molecular bio

genome

all genetic info, almost every cell in body contains entire genome

genes hold code to create proteins

transcriptome

all RNA

may vary over time

made up of

• mRNA (1-2%)

• ribosomal RNA (80%)

• tRNA (15%)

• micro RNA

• small nuclear RNA

proteome

the protein

each cell has varied requirements so proteins expressed can vary

flow og genetic info

DNA → RNA → Protein

DNA replication

DNA → DNA

transcription

DNA → RNA

translation

DNA / RNA → Protein

types of proteins

proteins fold into various structures

• ion channels

• enzymes

• trancription factors

• receptors

• antibodies

nucleic acid purines

adenine, guanine

have 2 rings

nucleic acid pyrimidines

cytosine

thymine (DNA)

uracil (RNA)

have 1 ring

chargaff’s rules

purines bind to pyrimidines

base pairing (at physological pH)

A - T (2 H bonds)

G - C (3 H bonds)

base pairing (at low pH)

A - T ( 1 H Bond)

G - C (2 H bonds)

extra protons acidify some areas, preventing binding between bases

sugar component (RNA, DNA)

1’ = N-glycosidic bone attaches

2’ - OH for ribose, H for deoxyribose

3’ = hydroxyl (OH)

5’ = phosphate attachment site

sugar-phosphate backbone

• overly negative due to phosphates

phosphodiester bond - between the sugar and phosphate

N-glycosidic bond - between the sugar and base

backbone structure

base attaches to 1’ of sugar = N-glycosidic bond

phosphate attaches to 4’ and 3’ of sugar = phosphodiester bond?

phosphate end = 5’

hydroxy end = 3’

interactions forming DNA helix

• H bonds

• ionic interactions

• van der waals

• hydrophobic interactions

H bonds - DNA helix

between complimentary bases

• 2 for A-T

• 3 for G-C

ionic interactions - DNA helix

backbone has many -ve phosphates, repel to max distance creating helix twist structure

hydrophobic interactions - DNA helix

pyrimidines and purines are central, while hydrophobic sugars and phosphates remain on outer side

base stacking/vaan der walls - DNA relix

van der waals favours base stacking

the distance between bases is 3.4 A (radius of C = 1.7A) leading to interactions

helix properties

• all bases absorb at 260 nm

• [DNA] of 50 ng/mL = absorbance of 1

• [RNA] of 4 ng/mL = absorbance of 1

• aromatic protein rings absorb at 280 nm

• ratios used to determine purity + contamination

hyperchronic effect

due to base stacking

double stranded DNA absorbs less at 260 nm compared to RNA

major + minor grooves

due to the way the bases bind to the backbone

base pairs further for major groove

DNA groove interactions

• binding of zinc finger transcription factors (or other proteins) to major groove

• smaller things such as DAPI (DNA dye) can bind to minor groove

storing genetic info

hydrophobic bases contain info, protected inside helix

DNA is double stranded so a backup code is accessible

DNA vs. RNA

• 2’ of sugar, H for DNA, OH for RNA

• uracil = RNA, thymine = DNA

• RNA in acidic pH may degrade

• RNA stability varies between cells

uracil in DNA - problem

cytosine may spontaneously deaminate into uracil

this can be recognised and removed in DNA

this issue is not corrected in RNA, but may lead to mutations

occurs 100-500 times per cell per day

E. coli

• grows very quickly, very little needs

• used often in molecular bio

• one circular chromosome of 4.6 million bp

• must copy entire genome to replicate (replication), occurs in less than 40 mins

DNA → DNA (briefly)

1. separate DNA strands

2. primers bind to complimentary sequence

3. complementary base H bonds

4. nucleotide added to strand

5. optional = proofreading

DNA replication rules

• nucleotides added in 3’OH to 5’P direction

• DNA polymerase adds nucleotides to the strand

• result = 2 double stranded DNA molecules

  • each contains 1 OG strand, 1 new strand

  • “semi conservative replication”

copying DNA in E. coli

1. separate DNA strands (starting at ORI site)

2. primers bind to complementary sequences (primer = short RNA strand)

3. complementary base H-bonds

4. nucleotide added

5. proofreading

continues along strand until complete

DnaA

• bacterial protein that recognises oriC site (origin of replication)

