Semi-conservative method: one parental strand used as template for synthesis of new daughter strand and each new DNA comprise of the parental template and the new strand. Preamble: DNA has to be replicated in high fidelity before cell division.

Process:

• Occurs during interphase (S phase)
• Helicase (6 proteins in ring shape) binds to origin of replication(s) [multiple sites on DNA with similar DNA sequences] and unwinds DNA molecule by breaking hydrogen bonds, creating replication fork
• Single-stranded binding proteins (SSBs) bind to the DNA to stabilise it and is assisted by topoisomerases (exonuclease cum ligase) to remove supercoiling stress during unwinding by cleaving the phosphodiester bonds (Topo I cleaves at 1 strand while Topo II cleaves on both strands and later "glues" them back as they have sequence specificity. Supercoiling stress may cause irregular strand breaks)
• No ATP is required for topoisomerase I as it uses the tension from the supercoiling to unwind DNA and the tyrosine residues bind to the free phosphate end (after cleaving) to stabilise and move it to opp side. 2 ATP molecules required for topoisomerase II as it needs to cleave both strands and involves binding of 2 DNA segments for cleavage of one of the first bound segment (involving Mg2+) and the formation of phosphotyrosine bonds for stability
• Primase (RNA polymerase) synthesises short RNA primers (5 nucleotide sequence) from free RNA nucleotides to create free hydroxyl group at 3' end.
• DNA polymerase III (right-hand shaped enzyme) extends the DNA strand by adding free DNA nucleotides to the 3' end and catalysing the phosphodiester bonds between 5' phosphate group of new DNA nucleotide triphosphate and existing 3' hydroxyl group.This is faciliated by Mg2+ ions (deficiency results in brittle nails & leg cramps in morn) and accessory proteins eg sliding clamp to hold polymerase onto strand
• Due to nature of directionality, there is a lagging and leading strand formed at both ends of replication bubble.
• RNAse H aids in excising the RNA primers (recognise RNA-DNA hybrid helices & degrades RNA by hydrolysing phosphodiester bonds. DNA Polymerase I replaces the gaps with DNA nucleotides and ligase is used to catalyse the formation of phosphodiester bonds (using ATP) between the Okazaki fragments (1000-5000 nucleotides) to form long continuous DNA

Central Dogma:
DNA used as template for complementary RNA synthesis which is translated to produce the corresponding peptide chain. Each codon codes for a specific amino acid and the transfer of biological info from gene to protein is termed Central Dogma.

For eukaryotes: Occurs in nuclear membrane.

• [initiation] Transcription Factor (TF) 2D contains TATA box binding protein (TBP) that recognises the promoter sequence (TATA box/10 A-T rich seq (3' AAATAT 5' seq found 26 nucleotides upstream of initiation site) & initiation site is at +1 position) on antisense/non-coding strand and binds, unwinding the DNA helix. TF2B and TF2A then binds to form the multi-protein complex which RNA Polymerase II recognises (low degree of specificity compared to prokaryotes) and binds to. TF2E, TF2F and TF2H then binds to form a stable pre-transcription initiation complex (helps direct & position RNA polymerase for transcription)
• [elongation] RNA polymerase II unwinds DNA strand (to form transcription bubble) and free RNA nucleotides form complementary base pair to the DNA template and mRNA synthesised from 5' to 3' and topoisomerase involved to reduce supercoiling stress
• [termination] Near the end of the translated sequence, the polymerase transcribes the polyadenylation signal [AAUAAA] in the sequence and endonucleases cleave the transcript ~ 10-35 nucleotides downstream from the signal & transcription ceases. The mRNA is then released.
• [Post transcriptional modification] Pre-mRNA will contain a 7-methylguanosine cap at 5' end [joined in reverse orientation to mRNA, creating a 5' to 5' triphosphate bridge and catalysed by mRNA guanyltransferase] (protect from 5' exonucleases, exporting mature mRNA from nucleus to cytoplasm & attachment to ribosome), a polyadenylate/polyadenine tail (100-200 adenylate residues) added to 3' end [after the polyadenylation signal] by poly (A) polymerase to increase half-life/metabolic stability and the introns (non-coding regions for alternative splicing and usually contain "GU" and "AG" sequences [splice sites]) are excised (and exons spliced), to form the mature mRNA that is exported to cytoplasm
  Silencer & Enhancer regions found upstream of promoter/distal to regulate gene expression rate by interacting with the pre-transcription initiation complex when the DNA strand loops onto itself at a right angle
  Each gene is under control of single promoter sequence with the specific distal enhancer & silencer regions (multi-point control) [absence of operator & polycistronic mRNA unlike in prokaryotes]. TATA box (2 H bonds between T and A, allowing for rapid unwinding) may also be absent in eukaryotic promoters with CG rich regions as rate of transcription is generally lower
  Splicing process is initiated by 5 small nuclear ribonucleoproteins that bind to the splice sites and complex to form spliceosome, bring the exons upstream & downstream close together for splicing (and introns excised)

