Information and Inheritance

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  • Created by: jessica
  • Created on: 09-04-13 15:39
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  • Information and Inheritance
    • DNA
      • bases have tautomerism, electrons are time shared against adjacent electrophilic atoms
      • secondary structure is the helix, strands running antiparallel, seen in crystallography
      • The sequence is always written and read in the 5' to 3' direction
      • Visualize DNA by staining with ethidium bromide and capturing image on UV agarose gel.
      • topoisomerases relieve the tension of the DNA coils by breaking transient DNA strands
      • stacked bases are 0.34nm apart, complete turn every 3.4-3.6nm. forms a major and minor groove
      • major and minor groove are where the DNA binding proteins read the base sequences
      • Nuclear envelope protects the DNA from physical and chemical damage
      • Nuclear pores are used to regulate the transfer of RNA and proteins in/out of the nucleus
      • Chromosomes are formed from the formation of nucleosomes
        • nucleosomes formed from histone core octamer, the DNA then wraps around the octomer
          • histone octamer formed from H2A, H2B, H3, H4, with 2 copies of each,
    • Protein synthesis
      • different pepties formed depending on where the transcription frame is started
      • mRNA reading frames used to identify triplet codons
      • tRNA have specific bases in amino acid acceptor stem and in the anticodon loop.
        • correct amino acid fits well in the synthesis site
      • tRNA can recognize various codons for the same amino acid via wobble base pairing
      • imosine is only present in wobble base pairing
      • ribosomes have three sites, A, P and R sites
        • A site is the acceptor site, P site is the peptidyl tRNA site for peptide growing chain on mRNA, E is the exit site for releasing used empty tRNAs from the ribosome
      • initiation complex formed on the small ribosome subunit
        • the initiation factors are released on binding of the large subunit
    • Cell differentiation
      • stem cells are undifferentiated cells, totipotent stem cells can form any cell type
      • progenitor cells give rise to a single type of differentiated cell
      • Stem cells can divide symmetrically, producing identical daughter cells or asymmetrically generating an identical duaghter cell and another cell that has more restricted capabilities.
    • Transcription
      • RNA polymerase
        • is a single beta barrel polypeptide, has a sigma subunit that is lost from the RNA polymerase on binding to DNA
        • can back track and hydrolyse the previous base, the orientation determines the template strand
          • RNA polymerase moves from left to right-using bottom strand as template, and vice versa
        • one for each class of RNA in eukaryotes,
          • pol 1 for rRNA, pol 2 for mRNA, snRNAs, miRNAs, Pol 3 for tRNA,
      • promoter sites are on the DNA and direct the RNA polymerase to the start of transcription
        • located upstream of the genes, with optimum frequency of 17 bases, called the TATAA box
      • terminates when there are no phosphodiester bonds left, the melted DNA rewinds and the core enzyme is released
      • the components that are needed to start are the template strand, activated precursors, Mg2+ ions, activators and RNA polymerase
      • DNA footprinting carried out by DNAase degrading DNA with/without RNA
      • is the first stage in gene expression
      • in eukaryotes the introns are removed after transcription
      • repressors
        • proteins that bind to operator sequences that are overlapping or lying adjacent to promoters
        • inhibit transcription initiation, modulated by small molecule ligands
        • allows transcription regulation of specific genes in response to environmental changes
      • TATA boxes are conserved sequences found 26-31bp upstream of the transcription start site
        • initiator sequences are similar to the TATA box
          • have a cytosine at the -1 position and an adenine residue at the transcription start site
      • promoter regions have a high number of CG bases where transcription factors bind to
      • transcriptional controllers
        • repressors and activators, co activators needed to help switch on genes and co repressors needed to help switch off genes
    • Nucleus
      • contains a variety of subnuclear compartments e.g. sprekle, cajal body, nucleolus,
      • nucleolus is the processing site of rRNA and ribosomal subunit assembly
        • not bound by a membrane, is just an aggregate of rRNAs
      • cajal body is where snRNP are recycled
    • RNA structure and function
      • tRNA is the information adaptor, has 4 stems and 3 loops
        • translates codons in mRNA into amino acids
        • anticodon on tRNA ensures that the right amino acid enters the chain at the right point as the anticodon is complementary to the mRNA codon
        • forms an aminoacyl-tRNA bond before it can base pair with the codon on mRNA
      • ribosomal RNA are part of the protein synthesis.
        • have large and small subunits, join together for each translation cycle into a more compacted structure
          • ribosomes have 3 or 4 different RNA molecules and up to 83 proteins.
            • small subunit has one small rRNA and the large subunit has one large rRNA molecule, plus one molecule of 5S rRNA and in vertebrates a molecule of 5.8SrRNA.