• acts as replication initiator by binding to a specific sequence (ORI) and initiating unwinding of DNA

• prokaryotic replication

helice (aka DnaB)

• moves 5’ to 3’ unwinding DNA

SSBP

single stranded binding proteins

keep DNA strands separated

primase (DnaG)

an RNA polymerase that makes short RNA primers

• all DNA polymerases need a primer to add the nucleotides to

• primase provides the 3’OH that nucleotides will bind to

note:

• RNA polymerase do not need primers for synthesis

• DNA polymerase used RNA primer for DNA synthesis

nucleoside

base + sugar

• substrate for nucleic acid synthesis

• for DNA, H instead of OH at 2”

• generalize to NTP or dNTP

nucleotide

nucleoside (base + sugar) + phosphate

forming a phosphodiester bond (DNA)

1. base pairing w/ template strand

2. DNA polymerase catalyses formation of bond

3. pyrophosphate released

4. pyrophosphate further breaks down into 2 phosphates

released phosphates provide energy for next reaction (coupled)

base pairing must be…. during replication

checked/proofread

must be A-T/U or C-G

RNA/DNA syntheisis if thermodynamically unfavourable so

free energy released during the formation of phosphodiester bond provide the energy for DNA synthesis

forming a phosphodiester bond (RNA)

same as in DNA replication, although RNA polumerase used to catalyse bond formation rather than DNA polymerase

proofreading of base pairing

double check what base was just added, was it the correct

if not - can cut of by 3’ to 5’ (opposite) exonuclease

nucleases

enzymes which cut phosphodiester bond

• 3 to 5’ exonuclease cuts from 3’ end

• 5’ to 3’ exonuclease cuts from 5’ end

• endonucleases cut somewhere in between

replication fork

A replication fork is a structure that forms during DNA replication, where the double-stranded DNA molecule is unwound and separated into two single strand

• as helicase moves along each strand, the replicationbubble grows

• DNA synthesised on both strands as each replication fork as it opens

• issue - synthesising in the other direction

the lagging strand

synthesized as discountinuous okazaki fragments

DNA pol III

• core - adds nucleotides + proofreads

• beta sliding clamp - holds tightly onto DNA - synthesis stops when it disassociates

• clamp loader - grabs lagging strand + moves it to the polymerase

challenge - supercoiling

as DNA strands separate, it over winds = positive supercoiling

solutions = DNA topoisomerase 3

positive supercoling

twisting in the same direction as double helix, becomes tighter + harder to spearate

negative supercoiling

twist in opposite direction to the double helix - makes DNA easier to separate

topoisomerase 3

moves ahead of rep bubble + introduces negative supercoils

• twists in opposite direction

cuts both strands, reorients ends + sticks them back together

• requires ATP

removing primers

at end of replication, primer must be removed and replaced with DNA

done by DNA pol 1

DNA pol 1

• removes RNA primer

• has proofreading ability

• has 3’ to 5’ exonuclease and 5’ to 3’ exonuclease abilities

• can degrade RNA and incorporate DNA

DNA ligase

forms bond in backbone after primer removes, connects strands

termination of replication (prokaryotic)

• it is important entire chromosome only copied once

• mechanisms (TerA to TerK) prevent further synthesis

• the 10 Ter sequences bind to a protein (Tus), creating trap for the fork

chromosome separation

• another topoisomerase plays a role (type IV)

acyclovir - antiviral

• treats herpes simplex virus infections

• nucleoside analogue (modified nucleoside)

• viral thymidine kinsae phosphorylates the drug to form nucleotide incorporate into growing strand in infected cells

azidothymidine - antiviral

• HIV/AIDS streatment

• nucleoside analoguse

• viral reverse transciptase will incorporate drug into growing strand

molnupiravir

• COVID-19 treatment

• ribonucleoside analogue

• once phosphorylated, viral RNA dependent RNA polymerase incorporates into the growing strand and keeps going

