DNA STRUCTURE ○​ Nitrous acids ●​ Spliceosomal introns by spliceosomes (snRNPs)

Double helix ○​ Alkylating agents ○​ GU at 5’ end + AG at 3’ end = splicing

●​ 2 strands, anti-parallel, major + minor groove ○​ Oxidative damage = DNA oxidised by site
●​ B-form DNA = right-handed direction, large + small oxygen radicals ○​ Lariat-like intermediate = bulge/gap
groove DNA repair systems: formed
●​ 10 bases need for 1 full turn (36Å) ●​ Nucleotide-excision repair = DNA helicase removing ○​ tRNA introns = spliced by protein-based
●​ Intrinsic direction → 1 free 5’ phosphate end + 1 free lesions/base mis-match + DNA pol I fills gap enzymes
3’ OH end ●​ Base-excision repair = DNA glycosylase removing
●​ Nucleotides damaged base → AP endonuclease creating gap → GENE EXPRESSION REGULATION (IN GENERAL) → level of
○​ Nucleotides: base + sugar + phosphate DNA pol I filling gap with new bases transcription initation
○​ Nucleosides: base + sugar 1) Using regulatory proteins → affecting interaction between RNA
Complementary base pairing DNA CLONING pol and promoter
●​ A = T (2 H-bonds), C = G (3 H-bonds) → Creation of identical copies of a piece of DNA (gene) ●​ Repressors - block RNA pol binding to promoter ⇒
●​ Purines: A, G - 2 rings → Recombinant DNA = artificially created DNA that combines negative regulation
●​ Pyrimidines: C, T, U - 1 ring sequences ○​ Bind to operators (near promoter)
RNA = OH group, DNA = H - at 2’ carbon → Plasmid = small circular pieces of DNA (from bacteria) ○​ Effector binds to repressor →
→ Process: conformational change →
1)​ Cut source of DNA with restriction endonuclease binding/unbinding of repressor from
2)​ Select suitable carrier DNA (vector) operator → ↑/↓ transcription
3)​ Insert the gene into the vector (joining with DNA ●​ Activators - enhance RNA pol-promoter interaction ⇒
ligase) positive regulation
4)​ Antibiotic selection to check if the cloning is ○​ Binding sites near promoter
successful ○​ Molecular signal can ↑/↓ transcription
5)​ Insert the recombinant vector into host cell ●​ 𝝈 subunit of bacterial RNA pol - mediates promoter
6)​ The host producing multiple copies of recombinant recognition and binding (𝝈70)
CHROMOSOMES DNA ○​ Heat shock response: replaced to 𝝈32 →
●​ DNA wrapped up as chromosomes in nucleus → PCR (polymerase chain reaction): DNA amplification RNA pol directed to different set of
●​ Centromeres = holds chromatids together 1)​ DNA strands separated by heating promoters with new consensus sequence9
●​ Telomeres = rich in GC bases + large repeats of DNA 2)​ Annealing of primers to each strand for new strand → transcription of new products +
sequence synthesis chaperones that keep proteins in correct
●​ Supercoil = allow for entire length of DNA to fit 3)​ Extension of new DNA strands by DNA pol (Taq conformation in heat
within the nucleus DNA pol) 2) DNA-binding domains and protein-protein interaction domains
●​ Genome = all genetic material of an organism in regulatory proteins
●​ Haploid genome = 3.2 billion bp RNA METABOLISM (PROKARYOTES) ⇒ Amino acid residue-base pair interactions:
●​ Human - only 1.5% of gene is protein-coding genes RNA is single stranded (DNA is double stranded) ●​ Gln/Asn (A-T) → form H-bonds with A’s N-6, N-7 in
(total of 25,000 genes) ●​ Ribonucleotides = monophosphates major groove
●​ 55% of genome is repetitive DNA (45% of this DNA ●​ mRNA = encodes amino acid sequences ●​ Arg (G-C) → form H-bonds with G’s N-7, O-6 in
is interspersed elements) - Alu is 10% of genome (transcription) major groove
●​ VNTRs = name for all single copy satellite DNA loci ●​ tRNA = matches anticodon to mRNA, used for protein → DNA-binding domains:
