Molecular Biology: Eukaryotes

Eukaryotic chromosome organization

  • Chromosomal proteins
    1. Histones: responsible for the compact packing and winding of chromosomal DNA. DNA winds itself around histone octamers.
    2. nonhistone chromosomal proteins: all the other proteins are lumped together in this group. Responsible for various roles, such regulatory and enzymatic.
  • Single copy vs repetitive DNA
    • Tandem repeats (repeats right next to each other, eg: CAGCAGCAG)
      • Trinucleotide repeats: too much can cause diseases. Eg: Huntington disease and fragile X syndrome
      • Satellites, minisatellites and microsatellites: unique pattern of repeats identifies an individual (paternity testing, forensics) or a particular transplant donor (bone marrow engraftment studies)
    • Interspersed repeats: mobile elements/transposons have repetitive DNA at both ends of the sequence
  • Supercoiling: a coil on top of a coil, helps to compact very large amounts of genomic DNA to fit into a compact chromosome
  • Telomeres, centromeres
    • Telomere: the 2 ends of the chromosome.
    • Centromere: a region on the chromosome, can be at the center or close to one of the ends. After replication, sister chromatids are attached at the centromere. During mitosis, spindle fibers are attached at the centromere and pulls the sister chromatids apart.
  • A common question is what is the difference between chromatin and chromosome. The answer is chromatin is the "stuff" that chromosomes are made of. If the chromosome is a cotton shirt, then chromatin is cotton.

Control of gene expression in prokaryotes

  • Transcription factors (proteins) binds to enhancers or silencers (DNA) to affect transcription. Enhancers increase transcription when bound by transcription factor, silencers decrease transcription when bound. Enhancers and silencers in prokaryotes are close to the core promoter, and is part of the extended promoter.
  • Operons are groups of genes whose transcription can be regulated by binding of either repressors or inducers onto a stretch of DNA on the operon called the operator. Repressors reduce transcription, inducers increase transcriptions.
  • Jacob-Monod model describes the lac operon: repressor binds operator (DNA near promoter), blocking transcription
  • Sometimes you come across the term co-repressors and co-inducers. When a co-repressor binds to its target, the resulting complex becomes either an active repressor or an inactive inducer. When a co-inducer binds to its target, the resulting complex becomes either an active inducer or an inactive repressor.
  • alpha factors: these are how phages control transcription inside their bacterial host. By making different α factors at different times, the phage can control the correct transcription sequence of early, middle, and late genes. For example, the α factor for late gene is not made until last.
  • Transcription attenuation: works in the trp (tryptophan) operon. When tryptophan is scarce and needed, transcription occurs normally. However, if there's already a lot of tryptophan present, then transcription terminates prematurely.
  • Negative control = repressor, anything that decreases transcription
  • Positive control = inducer, enhancer, anything that increases transcription