• rationale - introduces mutations to point it can no longer work

5-fluorouracil (adrucil) - cancer drug

• sugar + phosphate added in cell

• thymidine synthetase methylates C-5 to convert dUMP to dTMP

• when F5dUMP gets stuct, no dTMP is made

• run out of substrate (dTTP) for DNA synthesis

• only affects rapidly dividng cells

eukaryotic vs. prokaryotic replication (DNA)

mostly similar

• initiation regulated - entire genome must be copied during replication

• bi-directional

• primase lays down RNA primers

• highly processive polymerases do most of synthesis (3’ to 5’ exonucleoase activity)

• leading + lagging strands

differences

• 1 circuler chromosome (pro)

• many linear chromosomes (eu)

human genome

~3 billion x 2 bp

23 linear chromosomes

e. coli genome

4.6 mill bp

1 circular chromosome

controlled cell division

in eukaryotes

• processes only occur if there are signals telling things what to do

e. coli

• can divide quickly if provided with correct media + necessary nutrients

the cell cycle

the life cycle of a cell, encompassing its growth, DNA replication, and division into two daughter cells.

It's a coordinated process that ensures the accurate distribution of genetic material and division of the cell. The cell cycle is broadly divided into two phases: interphase and the mitotic (M) phase. 

G1 (gap1) - the cell cycle

cells decide whether to divide (enter phase S) or stop (enter phase G0)

RNA + protein synthesis, no DNA synthesis

may last 6 - 12 hrs

G0 phase - cell cycle

cells not dividing

• % of cells in G0 increases w/ age, also differs between cell type

terminally differentiated cells withdraw from cell cycle indefinitely

fibroblasts + epithelium

almost never enter G0 phase

adult liver cells

enter cell cycle ~ 1/ year

adult brain cells

almost always in G0 phase

quiescent cells

can be induced to re-enter cycle by mitotic signals

senescent cells

cannot re-enter cell cycle

restriction point

a cell that passes this point is committed to pass into S phase

S (synthesis) phase - cell cycle

DNA synthesis - doubling amount of DNA in cell

RNA and proteins also synthesized

many signalling proteins involved

may last 6-8 hrs

G2 phase - cell cycle

no DNA synthesis

RNA and protein synthesis continue

preparation for M phase (mitosis)

may last 3-4 hrs

M (mitosis) phase - cell cycle

mitosis (nuclear division) and cytokinesis (cell division) yielding two daughet cells

final phase of cell cycle

CDKs + cyclin

activation of kinases

• levels may vary at phases

• active CDKs phosphorylate proteins involved in regulation of cycle

cell cycle checkpoints

1. G1 to S - check for DNA damage

2. S to G2 - check for DNA damage, and that synthesis is complete (no okazaki fragments etc)

3. M - check sister chromatids are correctly attached to spindles before division

cell cycle checkpoint 1 (G1 to S)

important to control progression through the cycle

• DNA must not be synthesized if damaged

• DNA must be complete before progression to G2

• check spindles before division

• phosphorylation of target proteins by active CDKs affect function

oncogenes

“accelerator” proto-oncogenes control cell growth

referred to oncogenes when mutated

mutations are dominant, one copy is enough

Oncogenes are mutated genes that can lead to cancer. They are derived from normal genes called proto-oncogenes, which play a role in cell growth and division. When a proto-oncogene becomes an oncogene, it can cause uncontrolled cell growth and division, potentially leading to tumor formation

tumour suppressor genes

“the breaks”

slows down division or induces apoptosis

mutations are recessive - problems occur when 2 alleles

cancer

DNA mutations leading to cancer may be inherited (Rb, BRCA1) or acquired (behaviours - sunbathing, smoking, radiation, random during cell division)

retinoblastoma protein

a tumour suppressor protein

retinoblastoma = retina cancer in children

those w/ condition have often inherited one mutated copy and acquire a mutation in the other copy in retinal cells → two copies

people with mutaiton also have increased risk of lung, prostate and breast cancer

eukaryotic replication unique differences

• much more genetic material to copy

• much slower

• many ORI sites - not all are activated in each round of replication, but enough need to be activated to copy all chromosomes entirely

• regulated to ensure rep only once per cell cycle

initiation of replication (eukaryotes)

