BIO130 first half

Week 1: cellular diversity: Cell Theory

• The cell is the basic unit of life

• All organisms 1 or more cells

• Cells arise from pre-exsisting cells

Prokarotic cell

• No membrane-bound organelles

• Smaller than eukaryotes

• Less DNA

DNA is compartmentalized but no membrane —> nucleoid

Eukaryotic cell

• Nucleus

• Membrane-bound organelles

• Larger and more complex

Origins of mitochondria

Entangle-engulf-endogenize (E³ ) model

Ancient anaerobic archeal cell and ancient aerobic bacterium

• started as ectosymbiote (outside)

• then engulfed as endosymbiote

• then bacteria membrane broken

(other model show predatory mechanism)

Ancient cell folding protusions

Give way for nuclear envelope and endoplasmic reticulum

Origins of eukaryotes graph

mitochondria first —→ eukaryotes

chloroplast second —→ plants

Endosymbiont hypothesis for mitochondria and chloroplasts — evidence

1. Both have remnants of own genome which resemble modern prokaryotes

2. Both have kept some of own protein and DNA synthesis components, also resemble prokaryotes

3. Membranes are similar to prokaryotes and derived from bacteria ancestor

Model organisms and Humans — general attributes of model organisms

1. Fast development and short life cycles

2. Small reproductive (adult) size

3. Readily available

4. Tractability (manipulation or modification)

5. Understandable genetics

Examples of model organisms

• Ecoli

• Brewer’s yeast — simple eukaryote

• Arabidopsis — plant

• Nematode, drosophila, zebrafish, mice — animals

Lec 2: The Central Dogma of Molecular Biology

Information flow is always in one direction

DNA (transcription) —→ RNA (translation) —→ Protein

.

Refined:

• messengerRNA: translation for protein

• transferRNA: transport amino acids

• rRNA: heart of ribosome, breaking and forming of bonds

Antiparallel and genetic code

• DNA, RNA, and proteins are synthesized as linear chains of info with intrinsic direction

• RNA translating to amino acid is universal through the genetic code

What are nucleic acids?

1. Genetic material in a cell

2. DNA & RNA

Three parts of a nucleotide

1. Pentose sugar

2. Phosphate group (1, 2, or 3)

3. Nitrogenous base

Nucleotide bases

Pyrimidine

• Cytosine

• Thymine

• Uracil

Purines

• Adenine

• Guanine

Differences between DNA and RNA

RNA:

• ribose

• OH on 2’ carbon

• uracil

DNA:

• deoxyribose

• H on 2’ carbon

• thymine (extra methyl)

Nucleic acid nomenclature

Nucleoside: sugar + base

Nucleotide: sugar + base + 1, 2, 3 phosphate

1. Nucleoside monophosphate

2. Nucleoside diphosphate

3. Nucleoside triphosphate

Nucleic acid chains (phosphates provide energy..)

DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs)

RNA is synthesized from ribonucleoside triphosphates (NTPs)

• Nucleotides linked by phosphodiester bonds

Phosphates provide energy for bonds

Molecular interactions

1. Electrostatic attrations (charges attract)

2. Hydrogen bonds

3. Van der waals attractions

4. Hydrophobic force

(Individually weak, sum to be strong)

Three forces that keep DNA strands together

1. H-bonds (base pairing, G-C stronger cause 3 bonds)

2. Hydrophobic interactions (phosphate backbone hydrophylic, bases hydrophobic

3. Van der waals attractions

DNA structure - energetically favourable

• DNA will naturally come together, energetically favourable

• Proteins can recognize and make contact with specfic sequences in major & minor grooves

Separating DNA strands

DNA can unravel and return to double helix

• use energy or enzymes to denature

• useful for replication, transcription, or PCR

Lec 3: intro to protein structure (primary, secondary…)

Quaternary: more than 1 polypeptide chain, subunits

Multiprotein complexes: many chains and subunits, machine

Amino acid structure

1. proteins composed of amino acids

2. side-chain/R group is variable and determines the type of amino acid

3. three major categories

   1. acidic ( - charge)

   2. basic ( + charge)