For eukaryotes:

• [Initiation] 40s ribosomal subunit interacts with eukaryotic initiation factors (ard 12 factors) to form the pre-translational complex and amino-acyl tRNA carrying methionine is recognised & recruited. The complex then binds to the 5' cap/internal ribosome entry site and travels downstream from untranslated region to Kozak seq (5' A/GCCACCAUG 3') containing the start codon (1st AUG codon). ATP is then utilised for release of the initiation factors and the 60s subunit binds to complex [GTP involved] and the tRNA is directed to P site.
• [Elongation] The complex then moves downstream and another aminoacyl tRNA carrying the next amino acid attaches at the A site and a peptide bond is catalysed between both a.a by peptidyl transferase (found on ribosomal subunit). Existing phosphoester bonds between methionine and tRNA is cleaved. The complex proceeds downstream again [GTP is hydrolysed in translocation process] and another aminoacyl tRNA then attaches at A site, while the second tRNA moves to the P site and the first tRNA is displaced at E site. The entire process is assisted with elongation factors that remain bound to complex and polypeptide is synthesised from amino to carboxyl end.

• [Termination] Process repeats until stop codon (UAG/UGA/UAA) is reached and there is no tRNA to bind to and release factors recognises seq and binds, promoting hydrolysis of peptidyl-tRNA link. The ribosomal subunits then dissociate and the mRNA is used to make another protein chain.

  
  

  Peptidyl transferase is a form of ribozyme (RNA molecule with enzymatic function)

  
  

  Process expedited with the formation of polyribosomal complexes, allowing a single mRNA to make multiple polypeptide copies simultaneously

  
  

• [Post-translational modification] Required to confer functionality eg tertiary folding (assisted by chaperone proteins), attachment of signal peptide to N-terminus (short a.a seq used to transport polypeptide into rER via channel proteins & cleaves by enzymes in cisternae), binding to co-factors (if any) or polymerisation eg helicase (hexamer of helicase monomers) which increase proteomic diversity [Refer to carbohydrates for specifics]

  
  

  Eg erythropoietin (produced in kidneys): increase r.b.c. production by inducing erythroid progenitor cells to differentiate & release of reticulocytes/mature rbc to general circulation from bone marrow and increased production usually stimulated by tissue hypoxia (insufficient O2 transport due to anaemia) EPO treatment used for anaemia treatment (chronic renal failure) or for patients after post-chemo/bone marrow transplant/surgery to replenish r.b.c. count and made up of 66 a.a and is heavily glycosylated with 3 N-linked carbohydrate chains (Mr increase from 18.4kDa to 30.4kDa) and 2 internal disulfide bonds between Cys residues. Synthetic recombinant EPO produced: epoetin a and epoetin b having same a.a. seq and almost similar glycosylation while darbepoetin (long-acting: injection once every week or alternate weeks) has 2 additional N-linked carbohydrate chains (increased Mr and sialic acid residues from 14 to 22), increasing half-life. [manipulation of glycosylation during drug designing process]

Alkylating agents: covalent binding of agents (electrophiles eg mechlorethamine/nitrogen mustard & cyclophosphamide (anti-cancer drug) binds to N in imidazole side chain of guanine & adenine to form a dimer (no base-pairing) and disrupt transcription/replication process.

Intercalactors: highly planar molecules that insert themselves between adjacent nucleotides due to pi-pi stacking interactions between aromatic rings and drug (strong interactions with DNA) eg doxorubicin (anti-cancer), disrupting the major & minor grooves.

Eg Camptothecin (CPT) that forms H bonds with DNA & topoisomerase I to form a stable ternary complex (ESI complex) and prevent DNA religation, thus damaging cellular DNA (accumulation of supercoiling and strand breaks upon collision with helicase/polymerase) during replication to arrest cell dvision and used as anti-cancer drug.
Eg Fluoroquinolone-class of drugs (nalidixic acid [Gen 1], oxolinic acid [Gen 1], norfloxacin [Gen 2], Ciprofloxacin [Gen 2], Levofloxacin [Gen 3], Moxifloxacin [Gen 3] and Gemifloxacin [Gen 4]) that inhibits bacterial topisomerase II (not human), resulting in accumulation of cleavage complexes that has bactericidal effect.