○​ Types: minisatellites, microsatellites synthesis (translation) ●​ Helix-turn-helix (common lac repressor) → 20 aa = 1
●​ rRNA = large and small ribosomal subunits in alpha helix → beta turn → recognition helix for DNA
DNA PACKAGING (EUKARYOTES) translation (tetramer recognition) near major groove
DNA wrapped around histones RNA pol: generates RNA transcript (binding to NTPs) ●​ Zinc finger (eukaryotes) → 30 aa = Cys + His + loop
●​ Histone = H1, H2A, H2B, H3, H4 ●​ X proofreading capability (lacks 3’-5’ exonuclease cross-lined by Zn2+ → weak interaction with
●​ Arginine, lysine = main amino acids in histones activity) DNA/RNA → often act in tandem (직렬로)
Nucleosome = DNA + histone complex ●​ 2 𝛼 = assembly + binding to upstream promoter → Protein-protein interaction domain:
●​ Contains of 2 copies of H2A, H2B, H3, H4 ●​ 𝜷 = main catalytic subunit, 𝜷’ = DNA binding ●​ Leucine zipper (dimerisation) → homodimer of
●​ Linker DNA binds to H1 ●​ 𝜎 = directs RNA pol to the promoter amphipathic alpha helices + DNA-binding region +
DNA → nucleosome → twisted 30 nm fibre → loops around ●​ 𝟂 = protects RNA pol from denaturation every 7th aa is Leu (zipper region) + Lys, Arg
protein-based nuclear scaffold (6 nucleosomes) → rosette (6 loops) → Transcription: (not require primer to bind RNA pol) (DNA-binding region)
→ coil (30 rosettes) → chromatid (10 coils) ●​ RNA pol (𝜎 bound3) + 2Mg2+ = binds to promoter → Eukaryotic RNA-binding domain in gene activators:
region (TATA sequences4) → initiates transcription → ●​ RNA recognition motifs (RRMs) → 4 antiparallel beta
DNA REPLICATION 𝜎 replaced by protein NusA5 sheet + 2 alpha helices → gene activators bind to
●​ Occurs at replication forks ●​ NusG binds to RNA pol, ribosome → directly DNA, RNA
●​ Both strands serve as a template affecting rate of transcription ○​ DNA binding → gene transcription
○​ Each strand - synthesis of 2 new dsDNA ●​ Template strand = non-coding strand (3’ to 5’) → used ○​ lncRNA binding → ↓ gene transcription
○​ Both strands replicated simultaneously to form RNA transcript
●​ Semi-conservative ●​ Coding strand = non-template strand (5’ to 3’) → GENE EXPRESSION REGULATION IN BACTERIA (LAC
●​ Bi-directional (with RNA primer) same sequence as RNA transcript (T → U) OPERON) → lactose metabolism
●​ Semi-discontinuous (5’ to 3’) → Termination: when RNA meets terminator → Lac operon = gene cluster (lacZ, lacY, lacA) + promoter +
○​ Lagging strand (Okazaki fragments) ●​ ρ-independent = form hairpin (due to U) → RNA pol regulational sequences
dissociated + mRNA released → Lac operon is turned on when: lactosese is available, and
ENZYMES IN DNA REPLICATION ●​ ρ-dependent = ρ helicase binds to 6rut site on mRNA glucose is not available
●​ Topoisomerases = stabilise ssDNA, relieving transcription and releases mRNA (NusG) → Lac repressor, CAP bind to DNA of lac operon → regulates
topological stress transcription rate
●​ Primase = RNA pol that synthesises RNA primer RNA METABOLISM (EUKARYOTES) ●​ Lactose level detected by lac repressor - binds to
(10~20 bases) → Transcription: operator
●​ Helicases = unravel DNA ●​ RNA pol I = transcribes rRNA ●​ Glucose level detected by catabolite activator protein
●​ DNA pol - requires template + RNA primer, ●​ RNA pol II7 (12 subunits) = synthesis of mRNA (CAP) - binds to activator binding site
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proofreading (3’-5’) (recognise promoters8) 1) Lac repressor: Inhibits transcription of lac operon
○​ DNA pol I = proofreading (5’-3’) + ○​ Assemble of RNA pol II → TBP + TFIIH ●​ lactose is not available (high glucose): lac repressor
removing RNA primers and replacing = helicase (unwinds DNA at promoter Inr binds to operator → preventing transcription