G1 phase

• origin sites selected

• pre-replicative complexes (pre-RCs) assemble

S phase

• active CDKs phosphorylate and activate pre-RCs → recruit DNA polymerases

• clusters of 2 to 80 sites initiated at a time

• CDKs also inhibit pre-RC formatio if replication already occured

repacking chromosomes

DNA is packaged in cell - wound around histones

existing histones can be reused

replication doubles DNA → more histones needed

• synthesis controlled at transcitional and post-transcriptional levels

• coupled to the cell cycle (mostly during S phase)

histone synthesis

many copies of histone genes in the genome. can be quickly transcribed

hostone genes = no introns - no need to spice

hostone mRNA are not polyadenylated - mRNA does not last long

telomeres

ends of chromosomes made up of repeating sequences known as telomeres

protects genetic infor

in humans

• repeat is 5’ - TTA GGG -3’

• can extend as much as 10kb

• double stranded except end where 3’ extends beyond 5’end

eukaryotic DNA replication - step by step

1. start at ORI (many sites)

2. RNA primers bind at ORI

3. DNA synthesized in 5’ direction

4. etc etc

end problem = overhangs in the 3’ end of the parent strand

telomerase

recognises overhand and synthesizes complementary strand

• low activity in somatic cells

• high activity in germ line cells zygote starts with full length telomeres

• high activity in highly proliferative stem cells

Telomerase, on the other hand, is the enzyme responsible for adding telomeres to the ends of the chromosome. Telomerase has a single-stranded RNA segment, which serves as a template for a single-stranded DNA. These single-stranded DNA fragments are repeated and added to the 3-prime end of the chromosome.

ribonucleoprotein

RNA + protein

• RNA: ~1.5 copies of the complement telomere sequence - the template

• protein: reverse transcriptase - DNA polymerase that uses RNA template to make a copy of DNA

5’ end extends by lagging strand mechanisms

overrhang on the 3’ end tucks in and caps the end

capping proteins protect the ends from nucleases

immortal cells

• eg, cancer cells, cells continue to divide due to telomerase activity

• HeLa cells were the first immortal cell line, derived from cervical cancer patient - still commonly used as a model cell line

cells which can divide indefinitely in culture

cells/genome across lifespan

• human cells start w/ ~10k bp of telomeres are birth

• some cells can survive an entire lifespan of replication erosion without suffering cell death (eg, those that do not divide much)

• shortening telomeres could be a limiting factor in determining organism’s life span

hayflick limit

lack of telomerase activity - as seen in most somatic cells

• telomeres shorten after each round of mitosis

• when too short, cell is in danger of losing coding genes

• cells can no longer divide (senescence) - number of times it can divide = hayflick limit

telomerases - a drug target

telomerase is active in between 80-90% of all cancers

1. targeting the RNA component with antisense oligodeoxynucleotides and RNaseH

2. reverse-transcriptase inhibitors (eg, AZT) or inhibitors of the catalytic protein subunit

dolly the sheep

• cloned by taking nucleus from adult cell from mammary gland

• died 203 - age 6 (LE 10-20 yrs)

• shorter telomeres - suspected to be use of adult cells in cloning process

NOT CASE

• no evidence of health usses related to accelerated aging

• other clones had different telomere lengths + aged normally

• examples of syndromes related to telomeres - so telomere length is important

copying DNA in a tube - ingrediants

• template

• DNA polymerase

• Primers - forward + reverse

• dNTPs

• buffer (optimal Mg2+, pH and ionic strength)

DNA synthesis: tube vs. cell

much simpler than in cell, we can add all relevant enzymes, synthesis RNA primers that need to be removed later, make a substrate, maintain pH and ionic strength

PCR

polymerase chain reaction

PCR steps

1. mixutre heated to separate DNA strands - contrast to cell where helicase is responsible - heating separates weak (H bonds) forces between strands

2. annealing of primers - cool and force primers to anneal to sequence wanting to be amplified

3. extension - complementary bases hydrogen bond, polymerase catalyses formation of phosphodiester bond, adds nucleotide to growing chain (3’OH to 5’P direction)

4. cycling - repeat, amplicon (newly synthesized strand) becomes template for next cycle, doubling in each cycle. note in the cell, synthesis of entire genome only occurs once at a time, with PCR we can keep cycling as many times as desired