   3. uncharged polar

   4. nonpolar

classification system for amino acids

• half are polar

• 5 charged polar

• 5 uncharged polar

Amino acids and the genetic code

AUG — start codon methione

UAA, UAG, UGA — stop codon

Degenerative code: more codons than AA, more than one can code for the same AA

Flexibility: similar codons for similar AA, can tolerate mutations better

Unique amino acid: cysteine

• can form disulfide bonds — (oxidation form, reduction break)

  • both interchain and intrachain

• covalent bond creates stability

• “staple”

• often used in structural proteins

Primary structure: Peptide bonds

• catalyzed by ribosome

• peptide backbone of C-C-N-C-C-N

Polarity: always grow in the same direction, starting at N-terminus

Differences in primary AA sequence matter - vasopressin example

• both vasopressin and oxytocin are 9 AA long

• both are identical except at two locations

  • vasopressin controls urine production

  • oxytocin involved in birth, lactation, and pair bonding

Order of AA is important too - Leu-enkephalin

• natural opioid

• the opposite order of AA has no pharmalogical effects

• the amine-carboxyl orientation essential to function

Secondary structure: Alpha-helix

• Forms independantly of side chains

• carboxyl — h-bonds — amino of AA 4 after

• n — n+4

(helical structures are common in biology because they are stable

Secondary structure: Beta sheet

Forms independtly to R groups (but they alternatively point up and down, interactions)

• H-bond of carbonyl (C=O) with amide hydrogen (N-H) of neighbouring strand

• typically contain 4-5 strands but can have more

• can be antiparallel or parallel

  • anti only needs small sequence between

  • parallel needs more

Counting polypeptide chains

Always count from N-terminus (amino end)

Coiled coil

Alpha helices twisting together

• Only form with amphipathic — protein

  • repeating hydrophobic molecule every 4 peptide bonds — hydrophobic stripe

  • 2 helices will wrap together, push hydrophobic parts into middle

• very stable and strong

  • keratin in hair

  • myosin motor proteins

Amyloid structure

Beta sheets stacked together

Misfolded proteins can form amyloid structure — neurodegenerative diseases

• prions: converts properly folded molecules

Lec 4: Tertiary structure (3D)

Overall 3D structure of a protein

Held together by:

• hydrophobic forces

• non-covalent bonds

• covalent disulfide bonds

Hydrophobic force

• non-polar AA in interior of folds

• polar AA on exterior

Tertiary structure — continued (energetically favourable + chaperone proteins)

1. Proteins fold into conformation that is most energetically favourable — spontaneous

   • H-bonding in:

     • backbone/backbone

     • backbone/side chain

     • side chain/side chain

2. Chaperone proteins help make process more efficient and reliable in living cells

   • misfolded proteins cant function

Tertiary structure can have large variety of shapes

• globular, filament, etc

But few of the possible chains will be useful

• majority 50—2000 AA long

• well-behaved, stable

Models for proteins

• Backbone model — only backbones

• Ribbon model — shows folding

• Wire model — shows positions of bonds

• Space-filling model — contour map

Protein domains (still single polypeptide) (eukaryotic proteins often have 2 or more…)

Regions of proteins that have specialized functions

single polypeptide

• each domain has own tertiary structure and function semi-independently

• connected by intrinsically disordered sequences (flexible regions)

.

eukaryotic proteins often have 2 or more

• domains are important for the evolution of proteins

Protein domains — extra example: Src protein kinase

Kinase — phosphorylate proteins (changes activity)

Src protein kinase has 3 domains

• SH2 and SH3 regulates kinase

Protein families (similar AA sequences & tertiary structures)

Way to organize proteins — a protein can belong in more than one

• Similar AA sequences and tertiary structures

• Members have evolved different functions

• Most proteins belong to families with similar structural domains

Quaternary structure (subunit)

More than1 polypeptide chain

• not all proteins

• subunit = separate polypeptide

• can get really big

Quaternary structure example: Hemoglobin

1. Each hemoglobin has 4 subunits (2 alpha, 2 beta)

2. Sickle cell anemia is caused by mutation in beta subunit

Multiprotein complexes and molecular machines

Can be:

• many identical subunits (actin filaments)

• mixtures of proteins and DNA/RNA (ribosomes)

• dynamic assemblies of proteins to form machines (DNA replication)

Multiprotein complexes and molecular machines (scaffold proteins)

conformational changes

• perform job

• often need ATP

.

scaffold proteins

• binds proteins together

How are proteins studied?

