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2010-11-10 12:28:01
Biomolecular Science

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  1. "central dogma"
    • Transcription Translation
    • DNA---------------> RNA------------->Protein
    • ^(DNA replication)
  2. nucleotides basic structure
    Sugar, Base and Phosphate group(s)
  3. Sugars of Nucleotides
    • sugars: pentose (5 carbon sugars)
    • B-o-ribose (ribonucleic acid)
    • B-o-2-deoxyribose (deoxyribonucleic acid)
  4. Difference between nucleotide and nucleoside
    • Nucleotide (has phosphate groups)
    • nucleotide triphosphate (3 p groups)
    • nucleotide diphosphate (2 p groups)
    • nucleotide monophosphate (1 p group)
    • nucleoside (no phosphate group)
  5. Examples of nucleotide/side
    • Base is adenine and sugar is deoxyribose with 3 phosphates: Deoxy Adenosine Triphophate, dATP
    • Base is adenine sugar is ribose and 1 phoshpate: Adenosine monophosphate, AMP
  6. Bases of Nucleotides
    • Purines (2 rings): Adenine and Guanine
    • Pyrimidines (1 ring): Cytosine, Thymine, Uracil (in RNA only)
  7. Polynucleotides
    nucleotide chains joined by phospho-diester bonds (3'-5')
  8. Watson-crick model of DNA structure
    3'-->5' andtiparallel double stranded helix with major and minor grooves (bases in centre- interacting with one another to form base pairs)
  9. DNA Base Pairs
    • ALWAYS!!!!
    • A-T (weaker bonds than C-G)
    • C-G
  10. Principal features of the structure of DNA
    • DNA consists of 2 helical polydeoxyribonucleotide strands
    • the sugar-phosphate backbones of the strands are antiparallel; they run 3'-5' and 5'-3'
    • the strands are joined non-covalently by hydrogen bonding between bases.
    • bases always pair according to A=T and G=C
    • base pairs show stacing interactions b/c they are flat and non-polar
    • stacking confers "stiffness" on the DNA duplex
    • the sequence of the strands are complementary
    • 2 types of base pairs have similar but signigicantly different dementions
    • DNA normally occurs in the B-form with the following dimensions: 10bp per turn of the helix, 3.4nm per turn of the helix, 2.0nm diameter
    • each nucleotide carries a Neg charge on the phosphate
    • stacking interactions between the flat aromatic bases stabilize the helix against electrostatic repulsion by neg charged phosphates
    • this stabilizing energy equals, or is greater than, that due to base pairing H bonds
  11. Importance of Primary structure of DNA
    • Primary sequence of DNA is important as it is transcribed into RNA
    • mRNA is produced from DNA by transcription of DNA by RNA polymerase II
    • mRNA is then translated into proteins
    • therefore the primary structure od DNA dictates protein sequence and is therefore of fundamental importance
  12. Importance of Secondary structure of DNA
    • base pairing gives it a template function
    • DNA can act as a template for synthesis of a DNA or an RNA strand
    • the copied strand is antiparallel and complementary to the template
    • copying of DNA gives replication, of RNA transcription
    • ingenomic terms replication is a global even, transcription a local one
  13. If thymine makes up 15% of the bases in a certain DNA sample, what % must be cytosine?
  14. The most common elements in living organisms are carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur. which of these is not found in DNA
  15. A certain segment of DNA has the following nucleotide sequence in one strand:
    5' ATTGGCTCT3'
    what must be the sequence in the other strand label its 5' and 3' ends?