with DNA, leaving slight gaps region), kinase activity (alters 2) CAP (activator): helps RNA pol binding to promotor
○​ DNA pol III = makes new DNA strand conformation of pol for binding) + etc ●​ low glucose level : cAMP is provided → cAMP
(insert dNTP2 in 5’ to 3’ direction) ●​ RNA pol III = makes tRNAs attaches to CAP → allowing CAP to bind DNA →
●​ DNA ligase = seals gaps with DNA + connects ●​ RNA pol IV (only in plants) CAP helps RNA pol bind to promoter → high levels
Okazaki fragments ●​ Elongation factors = bind to RNA pol II → enhance + of transcription
●​ Telomeres = adds DNA to end of chromosomes → coordinates elongation ●​ high glucose level: no cAMP made → CAP cannot
highly repetitive sequences (TTAGGG) + stabilise → Termination: RNA pol II released, dephosphorylated bind to DNA without cAMP → transcription occurs at
chromosomes → Post-transcriptional modifications: low level
1) mRNA processing: *THERE IS NEVER NO TRANSCRIPTION
DNA REPAIR ●​ Splicing (remove introns) → 3’-poly A tail by
●​ Mutations = change in DNA sequence polyadenylate polymerase → 5’-cap with 7-methyl GENE EXPRESSION REGULATION IN EUKARYOTES
○​ Ames test = using histidine lacking guanosine links to 5’-end via 5’-triphosphate link + → Most genes are subject to regulation by multiple transcription
bacteria forming rbs factors
●​ Proofreading + error checking mechanism → fidelity 2) Introns splicing: ⇒ combinatorial control: using different combinations of limited
○​ DNA pol III = 3’-5’ endonuclease + 5’-3’ ●​ Self-splicing group I and II introns repertoire of transcription factors at each gene
pol activity ○​ Group I = attack of guanosine 3’-OH on ●​ Positive regulation (genes regulated by activators)
●​ MutL-MutS complex = notice + correct errors in phosphodiester bond between U + A ●​ Mix + match of regulatory proteins to form dimers
replication of chromosomal DNA ○​ Group II = attack of adenosine 2’-OH on 1) Transcription activators
●​ Methylation = mismatch-repair enzymes use lack of phosphodiester bond at 5’ end of intron ●​ Activator binding to enhancer/UASs10 triggers many
methylation to identify + remove newly synthesised (lariat formed - splices 3’ end) promoters → ↑ transcription
DNA 2) Architectural regulators to facilitate DNA looping
DNA damage: ●​ Brings binding sites of activators + repressors +
●​
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Deamination = losing amino group by DNA base (U to 𝜎 factor converts closed complex to open complex promoters together → interact directly
C)
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Consensus sequences in -10 (TATA sequence = Pribnow box) and ●​ E.g. high mobility group (HMG)
●​ Depuration = losing purine via hydrolysis -35 regions for 𝜎 subunit binding 3) Chromatin remodelling (heterochromatin → euchromatin)
○​ UV light forming pyrimidine dimer
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NusA = stabilising RNA pol + moving along DNA + keep → Histone covalent modification:
●​ Damage from reactive chemicals: processing RNA ●​ Methylation: changes conformation
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Rut site = common CA-rich sequence
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Recruitment of RNA pol II initiated by TATA-binding protein
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Proofreading = removing incorrectly inserted bases (TBP) i.e. changing binding affinity of RNA pol
2 10
dNTP = deoxynucleoside triphosphate = base 8
Consensus sequence TATA box (-30), Inr sequence (+1) UASs = upstream activator sequences
 ○​ Binding to DNA → heterochromatin d.​ Proteins directly released into ER lumen ●​ R-groups not involved in H-bonding
