MBG 9 central dogma in molecular biology

• DNA replication

  • Process by which a double-stranded DNA molecule is copied to produce 2 identical DNA molecules
  • Essential as every newly divided daughter cell must contain the same genetic info as the parent cell
• location of DNA replication

  • Eukaryotes: nucleus
  • Prokaryotes: cytoplasm
• Semi-conservative model of DNA replication

  • Each new double strand consists of 1 parental strand, 1 daughter strand 
  • Semi means half, conserve means to keep

  1. Uncoiling of parental DNA molecule

  • Beginning at a predetermined location (called origin of replication; ori) 

  1. Unzipping of H bonds between base pairs

  • Resulting in 2 parental strands that act as templates

  3. Synthesis of 2 new DNA strands (pink) complementary to each template strand
• DNA replication requires 3 things

  1. Something to copy

  • Parental DNA molecule (templates)

  1. Something to help with copying 

  • Many enzymes 
  • Eg. helicase (breaks H bonds bounding complementary bases together)

  1. Building blocks to make a copy 

  • Free nucleotides eg.
  • Deoxyribonucleotide triphosphates (dNTPs)
  • Serves as building blocks for DNA
  • Always added to the OH at the 3’ end of the growing strand 
  • Catalysed by DNA polymerase III
• initation - DNA replication

  Before replication can take place in a prokaryote,

  At the ori, helicase unzips parental DNA by breaking h bonds between complementary bases

  • Ori is easily recognised as short sequences rich in Adenine and thymine, held together by 2 H bonds instead of 3
  • Much less energy needed to separate this area 
  • Helicase uses free energy from ATP hydrolysis to change shape, wedge itself into DNA, locally breaking H bonds between bases

  Replication fork- where 2 parental DNA strands separate

  • ‘Opening’ of circular DNA results in 2 replication forks 
• R coli replication rate

  E. coli replicates at 1000 bp per second, e. Coli divides every 20 mins
• enzymes involved in DNA replication

  helicase

  • Unzips DNA helix

  primase

  • Synthesises RNA primer

  DNA polymerase III*

  • Adds bases to new DNA chain, proofreads the chain for mistakes

  *Can only add nucleotides to existing DNA strand, requires primase to initiate

  • Can only add nucleotides to a DNA chain in the 5’ → 3’ direction
  • New bases added to 3’ end

  DNA polymerase I

  • Removes RNA primers, replaces gaps between Okazaki fragments with correct nucleotides, repairs mismatched bases

  ligase

  • Final binding of nicks in DNA during synthesis and repair
• Directionality of DNA replication

  1. Leading strand (synthesised continuously from 5’ to 3’)
  2. Lagging strand (synthesised discontinuously in short stretches, each one 5’ to 3’)
• elongation in DNA replication- leading strand

  1. Primase synthesises short RNA primer
  2. DNA polymerase III binds at the primer, initiates replication 

  • DP3 needs a primer to provide a 3’ -OH end to which new nucleotides can be added
  • DP3 adds complementary bases to the strand of DNA at the 3’ end 

  1. When replication completes, DP1 replaces RNA primer with DNA fragments
  2. Ligase then seals gap in the backbone 
  3. Entire leading strand consists of DNA only
• Elongation in DNA replication- lagging strand

  Numerous RNA primers made by primase 

  1. Primers bind to various points along lagging strand
  2. Multiple DP3 enzymes bind at the primers and initiate replication 

  • Short strands of DNA (okazaki fragments) made, each one 5’ to 3’

  1. DP1 enzymes remove old primers, replacing it with DNA
  2. Okazaki fragments finally linked by ligase  (forms phosphodiester bond)
  3. Lagging strand consists fully of DNA

  thus DNA replication is said to be semi-continuous
• termination in DNA replication

  DP1 removes primers, replaces it with complementary DNA

  Ligase joins the DNA fragments together to form 1 continuous length
• gene expression/central dogma of molecular biology

  Process by which info from a gene is used in the synthesis of functional gene product by the cell

  Info flows from DNA→(transcription)mRNA→(translation) protein
• Location of transcription and translation

  prokaryotes

  • no nucleus to separate processes, both take place in the cytoplasm
  • means that translation can start while transcription is still ongoing --> transcription & translation is said to be coupled
  • as bacterial genes are transcribed, transcripts immediately translated into proteins

  eukaryotes

  • transcription occurs in the nucleus, translation (of mature mRNA) in the cytoplasm
• Role of RNA molecules in protein synthesis

  Transcribed mRNA undergoes translation to form proteins

  types of RNA

  mRNA

  • Sequences of amino acids in a protein
  • Carries DNA master code (for protein) to ribosome for translation
  • used in translation
  • mRNA molecules transcribed from DNA, portions transcribed: structural gene

  tRNA

  • Specifying a given amino acid
  • Carries amino acids in the ribosome during translation 
  • used in translation

  rRNA

  • Several large structural rRNA molecules
  • Major part of a ribosome and is involved in protein synthesis
  • used in translation
• transcription to translation

