notes for module 3

• Gene


  A portion of DNA that codes for a protein or any functional unit
• Prokaryotic chromosomes


  • Usually have a single chromosome and one or a few plasmids
  • Chromosomes are almost always circular
  • They are either completely devoid of centromeres or carry the so-called "plasmid centromeres" which are not essential
• Prokaryotic DNA compaction


  • Supercoiling, aided by topoisomerases
  • Mesophilic prokaryotes use DNA gyrase to create a negative supercoil
  • Thermophilics uses reverse gyrase to create a positive supercoil
  • DNA topoisomerase I, DNA gyrase and other proteins help maintain the supercoil
  • Most bacterial genomes are negatively supercoiled
• Eukaryotic chromosomes


  • Usually have multiple chromosomes per karyotype, with a typical diploid number of between 10 and 100
  • Chromosomes are always linear and are always equipped with centromeres
• Eukaryotic DNA condensation and packing


  • DNA is wrapped around nucleosomes and is further organized by histones
  • Chromatin packing offers an additional mechanism for controlling gene expression (cells can control access to their DNA by modifying the structure of their chromatin)
  • Euchromatin - regions of chromatin where active transcription is taking place are less condensed
  • Heterochromatin - regions where transcription is inactive or is being actively inhibited or repressed
• DNA replication model


  • Semiconservative model - every new DNA double helix would be a hybrid that consists of one strand of old DNA bound to one strand of newly synthesized DNA
  • Conservative model - half of the new DNA double helices would be composed of completely old, or original, DNA, and the other half would be completely new
  • Dispersive model - every round of replication would result in hybrids, or DNA double helices that are made up of partly original DNA and partly new DNA
• Prokaryotic DNA replication


  1. Replication origin is a hundred nucleotides in length, have a defined zone, called the terminus, where converging replication forks fuse

  2. All replication origins in the same cell always fire at once (synchronously)

  3. DNA denaturation: the melting of double-stranded DNA to generate two single strands

  4. Replication bubble: the region formed after the separation of the 2 DNA strands

  5. Replication fork: the Y-shaped region on each side of the replication bubble
• Eukaryotic DNA replication


  • Eukaryotic chromosomes have multiple and alternative replication origins, generating up to hundreds of replication bubbles per chromosome
  • Undergo a single replication round at a time
• Reason for multiple replication origins in eukaryotes

  • Eukaryotes have more DNA to replicate
  • Eukaryotic DNA polymerase is slower than prokaryotic DNA pol
• Steps in DNA replication

  1. Unwinding of the double strands by helicase

  2. Creation of a replication fork

  3. DNA Polymerase III uses only single-strand DNA (ssDNA) as template

  4. Separation is maintained by SSB proteins, DNA helicase, and DNA topoisomerase/gyrase

  5. Start of synthesis of complementary strand by DNA Polymerase III

  6. Leading strand synthesis toward replication fork in 5'-3' direction

  7. Lagging strand synthesis away from replication fork in 5'-3' direction

  8. RNA primers synthesized by primase

  9. Removal of RNA primers and replacement with deoxyribonucleotides by DNA Polymerase I

  10. Joining of Okazaki fragments by DNA ligase
• Forms of DNA


  • B-DNA: right-handed DNA helix that is the most common form
  • A-DNA: right-handed antiparallel helical duplex, more compact along the helix axis and broader overall across the helix
  • Z-DNA: left-handed, the most underwound form of the double-helix
• Reasons for different DNA forms

  • Not enough room for DNA to be stretched out in a perfect, linear B-DNA conformation
  • DNA can shift from one form to another depending on the need and environment
• Other DNA structures


  • H-DNA: triple-stranded helices favored in negatively supercoiled DNA
  • Holliday Junction: essential to cellular processes like recombination-dependent DNA lesion repair, viral integration, restarting of stalled replication forks, proper segregation of homologous chromosomes
  • G-quadruplexes: four-stranded structures assembled from guanine-rich sequences
  • I-motifs: a transitory conformation that can form in sequences rich in cytosine, stabilized by acidic conditions
• Prokaryotic DNA polymerases


  • DNA Polymerase I: DNA repair, proofreading, replication
  • DNA Polymerase II: Main replicating enzyme, proofreading
• Eukaryotic DNA polymerases


  • DNA Polymerase α: Priming during replication, no proofreading
  • DNA Polymerase γ: Mitochondrial DNA replication, proofreading
  • DNA Polymerase δ: Main replicating enzyme, fill gaps after primer removal, proofreading
  • DNA Polymerase ε: DNA repair, proofreading
  • DNA Polymerase β: DNA repair, no proofreading
• Exonucleases