○​ Binding to histone (Lys-4/36) → e.​ Signal peptidase cleaves N-terminus (signal sequence) ●​ R-groups involved in backbone mediated structure
euchromatin + dissociates ribosome from its receptor 1) Alpha-helix (right-handed)
●​ Acetylation of Lys: E.g. Proteins imported into nucleus ●​ L-isomers, 3.6 residues per turn
○​ Neutralise +ve charged Lys in histone → a.​ NLS (nuclear localisation sequence) produced in ●​ Ala, Glu, Leu, Lys, Met = good formers
loose connection histone-DNA → nuclear protein ●​ Pro, Gly, Tyr, Ser = poor formers
euchromatin b.​ Binds importin alpha, beta subunits → binds to nuclear ○​ Glycine = too small
○​ Done by HATs, reversed by HDACs pore + translocates into nucleus ○​ Tyr, Trp = too large
→ Nucleosome restructuring: SWI/SNF family c.​ Dissociation of importin beta by binding of Ran-GTP ●​ Proline has imino group → kinks in transmembrane
●​ Nucleosomes become more irregulately spaced + d.​ Importin alpha binds to Ran-GTP + nuclear protein helices
stimulate transcription factor → ↑ transcription released ●​ H-bonds within polypeptide chain (intrachain)
→ Nucleosome remodelling: CHD family e.​ Importin alpha, beta transported out of nucleus 2) Beta-pleated sheet
●​ Binds to heterochromatin → unwinds DNA with 7) Protein degradation ●​ Anti-parallel (strong) - linear H-bonds
helicase → histones detach from DNA → ↑ → Eventually all proteins are degraded ●​ Parallel (weak) - bent H-bonds
transcription → Defective (unstable) proteins are short-lived ●​ Type I beta-turn - proline (kinking)
4) Coactivators → High activation energy is required for hydrolysis of peptide ●​ Type II beta-turn - glycine (too small + flexible)
●​ Mediate interaction between activators + RNA pol bonds ●​ H-bonds in crosslinkage/in between (interchain)
at/near promoter’s TATA box → mechanisms of degradation: → Tertiary structure: mixture of secondary proteins
●​ E.g. mediator binds to C-termianl domain (CTD) of ●​ E. coli: ‘Lon’ hydrolyses defective/shortlived peptides ●​ Weak interactions + sometimes covalent bonds
RNA pol II ●​ Eukaryotes: proteins with ubiquitin are cleaved by 26S (cross-links)
●​ E.g. TBA recognises TATA box proteasome complex 3D-conformation of proteins:
5) Post-transcriptional gene silencing → RNA interference ●​ Native fold = correctly-folded (functional) by weak
●​ miRNAs = prevent mRNA transcription by cleaving it [Prokaryotes vs Eukaryotes on translation] non-covalent bonds → favourable interaction
with Drosha/Dicer (e.g. stRNAs) ●​ unfolded/incorrectly-folded proteins → denatured
●​ siRNAs bind to mRNA and silence its translation PROKARYOTES EUKARYOTES ●​ Dependent on R-groups
6) RNA-mediated regulation of gene expression = ncRNAs 1) Fibrous (structural)
●​ HSR1 (heat shock RNA 1): functions in complex with 30S + 50S = 70S ribosome 40S + 60S = 80S ribosome ●​ Long + narrow
translation elongation factor (eEF1A) to regulate ●​ Repetitive of one secondary structure
trnalsaltion in stress cells fMet-tRNAfMet Met-tRNAi (not formylated) ●​ Generally insoluble in water
●​ 75K: binds to RNA pol II transcription elongation ●​ E.g. Alpha-keratin (hair) → heat (chemical reduction)
factor (pTEFb) to repress elongation Shine-Dalgarno sequence in 5’-cap in mRNA disrupts S-S + H-bonds in polypeptide chains
mRNA ●​ E.g. Collagen in tendons + bonds → provides strength
TRANSLATION → protein synthesis in connective tissue
→ polysome: clusters of ribosomes IFs + fMet-tRNAfMet + GTP eIFs + Met-tRNAi + GTP ○​ 3 left-handed type-2 alpha-helices joined
→ proofreading occurs in A site of ribosome bind to 30S subunit bind to 40S subunit as right-handed helix
