BI1014 - nucleic acids and edge of life

significance of DNA

- DNA (deoxyribonucleic acid) is the molecule that carries the genetic instructions

- It serves as the hereditary material in almost all organisms.

- DNA contains genes, which are specific sequences that code for proteins and functional RNA

- DNA ensures stability of genetic information and enables variation through mutation, supporting evolution

role of DNA

Protein synthesis (via transcription and translation)

Cell division and replication

Inheritance of traits from one generation to the next

structure of DNA

- DNA is made of nucleotides, which are the basic subunits

- The phosphate and sugar form the backbone of the DNA strand.

- The bases project from the backbone and are involved in base pairing.

structure of nucleotides

A phosphate group

A deoxyribose sugar (5-carbon sugar)

A nitrogenous base (one of four types):

nitrogenous bases

Adenine (A)

Thymine (T)

Cytosine (C)

Guanine (G)

formation of double stranded DNA

- DNA is double-stranded: two polynucleotide chains run in opposite directions (antiparallel).

- The strands are held together by hydrogen bonds between complementary nitrogenous bases

- Complementary base pairing ensures accuracy in replication and transcription.

- The formation of the double strand results in a stable helical structure.

base pairs

Adenine (A) pairs with Thymine (T) via 2 hydrogen bonds

Guanine (G) pairs with Cytosine (C) via 3 hydrogen bonds

key features of double helix

Antiparallel strands (one 5′ → 3′, the other 3′ → 5′)

Major and minor grooves: Allow proteins to interact with bases without unwinding DNA.

10 base pairs per turn of the helix (in B-DNA)

Diameter of helix: ~2 nanometers

key features of double helix

- DNA typically adopts a right-handed double helix known as B-DNA.

- The double helix is compact and stable, ideal for long-term storage of genetic information.

- The structure allows for easy unwinding during replication and transcription due to hydrogen bonding.

B-DNA

- Most common form under physiological conditions.

- Right-handed helix with ~10 base pairs per turn.

- Wide major groove and narrow minor groove

A-DNA

- Forms in dehydrated conditions or in DNA-RNA hybrids.

- Right-handed helix, more compact than B-DNA.

- ~11 base pairs per turn; deep major groove, shallow minor groove.

Z-DNA

- Left-handed helix with a zigzag backbone.

- Forms in regions rich in alternating purine-pyrimidine sequences (e.g., GC repeats).

- May play a role in gene regulation and chromatin remodelling

triplex and quadruplex DNA

Unusual structures formed in specific sequences or under special conditions

triplex DNA

Three-stranded structures often found in regulatory regions

other structural features

- Hairpin loops -> in palindromic sequences

- Cruciform structures -> from inverted repeats

- Triple-stranded DNA (triplex)

- G-quadruplexes

G-quadruplexes

- Found in telomeres and regulatory regions

- Involves stacked guanine tetrads

denaturation

separation of double-stranded DNA into single strands

key factors

- temperature

- GC content

- salt concentration

- pH levels

- chemical agents

temperature

- Higher temps → denaturation

- Melting temperature (Tm): temp at which 50% of DNA is denatured

GC content

- GC pairs (3 H-bonds) more stable than AT pairs (2 H-bonds)

- Higher GC content → higher Tm

salt concentration

- Stabilizes DNA by shielding negative phosphate charges

- Lower salt → easier denaturation

pH content

Extreme pH (acidic or basic) disrupts hydrogen bonding

chemical agents

Urea and formamide disrupt H-bonds → lower Tm

sugar

DNA: deoxyribose.

RNA: ribose (extra hydroxyl group at 2').

bases

DNA: adenine (A), guanine (G), cytosine (C), thymine (T).

RNA: uracil (U) replaces thymine.

strandedness

DNA: double-stranded (usually).

RNA: single-stranded (but may fold into secondary structures).

stability

DNA: more stable due to fewer reactive groups.

RNA: less stable, especially in alkaline conditions.

function

DNA: genetic information storage.

RNA: diverse roles (mRNA, tRNA, rRNA, regulatory RNAs).

location

DNA: mainly in nucleus (eukaryotes).