  16. Where in the cell cycle does DNA synthesis occur?
    S phase
  17. Semi-conservative nature of DNA replication
    parent strand--> 1st gen daugher molecules 1/2 parent strand each--> 2nd gen daughter molecules 2 have 1/2 original strand 2 do not contain any original DNA
  18. Chain elongation by DNA polymerase
    adds nucleotides to chain by following template strand grows in 5'-3' direction
  19. DNA polymerases synthesise DNA
    • DNA polymerases carry out the following reaction: (dNMP)n+dNTP=(dNMP)n+1 + PPi
    • synthesise chains ONLY in 5'-3' direction
    • DNA polymerases synthesise DNA with a high degree of fidelity
    • DNA is read in a 5' to 3' direction
  20. Bacterial DNA replication
    • Occur at a single origin of replication
    • unique sequences at the origin of replication
    • several proteins recognize the origin and separate the DNA strands
  21. Strand Separation of Bacterial DNA replication
    • DNA helicase unwinds the DNA ahead of the DNA replication fork
    • to sort out any tangling of the single stranded DNA, DNA topoisomerases are recruted (break DNA and rejoin to reduse stress)
    • Single stranded DNA binding proteins are also at the replication forks to protect the DNA cleavage
  22. RNA primer begins Bacterial DNA replication
    • An RNA primer synthesised by primase enables DNA synthesis to begin
    • a special type of RNA polymerase, PRIMASE, makes short 10 to 20 nucleotide chains of RNA that are complementary to one of the template DNA strands
    • Nascent DNA is covalently linked to the short stretch of RNA
    • Therefore RNA primes the synthesis of DNA (DNA polymerase III)
  23. Bacteria's 3 DNA polymerases
    DNA Pol I, II, III
    • can synthesize DNA
    • remove the RNA primer from the DNA (5' - 3' exonuclease activity)
    • proof read using 3' - 5' exocunclease activity
  25. DNA Polymerase II
    • synthesize DNA
    • proof read using a 3' - 5' exonuclease activity
    • probably involved in DNA repair
  26. DNA polymerase III
    • can synthesize DNA
    • Proof Read using 3' - 5' exonuclease activity
    • main enzyme responsible for the synthesis of DNA
  27. Length of DNA Problems:
    • interior of dbl helix, due to stacking of the bps resembles a stack of coins
    • therefore, DNA molecues are long, thin, and stiff and very susceptible to shearing. however they retain flexibility
    • DNA needs to be condensed and protection and structure
  28. Histones in Eukaryotic chromosomes
    • Histones consist of 1/2 of the mass of chromosomes, the other half being DNA
    • nucleoprotein chromosomal content is termed chromatin
    • there are 5 types of histones (H1, H2A, H2B, H3, H4)
  29. Histone facts
    • Histones are extremely basic: about 1/4 AA are basic (arginine or lysine)
    • basic content gives histones an overall positive charge allowing for interaction with negatively charge DNA
    • the AA sequences of H3 and H4 are nearly the same in all plants and animals
  30. Nucleosome
    • nucleosome core consists of 140bp of DNA wound around a histone octamer
    • nucleosomes are the repeating units of chromatin
    • the first stage in the condensaion of DNA
    • form a helical array making a solenoidal structure
  31. Chromatin
    made up of repeating units of 200bp; 140bp wound round the histone core (the nucleosome) with additional linker DNA
  32. Solenoids
    • Solenoids attach to a nuclear matrix (a protein scaffold)
    • this scaffold is then itself condensed, again potentially into a helical structure
    • all of this condensation allows the DNA to pack into chromosomes that we find in calls
  33. Organisation of DNA in Genomes
    • Bacterial genomes are composed of a single molecule of DNA, therefore genomes and chromosomes are identical
    • in eukaryotes, the genome is divided into a number of component parts; chromosomes
    • this is because of the amound of DNA eg: human genome has 3,000 million bp, e.coli a thousand fold less
  34. Euchromatin
    DNA condensed- dispersed appearance and occupies most nuclear volume
  35. Heterochromatin
    Densely packed DNA
  36. DNA Damage
    • Spontaneous damage (loss of purine base, pyrimidine base, deamination)
    • induced by externam agens (chemicals, UV light, tobacco)
  37. Spontaneous Damage to DNA
    • Loss of purines and pyrimidines: (hydrolytic loss of the base from the sugar)
    • mis-incorporation of a nucleotide during replication; e.g. a T is incorporated next to a G
    • Deamination (hydrolytic loss of the NH2 group from a base)- particularly with cytosine changing into uracil. this is potentially mutagenic as uracil will behave like thymine upon DNA replication
  38. External Damage to DNA
    • Physical agents including UV light (causes pyrimiding dimer formation) and x-rays (backbone breakage) resulting in inhibition of DNA replication and loss of genome integrity
    • chemical agents such as nitrous acid (deamination) resulting in mutation, and N-alkylation resulting in depurination giving a low mutation rate
  39. Consequences of DNA Damage
    • Changes in non-essential DNA
    • change in an essential part of the genome but does not alter the cell's information
    • the damage is repaired before it can exert any harmful effect
    • mutation is essential coding regions of the genome that are inherited by the daughter cells
    • change in protein sequence and potentially function- mutation
    • can result in several diseases, including cancer
    • cell death by apoptosis
    • tumour suppressor protin p53
  40. Example of deamination= mutation
    • G-C-----------> G-U---------> G-C and A-U----> A-T
    • Deamination Replication correction...
