which DNA strand will be transcribed

transcription in bacteria

initiation - RNA polymerase recognises promoter and starts the transcription

Elongation - RNA strand grows

termination - RNA polymerase stops synthesis

Lac operon components and mechanism

repressor gene - codes for lac repressor protein

promoter - where RNA polymerase binds

Operator - the binding site for lac repressor protein

Strcutural genes - genes that encode enzymes for lactose metabolism

when lactose is absent lac repressor protein binds to operator blocking RNA polymerase and preventing transcription.

when lactose present. it binds to lac repressor protein causing it to change shape and detach from operator. This allows RNA polymerase to bind to promoter and transcribe and break down lactose

what happens in TRP Operon

low TRP levels - repressor protein is inactive and cant bind to operator, RNA polymerase can transcribe structural genes

High TRP levels - activated repressor binds to operator blocking RNA polymerase and prevents transcription

RNA polymerases involved in eukaryotic transcription

RNA Pol I - Transcribes ribosomal RNA

RNA Pol II Transcribes all protein coding genes

RNA Pol lll - Transcribes transfer RNA

promoter sequences in Eukaryotic transcription

Promoter - A DNA sequence that initiates transcription

TATA box - core promoter element that is essential for the binding of general transcription factors.

Regulatory sequences in Eukaryotic sequences

Enhancers - DNA sequences that can bind activator proteins to increase transcription

Silencers - DNA sequences that bind repressor proteins to decrease or inhibit transcription

Transcription factors

General TFs - required for transcription, binds to core promoter and helps form transcription initiation complex

Promoter specific TFs - bind to enhancers or silencers and can activate or repress transcription

Chromatin remodelling - Eukaryotic DNA is packed into chromatin, for transcription to occur chromatin must be opened to make DNA accesible to RNA polymerase and transcription factors

Levels of Gene regulation in Eukaryotes

1. Chromatin level - condensed chromatin (heterochromatin) is inaccessible however relaxed (euchromatin) allows transcription

2. Transcriptional control - Regulation of whether RNA is transcribed from DNA

3. mRNA processing - capping, splicing, polyadenylation

4. post transcriptional modification - regulation of RNA stability

5. Translational control - Regulation of whether translation of mRNA into protein occurs

6. Post translational Modification - modification of proteins after synthesis can alter activity or stability