BIOCHEMISTRY

• Phosphodiester bond is a type of covalent bond formed between phosphate group of one nucleotide and hydroxyl group (OH) on sugar of another nucleotide which is essential for polymerisation of nucleotides into nucleic acids chains such as DNA and RNA
• Biosynthesis of Pyrimidine

  1. Pyrimidine ring is synthesised as free pyrimidine from carbamoyl phosphate and aspartate before ribose is supplied by PRPP

  2. Carbamoyl phosphate is derived from CO2 and glutamine

  3. Carbamoyl phosphate reacts with aspartate to form carbamoyl aspartate which undergoes further reactions to produce dihydroorotate

  4. Dihydroorotate converts into orotate and followed by addition of ribose-5-phosphate to form OMP

  5. OMP is converted into uridine monophosphate (UMP) through enzymatic reactions of decarboxylation, phosphorylation and amination
• Catabolism of purine base

  1. Mainly degraded into uric acid in liver

  2. Most common abnormality is hyperuricemia which is referred to as gout
• De novo biosynthesis of purine

  1. Major site of purine biosynthesis is the liver

  2. Begins with phosphoribosyl pyrophosphate (PRPP)

  3. Purine ring is assembled on ribose-5-phosphate molecule of PRPP

  4. Involves 10 steps
• Structure of DNA

  1. Double helix - two polynucleotide strands wind around each other in a right-handed manner

  2. Nucleotides consist of 4 nitrogenous bases linked together by phosphodiester bonds between 3’C sugar and 5’C sugar on another sugar
• Functions of DNA

  1. Gene: basic physical unit of hereditary that occupies specific locus of chromosomes where specific DNA sequence coding for a polypeptide

  2. Storage of Genetic Characteristics: genetic characteristics are stored as sequence of nucleotides in specific gene where size of human genome 3x10^9 b.p.

  3. Gene Expression: genetic information coded by the nucleotide in a gene is transcribed into nucleotide sequence of RNA where first step of gene expression is transcription

  4. Replication: biosynthesis of DNA where it provides with genetic information with high fidelity to maintaining genetic stability. Semi-Conservative (one old and one new)

  5. Transcription: DNA dependent RNA biosynthesis with 3 different types of RNA
• Eukaryotic DNA replication

  1. DNA helicase unwinds the DNA at origin

  2. SSN proteins keep DNA strands separated

  3. Topoisomerase prevents DNA supercoiling

  4. RNA primase places primers for DNA polymerase to build new strands

  5. Formation of leading and lagging strands with okazaki fragments

  6. DNA ligase seals gaps between okazaki fragments

  7. Resulting in 2 identical double helix DNA molecules which are semi-conservative (1 parent and 1 new)
• RNA

  • Exists as a single-stranded molecule
  • Sugar is ribose which contains hydroxyl group (OH) at second C where it is more reactive and less stable compared to DNA
  • Thymine will be replaced by uracil
• Cell Cycle Stages

  1. Interphase: G1, S, and G2 phases where cellular growth and DNA synthesis occur leading to duplication of cellular materials for 2 complete new daughter cells

  2. Mitosis: 5 stages resulting in formation of two separate daughter cells

  3. Cytokinesis: cytoplasmic division following nuclear division
• DNA replication

  1. RNA primase needs to keep adding primers for DNA polymerase to build new strands and form okazaki fragments

  2. DNA ligase seals the gaps between okazaki fragments

  3. At the end of replication, 2 identical double helix DNA molecules are formed which is semi-conservative (1 parent and 1 new)
• Nucleotide Excision repair

  1. Removes UV light-induced DNA damage

  2. Forms pyrimidine-pyrimidine (T-T) dimers
• Mismatch repair


  1. Corrects mismatches of bases due to failed Watson-Crick base pairing

  2. Repair proteins involved: MLH1, MSH6, PMS1, PMS2
• Transcription Elongation

  1. Sigma factors dissociate from RNA polymerase

  2. RNA synthesis in 5' to 3' direction

  3. RNA polymerase catalyzes phosphodiester bond formation between ribonucleotides
• DNA Transcription stages

  1. In prokaryotic cells: initiation, elongation, termination

  2. In eukaryotic cells: initiation, elongation, termination, pre-messenger mRNA formation, editing by splicing
• Prokaryotic Transcription Termination