  41. Mutated DNA can be repaired by:
    • Recognition: detection of the damage
    • incision: cutting into the damaged DNA
    • Excision: cutting out the damaged part
    • Polymerisation: gap filled by DNA polymerase
    • Religation: Joining of the resulting nick carried out by DNA ligase
  42. Base Excision Repair
    See notes!!!!!!!!
  43. Xeroderma pigmentosa: disease
    • Rare genetic disorder
    • acute sensitivity to sunlight
    • high incidence of skin cancer
    • caused by a defect in a gene involved in excision repair
    • there are other diseases that involve other genes of the excision repair process... (cockayne syndrome, brittle hair syndrome)
  44. DNA repair in transcribed DNA in Eukaryotes
    • is faster
    • the repair of transcribed DNA is 100 fold faster than that of the rest of the genome
    • there is transcription coupled repair
    • as the DNA must be repaired in order to produce the correct mRNA for translation into proteins
  45. Double stranded DNA breaks
    • can be repaired by homologous recombination
    • these can be repired by non-homologous end joining (NHEJ)
    • NHEJ can be mutagenic as it can link DNA together resulting in mutation
    • NHEJ can therefore contribute to human diseases including cancer
  46. Base deamination can cause single base pair mutations becuase...?
    Deaminated C can base pair with A (A-U)
  47. In E.coli, parental DNA strands are distinguishable from newly synthesized daughter strands b/c?
    parental strands are methylated and daughter strands are UNmethylated
  48. List the forces that hold the DNA double helix together as a stable unit
    • 2 types of bonds: covalent and hydrogen
    • covalent bonds form w/in each linear strand and strongly bound the bases, sugars, and phophate groups (both within each component and between components)
    • hydrogen bonds form between the 2 strands, between a base from one strand and a base from the other strand in complementary pairing.
    • these hydrogen bonds are individually weak but collectively quite strong.
    • there is also "stacking" interactions between the bases in the helix that are equally as important as hydrogen bonding
  49. Why are the 5' and 3' ends of DNA named this?
    the 5' and 3' refers to the position of the carbon on the pentose ring. remember that nucleotides join via their 3' and 5' residues. this means that at either end of a DNA strand there will be a free 5' and free 3'
  50. Why is DNA synthesis continuous on one template and discontinuous on the other?
    DNA polymerases can only synthesise DNA in a 5'-3' direction. as the 2 strands in a double helix are anti-parallel this means that as the helix unwinds one of the strands, the lagging, has to be made in small fragments (okazaki) and then joined together
  51. Mechanical differences between Prokaryote and Eukaryote RNA
    • Prokaryote- linked transcription and translation
    • Prokaryotic mRNAs- polycistronic (1 message => multiple proteins)
    • Eukaryote- transcription (nucleous) and translation (ribosome) separate
    • Eukaryotic mRNAs- monocistronic (1 message => 1 type of protein)
    • Eukaryotic mRNAs are spliced and undergo covalent modifications (Prokaryotic are not)
  52. Eukaryotic polymerases
    • Eukaryotes contain 3 DNA dependent RNA polymerases:
    • RNA Pol I- synthesise rRNA
    • RNA Pol II- synthesise mRNA
    • RNA Pol III- synthesise tRNA and small RNAs
    • (RNA polymerase do not need a primer)
  53. Prokaryotic polymerases
    Prokaryotes have RNA pol and a sigma subunit
  54. Promoter (Prokaryote)
    • RNA pol is a multi subunit protein (a,aB, b1) sigma and RNA pol= RNA pol holoenzume
    • the sigma subunit guides RNA to correct location
    • There is a pribnow box (TATAAT) about -10 from starting location, about -35 back (promotor sequence prior to transcription site)
  55. Eukaryote (promoter)
    • CAAT box(sometimes pressent) about -75, then a TATA box (TATAAA) about -25 back from start of RNA
    • TATAA becuase A&T have fewer bonds than C&G so easier to seperate.