1) Activation of amino acids (tRNA charging) ​ ○​ X S-S bonds, stabilised by hydroxylation
a.​ Aminoacyl-AMP formed from ATP hydrolysis Poly-cistronic mRNAs Mono-cistronic mRNAs of proline
b.​ Transfer of activated aa to tRNA by aminoacyl-tRNA 2) Globular (functional)
synthetase11 → aminoacyl-tRNA formed Antibiotics inhibit protein No effect on antibiotics ●​ round/sphercial
2) Initiation of translation synthesis ●​ Mixture of secondary strucutres
a.​ 30S subunit binds IF1, IF2-GTP, IF312 Transcription (nucleus) → ●​ Generally soluble in water
b.​ 16S rRNA in 30S subunit binds to SD sequence of Transcription + translation post-transcriptional ●​ E.g. PsaA - Zn increases thermal stability
mRNA occur at the same time modification → translation → Quaternary structure: assembled subunits
c.​ fMET-tRNAfMet binds to P site (start codon) of 30S (cytosolic ribosome) ●​ Stabilised by disuflide bonds (+ weak bonds hold)
subunit ○​ Broken by reducing agents
d.​ GTP hydrolysis (IF2) → dissociation of IFs → [Effects of antibiotics + toxins on protein synthesis] ●​ Protein denaturation: (disruping non-covalent bonds)
docking of 50S subunit → 70S initiation complex → antibiotics: ○​ Heat (disrupts H-bonds) - Zn>Mn
3) Elongation E.g. Puromycin is aminonucleoside antibiotic that blocks protein ○​ Extremes of pH (alter R-groups)
a.​ EF-Tu-GTP carries aminoacyl-tRNA to A site of synthesis in both prokaryotes + eukaryotes: ○​ Organic solvents (e.g. alcohol)
ribosome → hydrolysis of GTP + dissociation of the ●​ Premature termination of newly forming polypeptide ○​ Urea + guanidien hydrochloride
complex chains → degradation ○​ Detergents
b.​ fMET group transferred to aa in A site by 23S rRNA ●​ Binds to A site of 30S subunit → prevents ●​ E.g. Fully functional multimeric hemoglobin
(ribozyme13) → peptide bond formation translocation of peptidyl-tRNA [Determining protein structures]
c.​ Ribosome moves down 1 codon by EF-G (translocase) E.g. Tetracyclines (30S), streptomycin (30S), Chloramphenicol + ●​ X-Ray diffraction (crystalisation)
+ GTP hydrolysis → open A site cycloheximide (50S) ○​ +: no size limits, well established
4) Termination and ribosome recycling → toxins: (inhibiting eukaryotic protein synthesis) ○​ -: difficult for membrane proteins/lipids,
a.​ Stop codon in A site → RFs release peptide + tRNA ●​ E.g. Diphtheria (elF2), ricin (60S) cannot see hydrogens
from ribosome → subunits dissociation (IF3 bindings ●​ Nuclear Magnetic resonance (NMR)
to 30S subunit) PROTEINS ○​ +: no need to crystallise protein, can see
5) Post-transcriptional modification and folding → Primary structure: amino acid sequence many hydrogens
→ Adding of prosthetic groups to conjugated proteins Amino acids (20 aa): ○​ -: difficult for insoluble proteins, works
●​ Apo-protein = protein without prosthetic groups ●​ Central alpha carbon, carboxyl group, amino group, best with small + less complex proteins
●​ Holo-protein = protein with prosthetic groups alpha hydrogen, R group ●​ Cryo Electron Microscopy (EM) → take video
→ Proteostasis: maintaining stable state of proteins ●​ Proteins only contain L-isomers of aa ○​ +: no need to crystallise protein, can
●​ Cell employs chaperones which assist in ●​ Linked by peptide bonds handle large proteins complexes (virus),
post-translational regulation of protein synthesis, Isoelectric point (pI) of aa: suitable for membrane protein
folding, assembly, degradation processes ●​ pI = electrically neutral pH of aa = mean of pKa ○​ -: relatively low resolution
●​ Lower free energy = more stable complex ●​ Each aa has its own pKa14 - depending on local
→ Chaperones prevent misfolding and aggregation of proteins environment (e.g. ligand binding - own charge) PRACTICAL