  G A C T A T G C A T C A G G C --> template strand

  C T G A T A C G T A G T C C G -->coding strand

  C U G A U A C G U A G U C C G --> mRNA
• Requirements for transcription

  Copying of genetic info. From DNA to synthesise an mRNA strand 

  • Like DNA replication, governed by complementarity
  • Unlike DNA replication, only 1 DNA strand copied

  Requires RNA polymerase, catalysed polymerisation in the 5’ → 3’ direction only 

  • More multi-purpose than DNA polymerase, as it does not require helicase to unwind DNA, nor RNA primer to initiate

  Gene that is being transcribed is called structural gene

  • Within the gene, 2 DNA strands have dif. Names
• Transcription unit

  Sequence of nucleotides in DNA that code for a single mRNA strand, together with sequences necessary for transcription 

  Contains:

  1. Promoter 

  • Located upstream of the gene(5’ end of structural gene), a position where RP binds

  1. Structural gene

  • Located on the template strand
  • Codes for mRNA strand 

  1. Terminator 

  • Located downstream of the gene, stops transcription process
• Transcription process- initiation

  initiated by promoter

  • Acts as recognition and binding site for RNA polymerase
  • From upstream of the start site (template strand 5’ end)
  • Not transcribed into mRNA
  • Signal unwinding of DNA into 2 separate strands:
  • Template strand 
  • Coding strand
• transcription process-elongation

  RP adds nucleotides to mRNA strand using info provided in template strand in 3’ → 5’ direction

  • The new mRNA strand (transcript) elongates in a 5’ → 3’ direction as more nucleotides added to 3’ end

  Transcription bubble: contains RNA polymerase, DNA template, growing RNA transcript

  • Around 50 bps of DNA in the bubble
  • After bubble passes, now-transcribed DNA unwinds as it leaves the bubble
• transcription process- termination

  RP keep transcribing until it reaches terminator sequence on the DNA

  • signals transcription complete

  Terminator sequence located towards 3’ end (downstream of coding strand)

  Both mRNA and RNA polymerase detach from DNA
• codon definition

  3 - nucleotide sequence coding for a specific amino acid
• Genetic code for amino acids (mRNA codons)

  genetic code is:

  1. Universal
  2. Degenerate

  • Some amino acids specified by more than 1 codon

  1. non-ambiguous
• ribosomes

  Large subunit + small subunit 

  Each ribosome has multiple tRNA binding sites:

  1. P (peptidal) site

  • Binds the tRNA to the growing peptide chain

  1. A (amino acyl) site

  • Binds the tRNA carrying the next amino acid 

  1. E (exit) site

  • Binds tRNA that carries the last amino acid

  2 functions:

  1. Decode mRNA
  2. Form peptide bonds

  large subunit: tRNA binding site

  small subunit: mRNA binding site
• initiation of translation (prokaryotes)

  Ribosomal subunits assemble in a way that forms sites to hold mRNA and tRNA

  • Small subunit (SS) binds at 5’ end of mRNA, moves towards its 3’ end
  • Large subunit (LS) holds tRNAs, involved in peptide bond formation

  SS binds to start codon (AUG) on mRNA, aligns it with P site of LS

  Initiator tRNA molecule brings the first amino acid, methionine.

  • Bonds with start codon using its anticodon, UAC

  LS then binds to SS

  • Initiation complex for translation forms by assembly of the subunits and initiator tRNA (met-tRNA) at the start codon of the mRNA
• Elongation (of polypeptide chain) - translation (prokaryotes)

  After ribosome is assembled around initiator tRNA and mRNA, elongation begins:

  1. Appropriate charged tRNA binds to codon at empty A site 
  2. Peptide bond forms between adj. Amino acids

  • Catalysed by peptidyl transferase
  • tRNA holding the protein chain moves from A site to P site

  1. Empty tRNA moves to E site and is then released
  2. Ribosome then shifts to next codon and cycle continues

  Growing polypeptide lies at the P site, A site always open for the binding of next tRNA molecules
• termination of translation (prokaryotes)

  Elongation continues until ribosome encounters a stop codon (UAA,UGA,UAG) at A site

  Release factor then binds to stop codon

  • Polypeptide is released from the ribosome

  Ribosomal subunits separate from the mRNA, and from each other 

  Polypeptide undergoes folding to become fully functional protein
• Causes of genetic alterations

  Errors occurring during DNA replication

  • Leads to erroneous DNA sequences replicated
  • Evasion of the proofreading function of DPs at replication fork → spontaneous mutation

  exposure to mutagens

  ->induced mutation

  • exposure to mutagens that could increase frequency of mutations
  • eg. radiation (UV, XRAYs), chemicals (nitrous acid, ethidium bromide), any agent causing changes in genetic material, increasing frequency of mutation above natural background level

  reacting with parent DNA, causing:

