Meeting 14

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Meeting 14
2011-11-10 10:26:20

7.2 7.3 7.4
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  1. gene includes:
    • 1) transcribed sequence (into mRNA, so essentially mRNA transcript
    • -this INCLUDES both introns and exons
    • 2) regulatory sequences (promoters, enhancers, etc)
  2. eukaryotic regulatory elements
    • 1) basal promoter
    • 2) promoter-proximal elements
    • 3) enhancers
  3. basal promoter
    a DNA sequence that specifies where RNA polymerase binds and initiates Tx of a gene; can include a TATA box
  4. promoter-proximal elements
    a control region that lies within 100-200 base pairs upstream (in the negative [-] region of the start site; can be involved in 'tethering' more distal elements
  5. enhancers
    refers to a Tx control region GREATER than 200 base pairs from the Tx start site; most distal elements either upstream, downstream or within introns that control tissue specific and temporal expression

    • 1) work over long distances.
    • 2) work independently of position and orientation
    • 3) control timing/levels/patterns of gene expression
  6. upstream v. downstream
    upstream: opposite to the direction of Tx

    downstream: in the same direction as Tx
    • -there are 3 different RNA pol's
    • -care eluted at different salt concentrations (which shows their various net charges
    • -also differ in their sensitivity to alpha-amanitin

    • -by looking at this graph you can see:
    • 1) sensitivity to alpha-amanitin: pol I least sensitive, pol III most with pol II falling somewhere in the middle
    • 2) pol I = least acidic, pol III = most acidic
  7. prokaryotic v. eukaryotic Polymerase II
    • prokaryotic:
    • RPB1 and RPB2 (large)
    • w-like subunit
    • and two non-identical α-like
    • -UNLIKE bacterial polymerase, eukary. have 4 additional subunits

    • eukaryotic:
    • -β' and β subuits (large): machines that run the Tx (read a strand and incorporate dNTPs), (catalysis)
    • -two α subunits: tells the beta subunits where to go, assembles, directs, recognizes promoter, IZ IN CHARGE
    • -w subunit: (omega subunit) holds things together/stability

    (the order for each shows that one's corresponds to the other's); the similarity in the structures of these core subunits in RNA pol from various sources shows that they probably evolved early and was largely conserved (seems logical for an enzyme catalyzing a process so basic/necessary as copying RNA from DNA)
  8. just know this
  9. What are the differences between RNA I, II, and III?
    • -RNA pol I: the only enzyme that transcribes ribosomal RNA, a type of RNA that accounts for over 50% of the total RNA synthesized in a cell;
    • important for PROTEIN SYNTHESIS

    • -RNA pol II: enzyme found in eukaryotic cells that catalyzes the transcription of DNA to synthesize
    • precursors of mRNA (& most snRNA and microRNA); important for PROTEIN ENCODING, RNA SPLICING, POST-TRANSCRIPTIONAL GENE CONTROL

    • -RNA pol III: genes transcribed by RNA Pol III fall in the category of "housekeeping" genes whose expression is required in all cells/most environments; transcribes genes that encode small RNAs that are themselves functional (ex: tRNA); [the regulation of Pol III transcription is primarily tied to the regulation of cell growth and the
    • cell cycle, thus requiring fewer regulatory proteins than RNA polymerase I]; important for PROTEIN SYNTHESIS, RNA SPLICING, MANY FUNCTIONS UNKNOWN, SIGNAL RECOGNITION!
  10. CTD (carboxyl-terminal domain)
    • -SO the carboxyl (3' end) of the largest RNA pol II subunit (RPB1) has this stretch of SEVEN amino acids that is almost perfectly repeated a couple of times ---- neither pol I nor pol III ave these repeating units
    • -this heptapeptide IS the CTD
    • -yeasts have 23 or more repeats (there need to be at least 10 for the yeast to survive), while vertebrates have 52
    • -what's important about the CTD is that at the start/initiation of Tx, the residues are unphosphorylated
    • -however during the process of Tx, many [of the serine and tyrosine] resudyes ARE phosphorylated
  11. initiator
    a promoter element some eukaryotic genes have instead of of a TATA box; most have a C at position -1 and an A at Tx start site (+1)
  12. what happens when you change or delete the sequence BETWEEN a promoter (TATA box) and Tx start site:
    -changes between the TATA box and start site do not significantly affect the rate of Tx

    -however if some basepairs between the TATA box and the Tx start site are DELETED, what generally happens is that Tx starts ~25 bp from the TATA box (like a little shift)
  13. CpG island
    • CG-rich region individual to genes that are transcribed at low rates (ex. housekeeping genes); such genes do not generally have a TATA box or initiator
    • -CG rich region seems to indicate that there's a protein-coding region nearby because it's not found very often in the genome except for when it acts as/may contain a Tx-initiation region
    • -insert DNA with altered 5' ends into plasmid containing reporter gene (so you can see whether or not the gene is expressed)
    • -after replication occurs within the organim, you can prepare a cell extract and assay activity of a reporter enzyme
    • the results of this experiment indicate that there are two control elements:
    • -one between region 2 & 3
    • -one between region 4 & 5