Past

• purify proteins

• electrophoresis & affinity chromatography

.

Now

• Mass spectrometry

  • sequenced many genomes

  • find mass and match to predictions

• discover precise 3D structure with other techniques

• can also use AI to predict structure using only polypeptide

Protein separation

separate using size, shape, charge, hydrophobicity

Proteomics

Large scale study of proteins

• structure

• interactions

• abundance and turnover

• location

Lec 5: sequencing genomes

We are able to sequence genomes

Genomes can come in all sizes

Bacteriophage — 16.9 kb (kilobases)

E.coli — 4.6 mb (megabases: million)

Generating genetic variation

Tinkering not inventing

• Mutation of gene

• Mutation of regulatory DNA

• Gene duplication and divergence

• Exon shuffling

• Transposition (TE)

• Horizontal transfer of DNA (common in bacteria)

Purifying selection and genes

Purifying selection has maintained few hundred fundamentally important genes

• one gene in all species: gene for rRNA

Circular DNA

Prokayote DNA is circular

Organellar genomes are also circular

• Because of shared ancestry

• small size due to relying on host for protein production

• mitochondria — 16.5kb

• chloroplast — 121kb

Human genome (base pairs + diploid + karyotype)

• 3 billion base pairs per genome

• One genome from each parent = diploid (46 chromosomes)

• 20,000 protein-coding genes across 23 pairs of chromosomes

Karyotype: organized profile of full set of chromosomes

Comparing genome sizes

Size of genome is loosely related to complexity of the organism — but MANY discrepancies

• Bac and archea — smaller

• Mammals — bigger

• But:

  • newt, wheat, and amoebas have bigger genomes (amoeba 10X)

Human genome: repeated sequences and unique sequences

• Repeated sequences: ~50% of genome is repetitive DNA

  • Mobile genetic elements: transposons (TEs)

    • can move places

    • copies itself and gets stuck

    • can also create novel genes

• Unique sequences: Less an 1% encodes protein — exons

  • introns

  • other non repetitive DNA used for regulation — intergenic DNA

Packaging of DNA — prokaryotes

DNA is condensed through folding and twisting about 1000 folds using proteins

• Forms the prokaryotic Nucleoid

Packaging of DNA — eukaryotes: nucleus picture

Dark circle: nucleoli/nucleolus — parts of chromosomes that encode rRNA

Dark stains around the edge: heterochromatin (transcriptionally silent, most compact)

Light stains: euchromatin (gene rich, transcriptionally active)

Fluorescence In-situ Hybridization (FISH)

Uses single strand probes labeled with fluorescence or radioactive isotopes

• first denature

• then hybridize with probe that pairs

• can paint chromosome

• highlights shape, size and number

Packaging of DNA: Solution — Chromosomes!!

23 pairs in humans

• each chromosome contains a single, long, linear DNA molecule and associated proteins — CHROMATIN

• chromatin is dynamic — accessible or transcription, replication, and repair

Centromere

Special sequence that allows for attachment of spindles to split sister chromatids during mitosis

• tightly condensed

Chromatin organization

DNA double helix

Wrapped 1.7 times around histone protein — nucleosome (beads on a string)

Bent with linker histone H1 — chromatin fiber

Folded into loops with SMC proteins (condensins) — mitotic chromosomes

Nucleosome

Nucleosome is the basic structural unit (can be experimentally decondensed from fiber)

• each has 147 pairs

Histones (postive/negative charge)

• Small proteins rich in lysine and arginine — positively charged

• Matches with negative charge of DNA backbone

• Made up of 4 parts

  • 8 polypeptides into dimers

  • dimers into 4 cores — octamer

• One linker histone (H1)

  • kinks loose DNA into tigher fold for chromatin fiber

Lec 6: Packaging of nucleosomes: cohesins + sequence specific clamp proteins

Chromatin fibers are looped into chromatin loops using sequence-specific clamp proteins and cohesins

• sequence-specific clamp proteins recognize specific sites

• use ATP to pull proteins together

Packaging of nucleosomes: mitotic chromosomes — condensins

Condensins II replace most cohesins to form loops

Condensins I forms loops with loops

• More compact

• Stacked

• Most compact during metaphase

Mitotic chromosomes packing results

10,000 times shorter than extented DNA length

Chromatin modification

Chromatin remodeling complexes + histone modifying enzymes — changes chromatin structure and access to DNA for replication or transcription

• Used ATP

• Slides DNA on histones

• Evict histones

• N-terminal tails of histones modified (adding groups, providing docking sites for proteins)

Chromatin remodelling complexes + histone modifying enzymes act as a team (HME recruits CRC)

Heterochromatin (constitutive vs facultative)

Highly condensed chromatin

• Meitotic and mitotic chromosomes

• Time spent highly condensed varies

• Regions of interphase chromosomes that gene expression is suppressed

.