  56. Eukaryotic Initiation RNA transcription
    • (Pre-initiation complex) TF2D finds TATA box, brings in TF2B then PolII binds w/2F and other basil transcription factors. (Basil transcription process)
    • Kinase activity comes in to phosphorilate C-terminal tail
  57. Primary Transcript (hnRNA)
    • Initial RNA product
    • several processing steps: capping (7-met), polyadenylation (poly-a tail), splicing (euk. only!)
    • for protection
    • (as soon as transcription starts the 5'-cap is added)
  58. 3'-Polyadenylation (tailing)
    • RNA endonuclease recognises polyadenylation sequence (AAUAAA)
    • cleaved 10-30 bases beyond AAUAA
    • adenylate polymerase adds 100-300 AMP residues
    • 5'->3' direction
  59. Termination of transcription in prokaryotes
    • termination is clearly defined!
    • Rho-dependednt- Rho binds 5' end RNA. Scans RNA displaces RNA polymerase
    • Rho-independent- stem loop structure and run of U residues (the two fall away fromeachother w/o Rho)
    • the loop destabilizes RNA Pol and DNA. RNA pol slows down =, Rho catches RNA pol and displaces it
  60. Alternative splicing
    • Eukaryotes ONLY!!!
    • Exons stay and introns are removed to form different isoforms of the same protein
    • mRNA precursors 1y transcript or hnRNA=exons and introns splice away the introns, stitch together the exons- splice sites on modified pre-mRNA recognized by splicesosomes
  61. Regulation of Transcription
    • Accessibility to DNA (histones)
    • Transcription Factors
  62. Accessibility to DNA
    • Histones + DNA = Chromatin (like a bead on a string)
    • amino-terminal 'tails' modified
    • Euchromatin (very open/loose easier to access DNA)
    • Heterochromatin (heavily packed, silent chromatin, tightly packed together hard to access DNA)
  63. Forces that keep histones and DNA together
    • Helix-dipoles: H2B, H3, H4 + charge to accumulate at contact site with - phosphate groups on DNA
    • Nonpolar: histone and deoxyribose sugars
    • Salt bridges and H-bonds: between side chains of basic amino acids and phosphate DNA
    • non-specific minor groove insertions: H3 and H2B N-terminal tails into two minor gooves each on the DNA molecule
  64. Histone modification
    • common nomenclature
    • know histones have lots of modifications
    • (methylation, citrulination, acetylation, phosphorylation, SUMOylation, ubiquitination, etc...)
  65. Transcription Factors
    • DNA binding factors that alter the transcriptional activity- recognize major and mino grooves of DNA helix
    • activator and repressors
    • bind away from RNA pol site, can recrute protein and basil factors faster
    • Repressors: TFs block transcription
    • Activators: Activate transcription
    • TF's bind DNA sequences called control elements
    • Repressors bind control elements called silencers
    • activators bind control elements called enhancers
    • (very specific)
  66. Epigenetics
    • the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence
    • (genes have a "memory")
  67. DNA methylation and cancer (epigenetics)
    • gene promoters get hypermethylated- transcriptional silencing that can be inherited by daughter cells
    • methylation of DNA impede TFs
    • methylated DNA is bound by methyl-CpG binding domain proteins
    • (MBDs)- recruit histone deacetylases and other chromatin remodeling proteins forming compact, inactive chromatin
  68. Epigenetic differences during aging
    • DNA methylation patterns change during aging-ongoing
    • (ex. twins use DNA differently)
  69. Protein synthesis (apparatuss)
    mRNA, Ribosomes, tRNA
  70. Ribosomes
    • 2 main subunits!!