RNA: nucleus and cytoplasm.

genome

the complete set of genetic material in an organism

plasmid

small, circular DNA molecules separate from the chromosomal DNA

operon

a cluster of genes under the control of a single promoter (common in prokaryotes)

gene

a DNA sequence that codes for a protein or functional RNA

prokaryotic genome properties

- Typically circular, double-stranded DNA.

- Often haploid (one copy per cell).

- Compact - very little non-coding DNA.

- Genes often organized in operons

prokaryotic genome properties

- DNA is supercoiled and associated with non-histone proteins.

- Some have plasmids for antibiotic resistance, virulence, etc.

- No membrane-bound nucleus - DNA located in nucleoid region.

challenge

Eukaryotic cells contain large amounts of DNA (~2 meters per cell) that must fit inside a tiny nucleus (~10 μm)

levels of DNA packaging

1. nucleosome

2. chromatin fibre

3. looped domains

4. higher-order folding

nucleosome

(1st level):

- Basic unit of DNA packaging.

- ~147 base pairs of DNA wrapped around a histone octamer (2 each of H2A, H2B, H3, and H4).

- Resembles "beads on a string" under electron microscopy.

chromatin fibre

(30 nm fibre):

- Nucleosomes are coiled into a solenoid or zigzag structure.

- Stabilized by Histone H1.

looped domains

Chromatin forms loops attached to a protein scaffold (e.g., scaffold-associated regions or SARs)

higher order folding

- Loops further fold and condense, especially during mitosis.

- Results in metaphase chromosomes, the most condensed state

types of chromatin

- euchromatin

- heterochromatin

euchromatin

- Loosely packed.

- Transcriptionally active.

heterochromatin

- Densely packed.

- Transcriptionally silent.

- Includes constitutive heterochromatin (always inactive, e.g., centromeres) and facultative heterochromatin (can be activated or inactivated).

epigenetic regulation

- DNA packaging is dynamic and regulated by histone modifications (e.g., methylation, acetylation) and DNA methylation.

- These changes affect gene accessibility and expression.

purpose of DNA replication

To create identical copies of DNA before cell division.

key feature of DNA replication

Semi-conservative: Each daughter DNA has one old (parental) strand and one new strand

size of eukaryotic genome

- Much larger than prokaryotic genomes.

- Size varies widely across species (not directly proportional to organism complexity).

structure of eukaryotic genome

- Linear chromosomes (in nucleus).

- DNA packaged into chromatin with histones.

- Typically diploid (two copies of each chromosome).

coding DNA

- Genes

- Only ~1-2% of the human genome codes for proteins.

genes

- DNA sequences transcribed into RNA, many translated into proteins.

- Contains exons (coding sequences) and introns (non-coding regions within genes).

non coding DNA

introns, regulatory sequences, intergenic regions, repetitive DNA

introns

Removed from pre-mRNA during splicing

regulatory sequences

Promoters, enhancers, silencers - control gene expression

intergenic regions

DNA between genes; includes regulatory elements, repetitive DNA, and unknown functions

repetitive DNA

- Tandem repeats (e.g., microsatellites, minisatellites).

- Interspersed repeats (e.g., transposable elements like LINEs and SINEs).

- Over 50% of human genome is repetitive.

mtDNA

Mitochondrial DNA (mtDNA)

- Circular, double-stranded DNA.

- Encodes genes for components of oxidative phosphorylation, rRNAs, and tRNAs.

- Inherited maternally.

- Replicates independently of nuclear DNA.

chloroplast DNA

(in plants and algae)

- Also circular.

- Contains genes for photosynthetic proteins, tRNAs, and rRNAs.

- Similar to prokaryotic genomes, supporting the endosymbiotic theory.

unique features of eukaryotic genomes

alternative splicing, gene families, pseudogenes, epigenetic modifications

alternative splicing

allows one gene to produce multiple proteins.

gene families

similar genes with related functions (e.g., haemoglobin genes)

pseudogenes

former genes that have lost their function due to mutations.

epigenetic modifications

like DNA methylation and histone modification regulate genome accessibility and expression

function of DNA polymerase

DNA polymerases catalyse the synthesis of new DNA strands during replication

features of DNA polymerase

direction of synthesis, template requirement, primer requirement, substrates

direction of synthesis

always 5' -> 3'

template requirement

Needs a single-stranded DNA template

primer requirement

Needs a pre-existing 3'-OH group to add nucleotides (RNA primer synthesized by primase)

NOT IN HUMANS

substrates

Deoxynucleoside triphosphates (dNTPs)

catalytic activity of DNA polymerase

- Adds nucleotides by forming phosphodiester bonds between the 3'-OH of the growing strand and the 5'-phosphate of the incoming dNTP.