• Constitutive heterochromatin — most compact, compact all the time (Telomeres)

• Facultative heterochromatin — can be opened, to turn on/off (degree of compaction controls gene expression)

• Centromeres are compacted all the time

Euchromatin

Relatively non-condensed chromatin

• Degree of condensation varies

• Level of activity varies

• Areas where genes tend to be expressed

.

• Quiescent euchromatin — less active

• Active euchromatin — least compact, gene expression

Degree of chromatin condensation is reversible (modifications of histones + chromatin remodeling complexes + RNA polymerase)

Localized covalent modifications of histones + chromatin remodeling complexes + RNA polymerase = modulate the reversible switch along chromosomes

Interphase chromosomes in discrete regions of the nucleus

(shown using FISH)

• Chromosomes have territories

  • Some interact

  • Some are more on the sides or the middle

  • Nucleolus — carry genes that encode for rRNA

Expressed gene is re-orientated within the chromatin

OFF

• Within periphery chromosomes

ON

• Genes are now internalized, more in the middle

• Chromosomes are more unfolded

Cell nuclei is dynamic

Conservative vs Semi-conservative DNA replication vs Dispersive

DNA replication is actually semi-conservative

Direction of DNA replication - overview

Bidirectional growth from one starting point

• DNA is synthesized 5’ — 3’

• DNA is split using initiator proteins

• 2 replication forks

• DNA polymerase uses template to add deoxynucleoside triphosphates (dNTPs) to the 3’ OH end

  • phosphodiester bond

  • base pairing

  • energy comes from dNTP — pyrophosphate out

• Leading strand

• Lagging strand — okazaki fragments

Always start from the same location on DNA

• A -T rich sequences, easy to open

• Recognized by initiator proteins that bind to the DNA

How many origins of replication & speed

Bacteria - 1 (1000 pairs/sec)

Eukaryotes - multiple (100 pairis/sec in humans)

DNA replication in bacteria

Two forks that increase until two new circular genomes are formed

What happens at the DNA replication forks?

Replication fork is asymmetrical — antiparallel with parental strand

• Leading strand synthesized continuously

• Lagging strand discontinuously — okazki fragments

• Fork keeps unzipping as it goes

Procedure and Ingredients for DNA synthesis

Procedure:

1. separate DNA strands

2. synthesize DNA

3. proofread newly synthesized DNA

.

1. origin of replication — specific

2. primers — RNA primer

3. dNTPs

4. ATP (energy)

5. DNA polymerase

6. Accessory proteins

Many mechanisms are similar with eukaryotes and prokaryotes

Many mechanisms are similar with eukaryotes and prokaryotes

1. origin of replication

2. initiator proteins

3. helicase

4. single-strand (DNA) binding proteins

5. RNA primers made by primase

6. DNA polymerase

7. sliding clamp holds polymerase

8. nick sealing by DNA ligase

Initiator proteins for replication (E.coli)

Process highly regulated — want start to finish completion

• Bind to origin

  • A-T rich sequences (very specific)

  • attracts initiator proteins

  • destablize helix

  • has ATP but dont use it until synthesize begins (for regulation, no one else can get on strand)

.

• Helps helicase bind

  • attract helicases and bind

  • and helicase-loading protein

.

• Requires ATP

  • start hydrolyzing when replication is about to begin

  • no other can start replication until initiator proteins get more ATP

Unwinding by helicase

Two types of helicases exist — predominant moving along the lagging strand 5’ —> 3’

Requires ATP — breaking hydrogen bonds

(helicase has quaternary structure)

Binding of single-strand binding proteins

Keeps DNA from reannealing or getting tangled — a single strand can H-bond (form hairpins) (important for PCR too)

1. separates strand by binding ssDNA, coats/cups strand, particularly the lagging strand

   1. proteins start attracting more proteins (cooperative, multiprotein assembly)

2. prevents strands from H-bond with eachother or by itself

RNA primers made by primase

DNA polymerase needs RNA primer to start synthesizing

• primer only need nucleotide + template

• polymerase needs 3’ OH + nucleotide + template

.