    -it is at these regions where there's a change expression, indicating that some of the shorter 5' ends are missing some control elements (or parts of a control element)
  14. Microinjection
    ex. mouse egg microinjection; the process of inserting microscopic substances into a single living cell; process is frequently used as a vector in genetic engineering and transgenics to insert genetic material into a single cell
  15. what makes a good protein reporter?
    1) it's biologically inert (can't be broken down any further)

    2) not produced by the organism under study

    3) product is easy to assay/visualize

    4) doesn't interfere with any pathways

    • examples:
    • -lacZ, which makes the protein β-galactosidase, which IN TURN turns X-Gal (organic compound) blue
    • -GFP directly visualized by fluorescence under UV light
  16. DNA sequence conservation
    • ·spikes show how much of regulatory elements that make an eye is conserved between these animals and humans
    • ·chances are if there’s conservation of sequence, then there's also conservation of function
  17. EMSAs (Electrophoretic Mobility Shift Assays, Gel Shifts)

    describe what you see:
    • in this experiment, it is clear that at first both DNA fragments (mutant and WT) are run on a gel and settle in the position at the bottom
    • -then, the fragments are mixed with a [DNA binding] protein of interest
    • -you know there's a mutation in the mutant DNA fragment (duh) because the DBP only binds to the WT fragment as evidenced by the longer/heavier strand on the top of the WT gel (heavier b/c it's a larger complex composed of both DNA and protein)
  18. describe what you see in this picture:
    • -lane 1: control; only contains only DNA.
    • -lane 2: contains protein as well as a DNA fragment (of the same length as the control it seems) and based on its sequence, this fragment does NOT interact with said protein
    • -lane 3: because the fragment moves slower than the other two, lane 3 appears to contain a protein and a DNA fragment that have reacted; resulting complex is larger/heavier/slower-moving
  19. DNAse I footprinting
    • -can identify control-element sequences
    • -DNA fragment known to contain control element is labeled with 32P on one end
    • -portions of the DNA are then digested with DNase I (cuts DNA via its phosphodiester linkages)
    • -if the DNA does not have a protein bound, then the DNase I is added in a concentration so that each fragment is only cut once at random sites
    • -if the DNA DOES have a protein bound, then the DNase I still cuts the fragment but it cannot cut at the section where the protein is bound (AKA THE CONTROL REGION)
    • -after DNase treatment the DNA is seperated from the protein, it's strands are dissasociated from one another and the single strands are run on a gel
    • -the 'footprint' site means that no fragments of that particular length exist because the bound protein prevented cutting actvity; so a the length that corresponds to that gap region is the length of the control region (aka region where protein was bound)
    • protein binds to DNA and mark it
  20. sequence-specific DNA affinity chromatography
    type of affinity chromatography in whcih long DNA strands containing multiple copies of the tx-factor binding site (!) are coupled to a column matrix
  21. GAL4/UAS system
    biochemical method used to study gene expression and function; it has two parts: the GAL4 gene, encoding the yeast transcription activator protein Gal4, and the UAS (Upstream Activation Sequence, a section of promoter region where Gal4 binds to activate gene transcription)

    -showed that production of reporter gene depended on the presence of both DNA binding domain (N-term) AND the activation domain (C-term); GAL4 would bind to DNA if complete N-term was present, but would only show Tx activity if some or all of the C-term (activation domain) was present (p. 289)

    -if the area seperating these two regions of the protein was compromised/changed in any way, there was no affect on DNA binding or Tx; explains why alternations of spacing between control elements is well tolerated in eukaryotes: FLEXIBLE
  22. DNA-binding domain
    the N-terminal part of the transcription factor that binds to the DNA sequence
  23. activation domain
    the C-terminal part of the transcription factor that ineracts with other proteins to stimulate transcription from a nearby promoter
  24. mutation of an activator binding site leads to _________ expression of the linked reporter gene, while mutation of a repressor binding site leads to _________ expression of a reporter gene
    mutation of an activator binding site leads to DECREASED expression of the linked reporter gene, while mutation of a repressor binding site leads to INCREASED expression of a reporter gene
  25. DNA-binding domains (of Tx factors aka proteins) contain many structural motifs; the ability for DNA-binding proteins to bind with specific DNA sequences:
    results from NON-covalent interactions between atoms in an ALPHA-HELIX (recognition helix) of a protein's DNA-binding domain and atoms on the outside of bases in the DNA's major groove
  26. helix-turn-helix motif (bacterial?)
    • composed of two α helices joined by a short strand of amino acids; supported by hydrophobic interactions between the 2 helices
  27. homeodomain proteins
    conserved 60-residue DNA-binding motif found in many eukaryotic Tx-factors (that function in development); similar to bacterial helix-turn-helix motif
  28. zinc-finger domain/proteins
    • when proteins have regions that fold around central Zn2+ ions (2 types)
    • 1) C2H2 zinc finger: most common DNA-binding motif in humans; comtains 2 cysteine and 2 histidine residues whose side-chains bind to zinc ion