      • microRNA inhibits protein synthesis by degrading specific mRNAs
      • various RNA types, mRNA, tRNA, rRNA, microRNA
        • miRNAs control expression of more than 1/3rd of all human genes
          • they target specific mRNAs for degredation by argonaute/dicer silencing complexes
        • siRNAs interfere with translation by mopping out specific sense mRNAs
      • primary structure similar to DNA, but has uracil not thymine
        • the ribose sugar makes it more labile than DNA, can be cleaved into mononucleotides by alkaline solution
        • RNA can be single or double stranded
    • Genotype
      • the particular set of alleles for all the genes carried by an individual
      • wild type genotype is the term for standard genotype for breeding experiments
    • Control of gene operon
      • e.g. lac operon degrades lactose
        • needs induction-the inactivation of the lac repressor and the activation by catabolite activator protein and cAMP
      • are conordinated gene expression of bacterial gene clusters
        • adjacent genes are simultaneously switched on/off, behave as single transcription units, makes a polycistronic mRNA
      • polycistronic mRNA is read by ribosomes, producing a single polypeptide
        • adjacent genes are simultaneously switched on/off, behave as single transcription units, makes a polycistronic mRNA
        • undergoes proteolysis to give separate proteins
      • allows rapid production of all proteins needed in a process
    • Control of gene expression in eukaryotes
      • Pre-transcriptional regulation
        • modulation of access of genes in transcription by modification of the histones and DNA methylation
        • gene rearrangements changes the area from active to inactive and vice versa-heterochromatin and euchromatin
        • histones can be used as chromatin remodeling tools, bending the DNA
        • increase amount of protein by increasing gene copy number
        • decoders recognise/bind to modified histone tails
      • transcriptional control
        • transcription switches are specific DNA sequences  or chromatin modifications recognized by transcription regulators
        • silencers are localised nucleosome modifications that keep a chromatin region transcriptionally inactive
          • limit the enhancer activity to specific genes
            • insulators are DNA sequences that stop the action of an enhancer on the trancription of a downstream gene
        • insulators are DNA sequences that stop the action of an enhancer on the trancription of a downstream gene
        • enhancer between 2 genes can be used by activators activating both genes at the same time
      • Post transcriptional control
        • Alternative splicing can retain or discrad introns/exons depending on the mutations affecting the splicing
        • 2 main deamination based editing mechanisms
          • Cytosine to Uracil-edited sites in introns and exons
          • Adenosine to Inosine-the ADR enzymes bind to exon sites to be edited
        • Alternative polyadenylation in B lymphocytes
          • on first infection the full length of IgM mRNA is transcribed.
            • when the antigens bind, sub-optimal cleavage and polyadenylation of the site produces a shorter antigen specific IgM mRNA
      • Translational control
        • Post translational control of protein activity, targeting and protein degradation
        • Controlled via mRNA 5'UNT and 3'UNT regions and smalll non coding RNAs
        • the mRNA 5'UNT is important to bind the small ribosomal subunit for initiation
          • repressors bind here to interefere with initiation
        • the mRNA 3'UNT is important for mRNA stability, containing localisation sequences
          • repressors bind here and interefere with mRNA circularisation
        • 3 main degradation paythways
          • removal of 3' polyA tail
          • decapping and exonucleoltic decay of both endsor decapping and endonucleases breaking down the mRNA
          • deadenylation and initiation complex formation for 5' cap and 3' polyA binding
    • DNA replication
      • is a semi conservative process
      • requires DNA polymerases, dNTPs, dATP, dCTP, dGTP, dTTP, physiological pH and ionic conditions
      • primer is a strand of DNA/RNA with a free 3' hydroxyl and hybridised to the template strand
        • grows  in the 5' to 3' direction
        • RNA primers are extended by DNA polymerase III
          • DNA polymerases have a sliding clamp to hold the enzyme to the DNA and to allow it to move down the template
      • initiated when the initiator protein recognizes the replication origin
      • helicases bind to initiator protein on OriC and uses ATP to melt hydrogen bonds, unwinding DNA
      • single stranded binding protein prevetns strands from reannealing by cooperative binding to opened strands, keeping bases exposed
      • Replication fork is the asymmetric extension of the RNA primer by DNA polymerase
        • continuous synthesis on the leading strand and discontinuous synthesis on the laggin strand
          • Okazaki fragments that are made in the lagging strand are joined together by DNA ligase
      • DNA polymerases prrof read DNA during synthesis
    • Mutation and Repair
      • silent mutations, missense mutations, nonsense mutations and frame shift mutations
      • spontaneous, DNA replicatiion errors and environmentally induced errors are all types of mutations
      • transitions are when a purine is replaced for a purine
      • Transversions are when purine is replaced with pyrimidine base or vice versa
      • deletions occur when the template strand loops
      • insertions caused when newly synthesized strand loops
      • 5000 purine bases are lost through depurination each day
      • deamination of amino groups gives different bases with different pairings
      • mismatch repair proteins recruit endonuclease to make a nick and then the helicase can unwind the DNA. Exonuclease degrades the DNA removing the error
      • glycosidase for the mutated base flips it out of the helix then cuts it away
    • Mitosis and Meiosis
      • mitosis used for somatic cell division
        • prophase, metaphase, anaphase, telophase, cytokinesis
      • meiosis for germ line sex cell production
      • 3 chromosomal elements essential for chromosome duplication and segregation
        • replication origin, telomeres, centromeres
      • cohesions keep the chromatids together
        • cohesions and condensins are part of the scaffold

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