Control of gene expression in eukaryotes

  • Transcription regulation
    • Transcription factors (protein) bind to enhancers or silencers (DNA) to affect transcription. Enhancers increase transcription when bound, while silencers decrease it. The main difference in eukaryotes that sets them apart from prokaryotes is that enhancers/silencers can be very far away from the actual promoter, and can be upstream or downstream. The DNA must must loop back on itself so that the transcription factor bound to enhancer/silencer can actually make contact with the promoter. Intermediate proteins are involved in the process.
    • Eukaryotes lack the bacterial transcription regulation mechanisms such as the operon and attenuation.
  • DNA binding proteins, transcription factors
    • DNA-binding proteins bind to DNA.
    • transcription factors bind to DNA, so they have a DNA-binding domain.
    • DNA-binding domains interact with the grooves in the double helix (major grooves and minor grooves).
    • Advanced: common DNA-binding domains include helix-turn-helix (HTH), zinc finger, basic-region leucine zipper (bZIP).
  • Gene amplification and duplication
    • Gene duplication = 2x amplification
    • Mechanism: either a portion of DNA gets duplicated within a chromosome or the entire chromosome is duplicated
    • Certain genes are amplified in cancer such as MYC and RAS
    • Certain gene amplifications can be used as a target for treatment: Her2 amplification is treated with Herceptin in breast cancer
    • Certain gene amplifications affect the prognosis of a disease: intrachromosomal amplification of chromosome 21 = worse prognosis in acute lymphoblastic leukemia
    • Other gene amplifications have no effect at all other than to increase the size of the genome (eg. Selfish/parasitic DNA that serves no function but is amplified)
  • Post-transcriptional control, basic concept of splicing (introns, exons)
    • tRNAs and rRNAs modifications: some normal nucleotides are modified to control the structure of these RNAs.
    • mRNAs modifications
      • RNA splicing: sequences called introns are cut out, sequences called exons are kept and spliced (joined) together.
      • Alternate splicing: different ways of cutting up and RNA and rejoining the exons pieces make different final RNA products.
      • 5' capping and 3' poly-A tail: these help to protect the RNA from degradation so they can last longer.
    • After the correct modifications, RNA is transported out of the nucleus where they can function in translation.
    • After some time, RNA is degraded. The rate and timing of RNA degradation can be controlled by the cell.
  • Cancer as a failure of normal cellular controls, oncogenes
    • Failure of normal cellular controls:
      • Cancer cells continue to grow and divide in situations normal cells would not.
      • Cancer cells fail to respond to cellular controls and signals that would halt this growth in normal cells.
      • Cancer cells avoid apoptosis (self-destruction) that normal cells undergo when extensive DNA damage is present.
      • Cancer cells stimulate angiogenesis (cause new blood vessels to grow to nourish the cancer cell).
      • Cancer cells are immortal while normal cells die after a number of divisions.
      • Cancer cells can metastasize - break off and then grow in another location.
    • Oncogenes: genes that cause cancer when activated. The product of many oncogenes are involved in speeding up cell division. Before an oncogene is activated, it is a harmless proto-oncogene. Something occurs that changes the proto-oncogene to an oncogene. The classic example of oncogene is the src.
    • Tumor suppressors: if the oncogene is the "bad" gene, tumor suppressors are the "good" genes. The product of many tumor suppressors are involved in slowing down or controlling cell division. If something happens that cause the tumor suppressor to no longer function, then the cell becomes cancerous. The classic example of tumor suppressor is the p53.
  • Regulation of chromatin structure
    • Chromatin = DNA wrapped around histones
    • Chromatin can be regulated by modifying DNA (methylation) or histone (methylation, acetylation)
    • Euchromatin: less compact chromatin, more exposed DNA, actively transcribed
    • Heterochromatin: more compact chromatin, less exposed DNA, not actively transcribed
  • DNA methylation = CpG methylation (natural) or alkylating agent (chemotherapy) induced methylation
  • CpG islands = GC-rich DNA sequence near promoters that is the target for methylation
  • The methylation occurs on the cytosine nucleotide to make 5-methylcytosine
  • The enzyme is DNA methyltransferase (DNMT)
  • Methyltransferases can work in both ways (add or remove methyl groups), depending on the enzyme
  • Methylating CpG islands silences a gene
  • Clinical utility: MGMT promotor methylation status
    • Glioblastoma (brain cancer) is treated by an alkylating agent (chemotherapy) +/- radiation
    • MGMT = methyltransferase enzyme that removes alkyl/methyl groups from DNA = undo's the chemotherapy
    • Bad = active MGMT = unmethylated MGMT promotor = undo's chemotherapy
    • Good = Inactive MGMT = methylated MGMT promotor = effective chemotherapy
  • Non-coding RNAs = not translated into protein = anything other than an mRNA
    • rRNA = makes up the ribosome
    • tRNA = brings amino acids to the ribosome
    • snRNA = makes up the RNA splicing machinery
    • snoRNA = guides RNA modification
      • RNA is heavily modified in RNAs where a structural purpose is needed (rRNA, tRNA, snRNA)
      • 2'O methylation (protects against hydrolysis/degradation)
      • Convert Uracil to pseudoUracil (plays a role in tRNA structure and integrity)
    • miRNA = makes up the RNA silencing machinery (blocks mRNA translation or degrades mRNA)