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Molecular Biology Exam 1 (DNA (DNA as genetic material (structure (and…

- - - - Set of techniques
        
        classic methods
        
        study one gene at a time
        
        modern methods
        
        a "top down" approach
        
        first identify every gene in the genome, then figure out what they do/how they interact
      - advanced genetics
        
        more detailed study of DNA>>RNA>>protein and the regulation of these processes
      - study of life at a molecular level
        
        pick and choose your system of study (cell, gene, etc.) and track on "molecular biology"
    - - a set of principles and concepts that:
        
        Elucidate how genotype (sequence of organism’s DNA) gives rise to phenotype (observable traits)
        
        Have strong support from large experimental sets (i.e., molecular biology is integrative)
        
        Biochemistry and physiology (molecular organization/processes)
        
        Genetics (function)
        
        Biophysics (structure)
      - a discipline in transition
        
        Pre-genomic era: before mid 90s – study individual units; larger connections difficult
        
        Post-genomic era: after mid 90s – compile entire genomes and figure out function of all genes
        
        Finding the functional relationships between genes, taking into account all the genes utilized in a particular cell under given condition, will take a LONG time!
        
        Practically, molecular biology contributes to several areas of study (anthropology, etc)
  - - - Agriculture, forensics, microbiomes, personalized medicine...
    - - Ancient times through 19thcentury: Beliefs based on superstition, limited observation
        
        God’s Word is right and true, but you cannot selectively breed sheep using whittled tree branches
        
        Aristotle (~300 BC) posits physical nature to heredity; ties it to his philosophy of “causes”
        
        17th–19th century
        
        Observational science (1600s) - Leewenhoek and sperm; spermists vs. ovists (no way of conceiving of equal contribution of male and female)
        
        By the end of 19th century
        
        Vitalism challenged with synthesis of urea (Wohler, 1832)
        
        Chromosomes observed in the cell (Flemming, 1880s)
        
        Ideas of heredity based on some sort of physical component, but clueless as to its identity or how it worked
      - Backing up a bit: Mendel links genotype and phenotype (1865)
        
        Good scientist: chose research subject well with careful experimentation
        
        Good fortune: chose unlinked traits
        
        His results: particulate nature to inheritance that is predictable (consistent)
    - - The “Three Botanists” independently rediscover Mendel’s laws (1900)
      - Chromosomes linked to Mendel’s laws (Walter Sutton, with much help from Eleanor Carothers, 1902)
        
        Stained chromosomes - observed chromosome behavior consistent with Mendel’s genetic mechanism for heredity
      - Traits linked physically to chromosome (T.H. Morgan, 1910-20)
        
        dentified 4 linkage groups: all traits (eg, fly mutations they created) can be assigned to one of the four chromosomes, based on how they assort together
        
        Traits can be ordered linerally on the chromosome: Sturtevant (at age 19!) takes data on % recombination from many fly crosses and makes first linkage map
- - - - Why did Watson and Crick succeed?
        
        Chargaff’s rules (1949)
        
        Franklin obtains high-resolution X-ray diffraction data on DNA; Wilkins gives them access (1952)
        
        W&C obtain data from (at best) dubious means; perform not a single experiment themselves
        
        Donahoe tells them correct base tautomers for modeling
      - Watson and Crick propose structure (1953) – mechanism for replication obvious
        
        Contingencies of history may have helped W&C
        
        Did Pauling miss out because of a suspicious passport officer? Because of a professionally depressed Astbury?
      - The “most beautiful experiment of biology” in the 20thcentury – Messelson/Stahl
        
        Simple predictions for replication mech; elegant expt that was easily measurable
      - Modern DNA biology
        
        Details of DNA replication worked out during 1960s
        
        Recombinant DNA work begins in 1970s
        
        Genome sciences starts only twenty years after first DNA manipulations
        
        First living genome sequenced 1995 (H. influenzae; ~million basepairs); only a few years later the human genome is finished (3 billion bp)
        
        Advances in technology have fueled the dramatic increases in ability to collect data
    - - Work on DNA
        
        Discovered in 1869 (Miescher)
        
        Nucleotide structure determined (Levene, 1909), but incorrect organization inferred (the “tetranucleotide” which is functionally uninteresting) – this idea persisted for some time
        
        DNA gets more interesting as a cellular component
        
        DNA is a polymer (longer than the few nucleotides as previously thought - Caspersson, 1934)
        
        Crude structure of DNA as bases stacked in some fashion (Astbury, 1937), with work and input from Florence Bell
    - - Proteins have much more complexity (more amino acids, diverse sequences, longer polymers); DNA structurally and informationally boring!
      - So, prevailing thought: proteins are hereditary material and nucleic acids, since they’re in the nucleus, provide a scaffold or other kind of support
    - - Something is a transforming factor in changing R Streptococcusto S (Griffiths, 1928)
      - It took 25 years after Griffith’s experiment to conclude: DNA!
        
        “Pretty sure” it’s DNA - Avery et al discover transforming substance (1944)
        
        Still, skepticism even from the authors – could DNA simply be necessary for protein function?
        
