MEDS2003: Molecular Biology

Central Dogma of Genetics

DNA can make DNA, DNA and RNA can make each other, RNA can make RNA and proteins

Pneumococci Experiment

Mixing dead-pathogenic and non-pathogenic pneumococcus, determined that the DNA is what allows the non-pathogenic to transform

Waring Blender Experiment

Radioactive labels on P (for DNA) and S (for proteins) showed that phages inject their DNA into the bacteria

Computer analogy

DNA is the hard drive, mRNA is the RAM, proteins (and some RNA) are the devices

DNA Requirements

Stable, corruption free, protected, backed up

Transcription

Creation of RNA from DNA

Translation

Creation of protein from mRNA

Genome

All the DNA in a cell

Transcriptome

All the RNA in the cell

Proteome

All the proteins in the cell

Transcriptome Proportions

mRNA (2%), rRNA (80%), tRNA (15%), microRNA and small nuclear RNA

Nucleic Acid Parts

Nucleotides with sugars, phosphate and bases

Bases

Adenine, Guanine, Cytosine, Thymine, Uracil, nitrogenous and hydrophobic

Purines

Adenine, Guanine

Pyrimidines

Cytosine, Thymine, Uracil

Base Pairing

AT (2 H bonds), CG (3 H bonds)

Chargaff's Rules

A:T = 1, G:C = 1, purine:pyrimidine = 1, regardless of organism

Base Pairing (Different pH)

Some groups get protonated in low pH, some get deprotonated in high pH, makes bonds weaker

Nucleic Acid Sugars

Ribose in RNA vs Deoxyribose in DNA, 1' is N-glycosidic bond to base, 2' is oxy, 3' is where next nucleotide will join, 5' is where phosphate is bound

Deoxyribose

Missing OH on the 2' carbon

Sugar-Phosphate Backbone

Nucleotide backbone with alternating units of phosphate and sugar, joined with phosphodiester bonds

Double Helix Structure

Two antiparallel strands, bases in the middle bonding with H-bonds, backbone outwards

Double Helix Interactions

H-bonds between bases, negative phosphates create the twist (repulsive), hydrophobic bases stay central to avoid water, van der waals forces attract base pairs, result in base stacking (as the radius is 1.7 A, and their distance is 3.4 A)

Base Absorbance

Both DNA and RNA absorb at 260 nm, due to their base rings, so can't distinguish between them

Hyperchromic Effect

Double stranded nucleic acids absorb less than single stranded

Grooves of DNA

Major and Minor groove cause by phosphodiester bonds being angled

Major Groove

On the non-sugar side, can fit zinc fingers, large proteins

Minor Groove

On the sugar side, small less specific molecules can bind like DAPI (DNA dye)

DNA Info Storage

Info is stored in the bases, double stranded as a backup, info is buried safely inside

Impact of RNA/DNA Differences

The 2' OH group (RNA) can easily be attacked, and so RNA is more likely to degrade, and also cytosine deamination in DNA can be detected

Cytosine deamination

Cytosine can spontaneously deaminate into uracil, but enzymes can detect and fix it

Semi-conservative

DNA replication is semi-conservative, each generation keeps one old strand, makes one new strand

DNA Replication (General)

Separate DNA strands, bind primers to DNA strands, nucleotides are added with a polymerase, sometimes is proofread

DNA Replication Direction

New nucleotides are added to 3' OH (so made from 5' to 3')

DNA Replication (E. coli)

DnaA recognises oriC site, separates DNA, helicase (DnaB) moves 5' to 3' to unwind strands, SSB keeps DNA separated, add primers with primase (DnaG), DNA polymerase III adds nucleotides (breaking phosphate bonds). Two replication forks on the whole loop, each have a leading and lagging strand. Lagging strand uses Okazaki fragments. DNA polymerase I replaces primers with DNA, and DNA ligase removes nick. Stop replicating at Ter sequences. Strands are separated by a different topoisomerase IV

DNA Replication (Eukaryotic) >Helicase moves 5' to 3' to unwind strands, SSB keeps DNA separated, add primers with primase (DnaG), DNA polymerase III adds nucleotides (breaking phosphate bonds). One replication fork being unwound by helicase, has a leading and lagging strand. Lagging strand uses Okazaki fragments. DNA polymerase I replaces primers with DNA, and DNA ligase removes nick. Stop replicating at the end. Ends have telomeres added to ensure stability.