Types of RNA

• tRNA (15%): small nucleotide seq and transport a.a to site of protein synthesis
• rRNA (80%): variable nucleotide seq and combines with proteins to form ribosomal subunits
• mRNA (5%): variable nucleotide seq and used as template for polypeptide synthesis
• small nuclear RNA: small nucleotide seq for pre-transcriptional modification of mRNA to mature form
• small interfering RNA: affect gene expression (possible inhibitor?)
• micro RNA: affect gene expression and incolved in growth & expression

For prokaryotes: Occurs in cytosol

• [initiation] Binding of sigma factor to RNA Polymerase, allowing it to recognise the promoter sequence/Pribnow box [3' TAATAT 5'] on antisense/non-coding
  strand (3' to 5' direction) and bind to it for use as template for synthesis of primary transcript as well as confer stability in binding
• [elongation] RNA polymerase unwinds DNA strand (to form transcription bubble) and free RNA nucleotides form complementary base pair to the DNA template and mRNA synthesised from 5' to 3'. After about 10 RNA nucleotide seq, the sigma factor is released and the RNA polymerase undergoes conformation changes. Elongation proceeds rapidly (40 nucleotides/sec) and no accessory proteins/sliding clamp required (unlike DNA replication) but topoisomerase involved to reduce supercoiling stress
• [termination] Rho independent: Termination sequence (rich C-G regions) induces the formation of a RNA hairpin stem-loop structure with inverted repeated sequences located upstream of 6-8 uracil nucleotides near 3' end that disrupt RNA polymerase binding, resulting in dissociation & terminate transcription
• [termination] Rho dependent: RNA transcript contains binding site for Rho factor that "chases" after RNA polymerase and contains ATPase dependent helicase (unwind RNA strand from DNA) and termination ends when Rho factor displaces RNA polymerase at Rho termination site
  Rate of transcription/gene expression is dependent on affinity for Polymerase to promoter seq: TATA box (found on coding strand)/10 A-T rich seq (TATAAT seq found 10 nucleotides upstream of initiation site); TTGACA seq found 35 nucleotides upstream of initiation site & initiation site is at +1 position
  Operon consists of activator binding site (activator proteins bind to upregulate expression), promoter seq (containing TATA box), operator seq (repressor proteins bind to downregulate expression) and one/multiple different genes involved in same biochemical pathway (to be transcribed) with the mRNA termed as polycistronic (contains multiple genes)
  Negative Regulation: Lac operon with Lac Z (codes for b-galactosidase to metabolise lactose to glucose & galactose), Lac Y (lactose transporter), Lac I (repressor protein) and Lac A (transacetylase which may be used to detoxify lactose analogs) genes under control of single promoter and repressor protein (tetramer) to bind to operator seq at N-terminal, preventing transcription, in absence of lactose.
  Positive Regulation: In presence of lactose (and absence of glucose), the lactose matabolite (allolactose) binds to C-terminal of tetramer, causing conformation change and dissociate from operator seq, allowing for transcription

Eg Acyclovir (structural similiarity to viral nucleoside: deoxyguanosine but the deoxyribose lacks a complete sugar ring and is a prodrug, hence needs 3 step phosphorylation to form the active drug) and is incorporated into viral DNA strand and causes premature termination of chain (lack free OH group at 3' end for elongation) and used in antiviral (cream & oral forms with 5 times a day as viral replication is very rapid & drug has short half-life) & anticancer drugs

Eg Rifampicin (from Streptomyces mediterranei) binds to b-subunit of RNA polymerase, preventing RNA elongation (steric hindrance to addition of free RNA nucleotides to strand) but does not prevent initiation as the first few RNA nucleotides can still bind to the polymerase. Used in cocktail of drugs for TB treatment

RNA consists of pyrimidines (C/U - U is similar to T with absence of methyl side chain at C=C portion of ring) and purines (A/G) bonded to ribose sugar (consists of 2 hydroxyl groups)