  Cleave nucleotides one at a time from the end (exo) of a polynucleotide chain
• Endonucleases


  Cleave phosphodiester bonds in the middle (endo) of a polynucleotide chain
• Without primer, there will be no DNA synthesis
• Prokaryotic DNA primase


  Its own entity and works in a complex with the DNA helicase, comprising a primosome
• Eukaryotic DNA primase


  Associated with another polymerase, DNA polymerase α, which initiates the leading strand and all Okazaki fragments
• Replication errors


  • Addition of a nucleotide with an incorrect base or mispairing
  • Insertion or deletion
  • Strand slippage
  • DNA damage
• DNA damage


  • Chemical damage can alter DNA structure
  • Damage caused by radiation
  • Damage due to attack by water (cause removal of an amine group from the base group of a nucleotide or the loss of the entire base group)
• Excision repair system


  1. Base-pair excision repair: initiated by DNA glycosylases, which recognize and remove specific damaged or inappropriate bases, forming AP (apurinic/apyrimidinic) sites

  2. Nucleotide excision repair: repairs damage caused by the formation of bonds between adjacent pyrimidines

  3. Short-patch and long-patch excision repair: enzymes will recognize and remove "short patches" or longer segments of damaged DNA
• Mismatch repair


  Identify mismatch errors because such damage leads to a small distortion in the DNA backbone
• RNA as genetic material

  • A nucleic acid consisting of a chain of ribonucleotides
  • The only genetic material of certain viruses (virus RNA)
  • Cellular RNA is linear and single-stranded, but in the genome of some viruses is double-stranded
  • In cellular organisms, this is the molecule that directs the intermediate stages of protein synthesis
• Types of RNA


  • Messenger RNA
  • Transfer RNA
  • Ribosomal RNA
  • Noncoding RNAs (including intron RNA and ribozymes)
• Reason DNA is preferred as genetic material


  It is more stable, more easily repaired, its information is better protected (because of double strands)
• Gene-enzyme relationship


  A gene codes for a specific product, usually an enzyme, that catalyzes certain chemical reactions in a metabolic pathway
• One gene-one enzyme hypothesis


  Each gene is responsible for directing the building of a single, specific enzyme
• Protein structure


  • Primary structure: sequence of amino acids
  • Secondary structure: alpha helix and beta pleated sheets formed by hydrogen bonding
  • Tertiary structure: ensemble of formations and folds in a single linear chain of amino acids
  • Quaternary structure: multiple polypeptide chains or subunits of complex proteins associate with each other to form a closely packed arrangement
• Colinearity of DNA and proteins


  Having corresponding parts arranged in the same linear order (e.g. a gene and the protein it codes for are collinear)
• Genetic code


  • Nucleotide base sequence on DNA which will be translated into sequence of amino acids of a protein
  • Each amino acid is represented by a triplet of bases, read in 5' to 3' direction
• Characteristics of the genetic code

  • It is a triplet code
  • It is non-overlapping
  • It is degenerate (or redundant)
  • It is almost universal
  • There are start and stop codons
  • There is a wobble in the anticodon
• Template strand and coding strand


  • Template strand or antisense strand: holds the information that codes for various genes (contains anticodons)
  • Coding strand or sense strand: the complementary strand that contains the codons
• Major enzymes involved in DNA replication

  1. Initiation: helicases, topoisomerases, SSB proteins, primase

  2. Elongation: DNA polymerase, ligase

  3. Termination: DNA polymerase, telomerase, nucleases
• Transcription


  1. Synthesis of RNA based on the nucleotide sequence on a portion of DNA

  2. Converting the DNA language into the RNA language that can be subsequently used to act as instructions in making proteins

  3. Takes place in the nucleus

  4. Promoter: A specific DNA sequence that is the binding site for RNA polymerase or its helper proteins

  5. RNA polymerase will open up a portion of the DNA which is called a transcription bubble

  6. In prokaryotes, RNA polymerase binds directly to promoter

  7. In eukaryotes, helper proteins called general transcription factors bind to the promoter first (initiation)

  8. RNA polymerase unwinds DNA by breaking H bonds (elongation)

  9. Template is read in a 3' to 5' direction but the RNA is synthesized from 5' to 3' end
• DNA replication


  1. Initiation (DNA polymerase, ligase)

  2. Elongation (DNA polymerase, primase)

  3. Termination (DNA polymerase, telomerase, nucleases)
• Transcription


  Synthesis of RNA based on the nucleotide sequence on a portion of DNA
• Transcription


  Converting the DNA language into the RNA language that can be subsequently used to act as instructions in making proteins
• Transcription takes place in the nucleus