Cell Division Control

Eukaryotes only divide with certain cellular signals; prokaryotes divide whenever they have nutrients

E. coli DNA Replication Termination

10 Temrinal sequences prevents DNA from being replicated more than once

Okazaki fragments

Repeatedly placing new primers and making fragments (for when replication is going opposite to the replication fork

DnaA

E. coli enzyme that separates DNA strands at oriC site

Helicase

Enzyme that moves along DNA separating strands

SSB

Single-stranded-DNA-binding proteins, keep the DNA strands apart

Primase

RNA polymerase that makes RNA primer to begin DNA replication

Nucleoside

Base + sugar

Nucleotide

Nucleoside + phosphate, e.g. dNTP is deoxynitrogenous base triphosphate

Correct Base Pairing

Before adding nucleotide, if wrong base pairing, won't fit well in active site, and also proofreading each time, cutting with 3' to 5' exonuclease

DNA Polymerase cofactors

Require Mg2+ to provide positive charge to allow negative backbone to come close enough

Exonuclease

Enzyme that cuts nucleic acid from the ends

Endonuclease

Enzyme that cuts nucleic acid from the middle

DNA Polymerase III (δ)

Enzyme that attaches new nucleotides to the strands, using a sliding clamp and clamp loader system to load each strand

Topoisomerase II

Cuts and unwinds DNA ahead of replication fork, introduces negative supercoils to counteract the positive ones being formed by the fork

Topoisomerase IV

Cuts and unwinds the two final loops of DNA in prokaryotic replication

DNA Polymerase I (α)

Replaces the RNA primers with DNA

DNA Ligase

Joins the gaps where RNA primers were, aka fixing the nicks

Acyclovir

Antiviral drug, a nucleoside analogue, viral thymine kinase phosphorylates drug into nucleotide, but then when it's used, stops the chain (no 3' OH to join to)

Azidothymidine

Antiviral drug, viral reverse transcriptase incorporates nucleoside analogue, can't extend due to no 3' OH.

Molnupiravir

Antiviral for COVID-19, ribonucleoside analogue, introducing mutations into the virus.

5-Flourouracil

Cancer drug, gets converted to dTMP, but the drug stops the process, so no dTMP can be made, cancer cells can't replicate DNA

Eukaryotic cell cycle

G1, G0 or S, G2, M, repeat, regulated by cyclin and cyclin kinases.

G1

Gap 1 in euk cell cycle, cells decide to go to S or to G0. Has a restriction point that once is past, cell will divide. Choose what replication origin sites to use. 6-12 hours.

G0

Cells exist here when not replicating, can enter G1 again (near the start), more cells in G0 with age. Brain cells almost always G0, fibroblasts almost never in G0.

Quiescent cells

Can be induced to leave G0

Senescent cells

Can't leave G0

S

Synthesis phase in euk cell cycle, DNA synthesis. CDKs activate pre-RCs, recruits DNA polymerases, 20-80 sites at a time, 6-8 hours. CDKs inhibit pre-RC formation if replication has already occurred.

G2

Gap 2, protein for new cell is made, prepare for M. 3-4 hours.

M

Mitosis, two daughter cells formed. 1 hour.

Cell Cycle Checkpoints

G1 to S, check for DNA damage. S to G2, check for DNA damage and that replication is complete. M, check sister chromatids are correctly attached to spindles.