●​ Hsp70 (Heat shock proteins) bind to unfolded ●​ Charge of aa change ability to interact with other aa → → plasmids:
polypeptides with rich hydrophobic aa influence protein structure ●​ pMICO = chloramphenicol resistant
○​ Protect proteins from heat ●​ ⇒ finding pI = helps identifying stability of protein ●​ pGLO = ampicillin resistant
○​ Protect during protein synthesis (solubility, structure, function, regulation) → Types of DNA fragments:
○​ Block folding if necessary ●​ aa (acidic pKa) = protonated (donate H+) in low pH → ●​ Partial digest = incomplete double stranded digestion
○​ X instructs protein folding carboxyl group (+1) of plasmid DNA
●​ Chaperonin (hsp60) ●​ aa (basic pKa) = protonated until high pH reached ●​ Linear DNA = plasmid DNA that has been cut at both
○​ Facilitate folding of proteins that don’t ●​ → amino group (-1) strands
fold spontaneously ●​ Charge on protein depends on ionisation of R-groups ●​ Nicked (open circled) DNA = plasmid DNA that has a
→ Protein folding → to lowest-energy fold → depends on pH single strand broken and runs less distance than linear
a.​ Local secondary structures form Grouping of aa: DNA of the same size
b.​ Super-secondary structures (dimers) form ●​ Supercoiled (closed circle) DNA = plasmid DNA that
c.​ Domains form → fold into stable globular units NON-POLAR POLAR
is undamaged and runs faster than linear DNA of the
d.​ Final tertiary structure forms same size
*Hydrophobic interaction facilitates protein folding Aliphatic/lipophilic: -ve (acidic), hydrophilic: ⇒ Supercoiled DNA > linear DNA > nicked DNA
[Protein misfolding] Gly*, Asp, Glu (fastests to slowest)
E.g. cystic fibrosis: one amino acid error in plasma-membrane Ala (hydrophobic), Pro, Val, → Ways to improve cutting ability of enzyme:
chloride transporter (CFTR) → incorrect folding → absence in Leu, Ile, Met +ve (basic), hydrophilic: ●​ Using appropriate buffer for the enzyme to cut the
epithelial cells Lys, Arg, His plasmid
6) Protein targeting Hydrophobic, aromatic: ●​ Use more units of enzyme in reaction and incubate
→ Proteins are targeted to specific places through N-terminal Phe, Tyr (-OH can form Uncharged, hydrophilic: restriction enzyme digest for a longer time
sequence (predominantly in eukaryotes) H-bonds), Trp Ser, Thr, Cys*, Asn, Gln ●​ Use appropriate temperature for the enzyme to cut the
E.g. Proteins directed to ER: → H-bonds = soluble plasmid
a.​ Signal sequence produced at N-terminus ●​ Use less DNA in reaction (if DNA fragments seem to
b.​ SRP (signal recognition particle) binding with *glycine = too small so neither of hydrophobic, hydrophilic be larger in size)
ribosome *cysteine = form covalent bonds (disulfide bonds) in oxidised state → isolation of plasmid DNA
c.​ SRP-ribosome complex interacts with SRP + ribosome *selenocysteine (make selenoproteins) act as nucleophile (donates 1) concentrate bacterial culture
receptor on surface of ER e-), antioxidant → encoded by UGA codon (stop) → produces 2) lyse cells
selonocysteine when Selenium (Se) is present 3) inactivate endonucleases
→ Secondary structure: conformation of polypeptide backbone 4) remove cellular debris
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Aminoacyl-tRNA synthetases cannot recognise different aa ●​ Stabilised by H-bonds 5) purify plasmid DNA
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IF3 keeps 30S and 50S subunits apart 6) elute plasmid DNA
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Ribozyme = RNAs with catalytic activity (23S rRNA is part of 7) determine purity and concentration of DNA
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50S ribosomal subunit) Lower pKa = stronger acid