  1. Rho-dependent termination: Rho factor climbs up RNA molecule to release it

  2. Rho-independent termination
• DNA repair mechanism

  1. Necessary due to exposure to environmental mutagens and spontaneous mutations during DNA replication

  2. In most cases, undamaged DNA strand is used as a template to correct mistakes, if both strands are damaged, sister chromatid is used

  3. Involves recognition, excision, repair by DNA polymerase, and sealing by DNA ligase
• Transcription Termination

  1. Occurs at stop sequence

  2. RNA polymerase releases DNA template, which unwinds back to double helical structure
• Double-stranded DNA repair

  1. Repairs damage from ionizing radiation, oxidative free radicals, or chemotherapeutic agents

  2. Homologous recombination: uses unaffected homologous chromosomes, involving BCRA 1 and 2 proteins

  3. Non-homologous end joining: joins ends without similarity, can introduce mutations
• Transcription Initiation

  1. Prokaryotes: initiation at promoter region, RNA polymerase binds with sigma factor, promoter sequence recognized by transcription factors

  2. Eukaryotes: RNA polymerase requires helper promoter, TATA promoter sequence recognized by transcription factors
• Base Excision repair

  1. Corrects spontaneous depurination and deamination

  2. Involves recognition and removal of nucleotides that have lost bases
• Components required for translation: amino acids, transfer RNA (tRNA), aminoacyl-tRNA synthetases, messenger RNA (mRNA), functionally competent ribosomes
• Role of different enzymes in RNA synthesis

  1. Major enzymes used in DNA transcription is RNA polymerase

  2. Prokaryotes use one type of RNA polymerase, while eukaryotes use 3 types

  3. RNA polymerase I synthesises rRNAs, RNA polymerase II synthesises mRNAs and miRNAs, RNA polymerase III catalyses the synthesis of tRNAs

  4. Formation of initiator complex, synthesis and elongation of RNA by adding nucleotide bases, formation of termination sequence
• Rho-dependent termination


  Rho factor climbs up towards RNA polymerase, pulls RNA transcript and DNA template strand apart to release RNA molecule
• Characteristics of genetic codes: specificity, universality, degeneracy, nonoverlapping and commaless
• Mature mRNA has open reading frames (ORF) which are translated into proteins
• Post-transcriptional (pre-translational) modifications

  5’ capping, polyadenylation, splicing
• Rho-independent termination

  RNA polymerase reaches a region rich in cytosine and guanine, RNA transcribed from this region folds back on itself forming a hairpin structure
• Mutations involved in genetic codes: silent mutation, missense mutation, nonsense mutation, frameshift mutation
• Untranslated regions (UTRs) at the ends of mRNA are not translated during protein synthesis
• Aminoacyl-tRNA synthetases


  Enzymes required for attachment of amino acids to their tRNAs
• Steps in Translation

  1. Initiation involves the assembly of translation components before peptide bond formation

  2. Elongation involves adding amino acids to the growing chain

  3. Termination occurs when a stop codon moves into the A site
• Repressible Transcription involves the inducer causing the repressor to have higher affinity to the promoter site, preventing RNA polymerase from sitting on it
• Post translational modifications

  1. Modifications may include trimming and covalent modifications

  2. Trimming involves removing parts of the protein chain to release an active molecule

  3. Covalent modifications like phosphorylation, glycosylation, hydroxylation, and addition of carboxyl groups
• Functionally competent ribosomes

  • Consists of two subunits: large ribosomal (catalyses the formation of peptide bonds) and small ribosomal (binds mRNA and responsible for the accuracy of translation)
• cids have more than one specific tRNA molecule
• Codon Recognition by tRNA

  1. Correct pairing of the codon in mRNA with anticodon of tRNA is essential for accurate translation

  2. Involves Anti parallel and Wobble hypothesis

  3. Wobble hypothesis allows a single tRNA to recognize more than one codon
• Antibiotics that target translation take advantage of differences in the initiation process between prokaryotes and eukaryotes
• Explanation of the lac operon when lactose and glucose are involved

  • E.coli can use either glucose or lactose, but lactose needs to be hydrolyzed first to form glucose
  • When lactose is absent, a repressor protein continuously synthesized sits on the operator site blocking the promoter site
• Inducible Transcription involves the inducer detaching the repressor from the promoter to allow RNA polymerase to sit on the promoter site
• Lactose utilization


  Lactose needs to be hydrolysed first to form glucose