1. To begin, DNA polymerase needs bound primer

2. Primase synthesize an RNA primer (joins ribonucleotides)

3. Primase synthesizes in 5’ —→ 3’ (antiparallel to template, moving 3’ — 5’ on template strand)

primer is only temporary, cell doesnt like

Primase is on helicase

DNA polymerase

Adds nucleotides to 3’ OH end

• correct base pairing

• clips off 2 phosphates — pyrophosphate

• catalyzes phosphodiester bond

• move onto next

DNA is synthesized complementary and antiparallel

Grows 5’ —→ 3’

Sliding clamp holds polymerase onto DNA (loaded by clamp loader)

Why? If DNA polymerase holds too tightly, hard to release quickly

Evolved DNA polymerase to not hold on tightly, sliding clamp will let it fall off

• Circular protein — doesnt impede polymerase progress

• loaded by clamp loader (also holds polymerases together, moves then together)

Replication fork picture

• lagging strand keeps making primers for okazaki fragments

• sliding clamp releases polymerase when reach other okazaki fragment 5’ end

• overall moving towards fork

Nick sealing by DNA ligase (ATP & AMP)

A special DNA repair system is responsible for removal of RNA primer and replacing with DNA sequence

• Nucleases remove primer

• Repair polymerase replaces DNA onto 3’ end of okazaki

• Nicks sealed by Ligase — seal phosphodiester bond (doesnt need more nucleotides)

  • uses ATP — hydrolyzes

    • attaches amp to nick (adenosin monophosphate)

    • release amp

    • phosphodiester bond

Summary picture (bac replisome + primosome)

whole thing: bacterical replisome — molecular machine

helicase + primase = primosome

Primosome

Helicase + primase

Lec 8: issues in DNA replication: Unwinding problem

• As helicase unwinds DNA, supercoiling & torsional strain increases

• Problem in circular chromosomes and large eukaryotic chromosomes

Solved by DNA topoisomerase

• binds to a location

• cut a nick in the backbone of 1 strand (does not cut H-bonds)

• allows for rotational freedom

Issues in DNA replication: Ends of linear chromosomes

On the lagging strand, after the removal of the last primer, nothing to replace lost segment — loss of sequence information on the 5’ end of daughter DNA

Ends of linear chromosomes solution: Telomerase & telomere replication (G rich ends…)

Repetitve sequence is added to the 3’ end of the parental strand (lagging strand template)

• added by telomerase

• complementary to an RNA template (telomerase RNA)

• resembles reverse transcriptase

• G-rich ends (stable)

Daughter strand will still be shorter but no important DNA information will be lost

Telomeres and cells

Stem and germ-line cells — indefinite, abundant

Somatic cells — limited, can stop

Senescent cells — not making telomeres, stop dividing when reaches signal

Cancer cells — high levels of telomerase (more than stem/germ), persistent growth

issues in DNA replication: Correcting mistakes

If mistakes are not corrected, it will lead to permanent mutations as the strand divides again

High fidelity of DNA replication

1. RNA polymerase have error 1 in 1000

2. DNA polymerase have error 1 in 10⁹

Human genome only changed by about 3 nucleotides per divide

DNA proofreading and repair: 3’ to 5’ exonuclease

DNA polymerase has proofreading exonuclease activity

• 3’ - 5’ direction

• same time as synthesis

• checks if previous is correct

• if not, clips off mispaired

• adds new nucleotide

(this is why DNA replication can only happen 5’ — 3’)

(extrude mistake to editing site, needs to unravel a little bit)

DNA proofreading and repair: Strand-directed mismatch repair (MutS and MutL)

If proofreading fails, error repair

• MutS recognize distortion in geometry caused by mismatched pairs

• Locks onto DNA

• Recruits MutL and scans DNA

• Reach sliding clamp, MutL nuclease activate

• Clips new strand twice

• Filled in with Polymerase δ

Recognizes new strand either by methylation on new strand or by clamp on nick