  1. DNA breakage
  2. structural change (affecting base pairing ability)
• effects of genetic alterations

  pathogenic effects

  • increased susceptibility to disease

  biological diversity

  • genetic variability
  • eg. phenotypic changes, evolution/adaptation to environmental changes

  some mutations have no noticeable effect on phenotype

  • mutation in DNA stretch with no function or
  • occuring in protein-coding region, but no amino acid sequence change
• types of genetic alterations

  1. Gene mutation

  • Alteration to nucleotide sequence of a gene

  --> point mutation

  • silent
  • missense
  • nonsense

  -->frameshift mutation

  -->triplet expansion mutation

  1. Chromosomal aberration 

  • Alteration of structure or number of chromosomes
  • More severe

  -->deletion

  -->duplication

  -->inversion

  -->reciprocal translocation
• point mutations

  silent mutations

  • Due to degeneracy of the genetic code
  • Amino acid coded remains the same

  missense mutation

  • Conservative missense → wrong amino acid coded does not differ in structure by much
  • Non-conservative missense → wrong amino acid coded has a very different structure --> Bad for protein function

  -->eg. sickle-cell anemia, glutamic acid exchanged for valine on chromosome 11

  nonsense mutation

  • Translation of protein stops prematurely
  • Protein is shorter than usual, non-functional and discarded by cellular mechanisms
• frameshift mutation

  • Either insertion or deletion of bases (1, 2, or 4) within an open reading frame (coding sequence)
  • Produces much more serious consequences than point mutations

  2 effects:

  1. Changes the whole sequence of amino acids downstream of mutation and/or
  2. Promotes premature protein chain termination 

  • Might result in a new protein (beginning portion identical, end portion differs) 

  eg. UV-induced thymine dimer formation
• UV-induced thymine dimer formation (frameshift mutation)

  • Formation of abnormal chemical bonds between adj pyrimidine molecules (mostly between adj thymines)
  • Disrupts normal pairing of bases with corresponding A bases on the opp. Strand 
  • Affects transcription and replication
  • UV light exposure is the primary cause of melanoma
• Triplet repeat (expansion) mutation 


  Triplet sequence of DNA (trinucleotide) is/are repeated

  Cause of many genetic diseases

  Depends on location in chromosome, unstable trinucleotide repeat can cause:

  1. Defects in protein encoded
  2. Change in regulation of gene expression
  3. Chromosome instability

  High no. of triplet repeats can cause disease ( a few may not)

  No. of repeats can increase when passed down from each generation

  • can result in worser condition or earlier onset
• chromosomal abberations

  numerical abnormalities

  • eg. trisomy 21 (down syndrome)

  structural abnormalities (extensive change to structure)

  Can arise spontaneously from errors in normal cell processes

  Serious health consequences resulting in poor health, sterility or diseases

  Structural abnormalities caused by:

  1. Deletion
  2. Duplication
  3. Inversion
  4. Reciprocal translocation
• structural abnormalitiesDeletion --> loss of large portion of chromosome, can be fatal

  1. Duplication --> region of chromosome duplicated, may or may not be fatal (outside a gene, no effect). tandem duplication=duplication next to OG region
  2. Inversion -> chromosome broken in 2 places, reversed, put together (outside gene= no phenotypic effect)
  3. Reciprocal translocation -> portions of 2 chromosomes exchanged, no net loss of info (usually harmless)
• structural abnormalities

  • Deletion loss of large portion of chromosome, can be fatal

  Duplication

  • region of chromosome duplicated, may or may not be fatal (outside a gene, no effect).
  • tandem duplication=duplication next to OG region

  Inversion

  • chromosome broken in 2 places, reversed, put together (outside gene= no phenotypic effect)

  Reciprocal translocation

  • portions of 2 chromosomes exchanged, no net loss of info (usually harmless)
• DNA repair


  collection of processes by which a cell identifies & corrects damage done to DNA molecules

  genomic mutations can be carried over to next generations of cells if mutation not repaired before mitosis

  specific DNA repair

  • Targets a single kind of lesion (damage to structure of DNA) in DNA and repairs only that damage 
  • For one particular form of damage caused by UV light

  eg. photoreactivation
• photoreactivation

  1. Recognition of damaged site
  2. Enzyme (DNA photolyase) absorbs energy from light in visible range
  3. Uses energy to cleave the bond between thymine dimers 

  • Found in all organisms
• control of gene expression

  Gene regulation in prokaryotes ensure that genes expressed only when their products are required 

  Prokaryotes utilise operons to perform gene regulation

  Operons are sections of DNA that contain:

  1. 1 or several structural genes involved in the same metabolic pathway 
  2. Operator → controls transcription
  3. Promoter → for binding of RNA polymerase

  • common in prokaryotes, rare in eukaryotes
• inducible operon

  • Lactose (lac) operon regulate metabolism of lactose in bacteria 
  • Inducible operon: 

  1. Absence of lactose → genes turned off
  2. Presence of lactose → genes turned on

  important note:

  Glucose is the preferred carbon/energy source for e. coli

  • Easily metabolised by cells (and produces more energy)
  • In the presence of glucose, secondary system ensures lac operon is inactive

  Therefore lac operon functions only in the absence of glucose