- Releases pyrophosphate (PPi) as a byproduct

proofreading

- Most DNA polymerases have 3' → 5' exonuclease activity for error correction.

- Enhances fidelity of DNA replication

different types of DNA polymerase

(example in E. coli):

DNA Pol I: Removes RNA primers and fills in gaps.

DNA Pol III: Main replicative enzyme; highly processive and fast.

dAMP

Deoxyadenosine monophosphate

dCMP

Deoxycytidine monophosphate

dGMP

Deoxyguanosine monophosphate

dTMP

Deoxythymidine monophosphate

supercoiling

- If DNA is underwound or overwound, it will become supercoiled - the molecule twists around itself

- Underwinding generates negative supercoils

- Overwinding generates positive supercoils

nucleosomes

- When interphase nuclei are lysed, and their contents viewed through the electron microscope we see this 'beads on a string' structure is sometimes known as the 10nm filament

- The string is DNA

- The beads are nucleosomes

nucleosome structure

A nucleosome us 145bp DNA wrapped around "core particle" containing

- 2 molecules of histone H2A

- 2 molecules of histone H2B

- 2 molecules of histone H3

- 2 molecules of histone H4

Nucleosomes can be thought of as subunits of chromosomes and chromatin

Meselson Stahl experiment

- Grew E. coli in growth medium containing a heavy isotope of nitrogen (14N) -> 15N, the DNA in these cells is therefore denser than normal

- Switched the E. coli to a medium containing 14N, all DNA synthesised after the switch will therefore be less dense than pre-existing DNA

- Measures the density of DNA after DNA replication

dna polymerases

The enzymes that synthesise DNA are called DNA polymerase

DNA replication in e coli

replication origin, initiation, elongation, termination

replication origin

- Starts at a single origin called oriC (~245 bp long).

- Recognized by the initiator protein DnaA.

initiation

- DnaA binds oriC → DNA unwinds.

- Helicase (DnaB) is loaded with help of DnaC → unwinds the helix.

- Single-stranded binding proteins (SSBs) prevent reannealing.

- Primase (DnaG) synthesizes short RNA primers.

elongation - enzyme 1

DNA Pol III holoenzyme:

- Leading strand: synthesized continuously in 5’ → 3’ direction.

- Lagging strand: synthesized discontinuously in Okazaki fragments.

elongation - enzyme 2

DNA Pol I:

- Removes RNA primers (via 5’ → 3’ exonuclease activity).

- Fills in gaps with DNA.

elongation - enzyme 3

DNA ligase:

- Joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone.

termination

- Replication ends at ter sequences.

- Tus proteins bind ter sites and block replication fork progression.

- Result: Two identical circular DNA molecules.

OriC

bacteria - single origin

eukaryotes - multiple origins per chromosome

replication rate

bacteria - fast

eukaryotes - slow

DNA polymerases

bacteria - fewer e.g pol I and pol III

eukaryotes - many types

initiator proteins

bacteria - DnaA, DnaB, DnaG

eukaryotes - ORC (origin recognition complex), MCM complex

primase

bacteria - DnaG (separate enzyme)

eukaryotes - Part of DNA Pol α complex

lagging strand processing

bacteria - Pol I removes primers

eukaryotes - RNase H and FEN1 remove primers

chromosome structure

bacteria - circular

eukaryotes - linear

end replication problem

bacteria - absent

eukaryotes - present (solved by telomerase)

termination

bacteria - ter sites with tur proteins

eukaryotes - no specific termination site, forks eventually meet and end replication

transcription in e coli

- Transcription = synthesis of RNA from a DNA template.

- Carried out by RNA polymerase.

- Occurs in cytoplasm (no nucleus in E. coli).

RNA polymerase in e coli

- Core enzyme: α₂ββ'ω – performs elongation.

- Holoenzyme: Core enzyme + σ factor – required for initiation and promoter recognition.

- Common σ factor: σ⁷⁰ (recognizes standard promoters).

stages of transcription in e coli

initiation , elongation, termination