• Universal: used by eukaryotes & prokaryotes with little exceptions
• Highly specific/non-ambiguous: one codon codes for a specific amino acid
• Degenerate/redundant nature: many codons code for same amino acid (except for Met & Trp) to mitigate effects of single base/point mutations, hence 20 aa but with 61 codons (last 3: UAA, UAG & UGA for stop) and usually is the degenerate nature of 3rd base/wobble base eg GGA/GGG/GGC/GGU all code for Gly
• Code is non-overlapping (i.e. read in sequences of triplet base) & punctuated (contains start/stop codon)
• Codons coding for same amino acids/amino acids with similar R group properties are similar in seq eg GAU/GAC code for Aspartic Acid & GAA/GAG code for Glutamic acid
  Codon bias with codons of C/G preferred over other degenerate codons due to more H bonds, conferring greater stability

Each tRNA contains an unique anticodon (that base-pairs to complementary seq on mRNA) and each tRNA has its specific amino-acyl tRNA synthetase that bonds the corresponding a.a to its tRNA at the 3' end (ATP involved), and is termed a.a activation. tRNA has a clover-leaf shape (stem-loop structure) with 5' end ending in G and 3' end ending with CCA (Asp)

For prokaryotes:

• [Initiation] 30s ribosomal subunit (contains complementary rRNA seq to Shine-Dalgarno seq) interacts with initiation factors (ard 3 factors) to form the pre-translational complex and amino-acyl tRNA carrying formylmethionine is recognised & recruited. The complex then binds to the Shine-Dalgarno seq (5' AGGAGG 3') which is 6 nucleotides away from 1st base of start codon (1st AUG codon). ATP is then utilised for release of the initiation factors and the 50s subunit binds to complex [GTP involved] and the tRNA is directed to P site.
• [Elongation] The complex then moves downstream and another aminoacyl tRNA carrying the next amino acid attaches at the A site and a peptide bond is catalysed between both a.a by peptidyl transferase (found on ribosomal subunit). Existing phosphoester bonds between methionine and tRNA is cleaved. The complex proceeds downstream again and another aminoacyl tRNA then attaches at A site, while the second tRNA moves to the P site and the first tRNA is displaced at E site. The entire process is assisted with elongation factors that remain bound to complex and polypeptide is synthesised from amino to carboxyl end.
• [Termination] Process repeats until stop codon (UAG/UGA/UAA) is reached and there is no tRNA to bind to and release factors recognises seq and binds, promoting hydrolysis of peptidyl-tRNA link. The ribosomal subunits then dissociate and the mRNA is used to make another protein chain.
  Transcription & translation can occur simultaneously as there is no nuclear envelope

Eg tetracycline binds to 30s ribosomal subunit and prevent recruitment of amino-acyl tRNA to RNA-ribosomal complex, inhibiting protein synthesis; Oxazolidinones bind to 50s ribosomal subunit and prevent binding to complex; Chloramphenicol blocks peptidyl transferase; macrolides & aminoglycosides prevents complex from moving downstream/translocation.

Gene synthesis control:
1) Chromatin remodelling/effect of chromatin packaging [for eukaryotes]: a) histone acetylation (by histone acetyltransferase) with addition of acetic acid to free lysine residues at N-terminal of histones, decreasing net positive charge (and lower affinity for DNA backbone), resulting in increased accessibility of DNA [less steric hindrance of RNA polymerase & transcription factors to promoter seq] and possible binding of additional gene regulatory proteins to control elements of gene; b) DNA methylation at CpG rich regions (cytosine), interfere with binding of transcription factors to these sites and reinforces histone deacetylation (reverse of acetylation)
2) Control elements: silencer & enhancer regions of DNA [for eukaryotes]
3) Operon control with operators & activator binding domains [for prokaryotes]
4) Post-transcriptional control [for eukaryotes]: a) Half-life of RNA determined by length of Poly (A) tail (determine rate of protein synthesis) and can be affected by certain hormones/binding proteins (to 3' untranslated region) to mark for degradation, b) 5' cap and c) repressor proteins/siRNAs binding to 5' untranslated region, acts as steric hindrance for ribosomal binding
5) Post-transcriptional control [for prokaryotes]: Stem-loop at 3' end (to prevent digestion by 3' exonucleases) and repressor proteins/siRNAs to inhibit translation
6) Post-translational control [for eukaryotes]: a) protein modification eg conjugation reactions, b) proteolytic cleavage eg removal of signal peptide, c) protein degradation through ubiquitinylation (covalent addition of ubiquitin) to N-terminus of protein to mark for degradation by proteasome

Groove binders: crescent-shaped molecules that fits into the grooves of the DNA double helix through Van der Waals interactions eg pentamidine (anti-microbial) or can affect the stability of DNA by interacting with the negatively-charged sugar-phosphate backbone.