    2) C4 zinc finger: four cysteines that contact zinc ion

    -important differences (lol): C2H2 prteins generally contain 3 or more repeating finger units and bind as monomers while C4 proteins only contain two and bind as homo/heterodimers
  29. leucine-zipper (b-zip) proteins
    -contains a hydrophobic leucineat every 7th position in the peptide chain; these proteins bind to DNA as dimers and the leucine is required for said dimerization

    -crystallographic analysis (of GCN4 protein) shows the dimeric protein has two extended alpha helices that 'grip' the DNA molecule (like scissors) and two adjacent major grooves
  30. basic (+) helix-loop-helix domains/proteins (bHLH)
    similar to basic zipper except that a nonhelical loop of the peptide chain separates two apha helical regions in each monomer

    -amino acid sequence is as follows: N-terminal alpha helix w/ basic residues that interact with DNA, a middle loop region, and a C-terminal region that has hydroPHOBIC amino acids
  31. Systematic Evolution of Ligands by Exponential Enrichment (SELEX)
    used to find specific binding site of Tx factor (or any protein)

    • -process begins with the synthesis of large oligonucleotide library with of randomly generated sequences of fixed length flanked by constant 5' and 3' ends that serve as primers (for a randomly generated region of length n, the number of possible sequences in the library is 4n; 4 nucleotides (A,T,C,G), with n possibilities).
    • - sequences are exposed to the target protein and those that do not bind the target are removed (using affinity chromatography)
    • -bound sequences are eluted/amplified by PCR
    • -these sequences are then subjected to subsequent rounds of selection in which the stringency
    • of the elution conditions is increased to identify the tightest-binding sequences
  32. Position-specific scoring matrix
    height of the letter indicates the importance of the letter to the transcription binding site

  33. homodimer
    a protein composed of two polypeptide chains that are identical in the order, number, and kind of their amino acid residues

    -heterodimer formed by 2 different peptide chains
  34. steroid hormone receptor
    generally intracellular receptors (typically cytoplasmic); initiate signal transduction for steroid hormones which lead to changes in gene expression over a time

    ·for the most part these don’t bind as dimers so they don’t have dimer-binding domain

    ·in the absence of ligand it can’t bind to DNA

    ·in presence of ligand it can bind to DNA (example of positive regulation)
  35. alone, these Tx factors have low affinity for respective binding sites, but interactions between the two proteins leads to an overall more stable complex so that the two proteins bind to the composite site cooperatively
  36. HGMI
    small protein associated with chromatin that makes the binding of Tx factors highly cooperative
  37. parts of Tx factors that are (or may be) necessaryfor Tx:
    • -DNA-binding domain (to recognize promoter)
    • -dimerization domains: b/c many factors occur as homo/heterodimers
    • -activation/repression domain: can be moved around
    • -*ligand-binding domains: allows regulation of Tx factor thought the binding of an accessory small molecule (ex. with steroid hormone receptors, hormones act as the small molecule)
  38. features characteristic of Tx ACTIVATION domains (AGP):
    • -acidic domains (acid blobs): ex.: yeast GCN4 & Gal4, Glucocorticoid receptor, herpes virus VP16
    • -glutamine-rich domains: proportion of Glut is more important than overall structure; ex. SP1
    • -proline-rich domains: found in Tx factors c-jun, AP2, & Oct-2
  39. poly-Glutamine diseases (Trinucleotide repeat disorder)
    when proteins in the brain have poly-glut. repeats, if during recombination transcripts don’t match up, excessive CAG repeats causes malformed proteins that can kill the cell; ex: Huntingtons

    -genetic disorders caused by trinucleotide repeat expansion, a mutation where trinucleotide repeats in certain genes exceeding the normal threshold, (this differs between genes). The mutation is a subset of unstable microsatellite repeats that occur throughout all genomic sequences
  40. features characteristic of Tx REPRESSION domains (aka how they function to repress):
    • -blocking the DNA-binding site of the activator (competing for the site)
    • -complexes with an activator to form a NON-DNA-binding complex (aka changing it's conformation so it can't physically bind to DNA); (ex. the Id (HLH) protein blocks bHLH protein-DNA interactions by dimerizing with bHLH)
    • -masking the activation domain even though activator can still bind to DNA; ex.: Gal80 masks the activation domain of the yeast Tx factor Gal4
    • -particular domain within the repressor inhibits transcription