    • Eukaryotic ribosomes (80s) 60s & 40S subunits (50 proteins & 33 proteins)
    • Prokaryotic Ribosome (70s) 50s & 30s (34 & 21 proteins)
    • (only pay attention to prokaryotic)
  71. Translation precursor...?
    • Nucleolus: rRNA genes transcribed by RNA pol I
    • proteins bind individual rRNAs in nucleolus
    • 45s rRNA precursor (pre-mRNA) cleaved to 18, 5.8 and 28s rRNAs before leaving the nucleus-equimolar amounts
    • ribose methylation
    • complete ribosomes and mRNA only assemble during protein synthesis
    • (5s RNA comes from elsewhere in nucleus not nulcleolus)
  72. tRNAs
    • adapter molecules
    • anticodons match codons
    • stem loop structure
    • aa 3'OH
    • aminoacyl tRNA synthetase proofreading activity
    • 20 aa tRNA synthetases
    • each aa has set of tRNAs except UAA, UAG, and UGA (stop codons)
    • tRNA precursor pre-tRNA
    • CCA replaces 2Uresidues
    • 16nt removed 5' end
    • introm removed anticodon loop
    • ribose methylation
    • base modification
    • "trap message"- pulls through like ticker tape. must have correct AA match to correct codon.
    • AA gets added to 3' end/ tRNA must match!!!
  73. Codons
    • trinucleotide sequence read 5'-3'
    • 20 amino acids in protein
    • multiple codons for each aa
    • low frewquency aa's have one codon
    • Reading frames: 3 potentioal reading frames
    • reading frame selected by AUG (start codon)
    • stop codon terminate translation
    • ORF (between start and stop)
  74. Start codon
    • AUG located near 5' end of an mRNA molecule
    • encodes methionine in eukaryotes and N-formulmetionine in prokaryotes
    • Eukaryotes- Kozak's consensus (purine-n-n-A-T-G-g)
    • Prokaryotes- Shine-Dalgarno sequence
  75. Prokaryotic translation
    • small ribosomes subunit binds to Shine-Delgarno sequence
    • Initiation factors also bind/help
    • (must bind at correct start)
    • Uses GTP but will only use energy if correct combo.
    • large subunit comes in with fMET- completing the 70s initiation complex generated.
    • AUG is in P location
    • next codon is read matched by tRNA, mRNA runs through complex
    • once hits stop codon- machine falls apart/release factor is introduced
  76. Antibiotics can inhibit bacterial protein synthesis
    • Chloramphenicol (blocks peptidyl transferase reaction)
    • erythromycin (blocks translocation from A- to P- site) inhibits large ribosomal subunit
    • Puromycin (mimics aminoacyl tRNA enters A site and terminates chain
  77. Protein Sorting
    • Protein targeting/sorting
    • (lumen or an organ) secretory pathway or cytosol
    • ribosomes initiate protein synthesis in cytoplasm
    • directed to ER signal peptide (5' end)
  78. RNAs that control transcription and translation
    • Small RNAs appear to be controlling a vast number of stress and developmental responses. these RNAs can target and degrade mRNAs
    • even if a cell has a huge amount of mRNA- this message may not get translated if it is targeted by regulatory RNAs
  79. Antisense RNA
    antisense RNA is a RNA strand which is a mirror image of the mRNA strand (sense strand) which is used to encoge proteins. the formation of dbl stranded RNA inhibits gene expression and/or can target message for degradation
  80. Post transcriptional control of gene expression
    • Dicers
    • miRISC
    • pimRNA
    • MicroRNAs
  81. problems with the sequence-mutation
    • substitutions
    • insertions
    • deletions
  82. substitution mutation
    • where in the codon?
    • a nucleotide is substituted leads to altered codon= may change the amino acid
    • silen mutations have no effect on aa, protein the same (GGG/GGA- both encode glycine)
    • transition (purine/purine)
    • transversion (purine/pyrimidine)
    • conservative substitution
    • radical substitutions
    • chain termination- mutation leads to a stop codon
  83. frame shift mutations
    • insertion and deletion mutations can change protein function
    • they may also result in a frameshift.
    • there are several outcomes when translating the RNA derived from a DNA that has suffered a frameshiftmutation
  84. functional/consequences of mutation
    • normal
    • loss of function
    • nonsense mutation (just wrong)
    • dominant neg (stops things from working- one copy can kill you)
    • gain of function