Cell Division Control in Eukaryotes

Oncogenes, accelerate cell division, and tumour suppressor genes, the brakes

Cancer DNA Changes

Mutations can be inherited or from the environment

Retinoblastoma Protein

A tumour suppressor protein, if two recessive copies gotten, then cancer

Differences in DNA Replication between euks and proks

Euks have a much larger genome, and have multiple chromosomes, with multiple origins of replication (not all of them activated each round). Euk DNA is also repackaged.

Histones

Proteins used to wind up DNA to be more compact.

Histone synthesis and regulation

Many copies of histone gene, can be transcribed quickly, no introns and not polyadenylated, so mRNA degrades quickly once used, histones can be recycled

Telomeres

Overhangs in eukaryotic replication, the ends of DNA has repeating sequences of telomeres, protecting the important information, it's okay if we lose some of these. Telomerase can extend them.

Telomerase

Enzyme with RNA template and RNA reverse transcriptase, can extend 3' end of telomeres, which allows for 5' end to be done by lagging strand method, then 3' end tucks in.

Hayflick limit

In somatic cells, lack of telomerase, so slowly telomeres shrink, and once they are gone, cells won't divide

Immortal cells

Like cancer cells, which have high telomerase activity

Telomerases as drug target

high activity in cancers, so degrading RNA component or reverse transcriptase inhibitors can be used.

PCR

DNA Replication in the lab, needs template, taq polymerase, forward and reverse primers, dNTPs, buffer (pH, Mg2+, ionic strength), melt>anneal>elongate

PCR Thermocycler

Melting phase, 95 C, 15 s, annealing phase, 65 degrees, 15 s, extension phase, 73 degrees, 10 s

PCR Melting Phase

Using high temperature (95 C) to break apart the two DNA strands. The temperature increases with GC, length and ionic strength

PCR Annealing Phase

Primers bind to specific target, need to carefully design them, use lower temperature (65 C)

PCR Extension Phase

Using heat stable polymerase to extend from the primer, does at (72 C)

Amplicon

Newly synthesised DNA made during PCR

Primer Specificity in PCR

A high Mg2+ concentration lowers specificity due to shielding of phosphates

Electrophoresis

Using charged terminals to separate DNA (or protein) fragments by size. Larger fragments move less due to increased drag. Results are visualised by staining.

cDNA

Product made from RNA (using reverse transcriptase), which can stably store RNA samples. Made with PCR.

Ribonucleases

Enzyme that breaks down RNA, found on skin and easily contaminates samples

Sanger sequencing

Uses fluorescent labelled ddNTPs (no OH on 3'), which then gives you DNA fragments of different sizes and with specific fluorescence for each base: sequence, limited by length and need knowledge of primer

Next generation sequencing

Sequences many different strands at the same time, computer program looks at overlaps and puts them together

Human Genome

22 pairs of autosomes, 1 pair of sex chromosomes

Human Protein Numbers

20 000 proteins

Introns and Exons

Introns are pieces inside a gene that needs to be removed during processing; exons get put together to make the mature mRNA

Gene family

Duplication of a single gene, creates multiple similar genes

Chromatin

Complex of DNA and proteins, which can be tightly packed (heterochromatin) or loosely packed (euchromatin) (more transcription on euchromatin)

Chromosomes

One bundle of chromatin, has a centromere, where sister chromatids attach, and has a p arm (short), q arm (long), ends are telomeres

Histone DNA interactions

Histone tails have a lot of arginine/lysine (+++), so binds to backbone, shields charges to allow for bending, restricting transcription

Histone Modifications

Acetylation (increases transcription), methylation (provides targets for proteins)

Histone Acetylation

Histone acetyltransferases (HATs) acetylate lysine, remove +, so the DNA would be more accessible to transcription, also increases protein binding. Histone deacetylases (HDACs) reverse this

Histone Methylation

Histone methyltransferases (HMTs) methylate lysine, some proteins bind to methylated residues, could activate or repress, histone demethylases remove methyl