        Confirmation: Hershey/Chase (1953)
  - - - Genes are responsible for activity of enzymes
        
        A. Garrod (1909) describes “inborn errors in metabolism” and associates biochemistry (enzyme activity) with Mendelian traits
        
        Work by G. Beadle and E. Tatum (1930s-40s)
        
        Wanted a model system to directly tie enzyme activity to gene function
        
        First tried to link traits in Drosophila(eye color) biochemistry using larval transplants; too intractable due to model system complexity
        
        “One gene, one enzyme” experiment
        
        Neurospora>>X-ray induced mutations; amino acid biosynthesis mutants; AND biochemical pathway mutation can be passed to progeny
        
        Conclusion: mutation in one gene leads to loss of one enzyme’s activity
      - Gene mutation correlated to change in amino acid sequence
        
        Work done with hemoglobin from sickle cell anemia
        
        L. Pauling: Disease linked to hemoglobin defect; inherited in Mendelian fashion
        
        V. Ingram, 1957
        
        Fragmented Hbnormaland Hbsickle celland ran chromatography
        
        Sequenced protein fragment that differed between the two Hb forms and found single amino acid difference
      - Linear correspondence between DNA nucleotides on gene and amino acid sequence in protein (C. Yanofsky, 1963)
        
        Isolated several trpA- mutants in E. coli, then performed genetic crosses to order mutations relative to each other >>genetic map of position of mutations in trpA
        
        From each trpAmutant strain, isolated mutant TrpA protein and sequenced it; showed colinear relationship
    - - By 1950s, it was assumed that RNA from ribosomes (rRNA) was the link
        
        Ribosomes used for protein synth – move from nucleus to cytoplasm, and the RNA was right there at the location of protein synthesis!
        
        It was constant sized and stable; perfectly suited to take info from DNA to cytoplasm
      - RNA discoveries (1950s, 60s)
        
        Francis Crick was instrumental in theoretical backdrop
        
        Correctly posited there must be a nucleic acid template that moves from nucleus to cytoplasm
        
        Correctly predicted “adaptor molecule” (tRNA) that brings amino acids to the template
        
        Volkin and Astrachan 1956
        
        Showed that in bacterial system, variable sized RNA that was unstable is produced
        
        Called it “DNA-like RNA,” but not much attention from their work
        
        Jacob and Monod - 1960
        
        Working on another bacterial system, had a hard time explaining their data based on the ribosomal RNA idea
        
        Realized the Volkin/Astrachan explained their data perfectly; win the Nobel prize putting it all together into “mRNA”
      - The last frontier: cracking the genetic code
        
        Two ways to approach the problem: theoretical musings (put on a ridiculous tie and talk it over) or get in the lab (M. Niremburg at the NIH, 1961)
        
        Used E. coli based system to assign codons to their corresponding amino acid
    - - This basic “backbone” forms the basis for much of the discipline
      - Has been updated in recent years to include various amendments
        
        Epigenetics, transposons, reverse transcription, non-coding RNA, alternative splicing
  - - - some definitions
        
        Philosophically, DNA = information
        
        What is the connection between physical molecule and abstract idea of “information”?
        
        Biologically, DNA is “two H-bonded polynucleotide chains”
      - fun facts
        
        20 A width (1/50 of a human hair)
        
        ~10bp/turn
        
        Very dynamic molecule (bases can be flipped out to be covalently modified)
    - - The two sugar-phosphate backbones are in a 5’ >>3’ antiparallel configuration
        
        Please know how to draw two nucleotides joined together as part of the backbone
      - Four nitrogenous bases
        
        Please know how to draw all four bases and how each H-bonds to its partner
      - Why is DNA helical?
        
        Achieving lowest energy state leads to
        
        1) base stacking (exclude water from hydrophobic bases) and
        
        2) slight twist in stacked bases (avoids steric hindrance of electron densities)
        
        Stacking/twisting is allowed by free rotation around glycosidic bond (bonds base to ribose sugar)
        
        Result of these properties is a helical molecule, and from a functional perspective:
        
        1) formation of major and minor grooves
        
        2) major groove (unlike minor groove) has unique information, since each base pair configuration presents a particular “molecular signature”; so in one turn of DNA (with 10 base pairs), very specific information is available for DNA-binding proteins
      - Information content
        
        Nucleotide sequence determines RNA/protein sequence
        
        the major groove (unlike minor groove) has a unique signature since each possible base pair configuration presents a unique bonding and steric environment to DNA-binding proteins
        
        Compaction state is not only structural, but regulatory
        
        Modifying DNA (epigenetics) imparts an additional code
      - DNA Forms: A, B, and Z
        
        A and B differ in form due to degree of water solvation of the molecule (less H2O in A form; B form is the “physiologically relevant” form)
        
        Z has zig-zag backbone due to alternating glycosidic bond angle in backbone
  - - - eukaryotes replicate only once at specific time: during s phase
        
        steps towards replication firing
        
        1) ORC (large protein complex) is associated with the DNA replication sequence, then recruits cdc6, which then recruits helicase, to form the pre-RC
        
        2) This activity happens during G1 phase of cell cycle (before S phase); the pre-RC is set up to simply require the signal to allow replication to commence (turn helicase loose!)
        
        kinase activity in the from of cyclin-dependent kinases, cdk required to initiate origin firing and commencement of s phase
        
        cdk's only active starting at G1>>S phase transition and the continuing through G2 and M phases
        
        In G1, they are not active, so pre-RC can form but is not activated
        
        In S, cdk’s are active >> phosphorylate cdc6 (allows helicase to unwind DNA and replication begins); and they phosphorylate ORC (prevent another pre-RC from forming until G1 phase reached again, when another pre-RC can form)
        
        Cyclin E, which is active at G1 >> S transition, activates the cdk’s to do their job
      - Required sequences--origin of replication ORIs
      - DNAA initiates replication at bacterial ORIS
        
        In E. coli, ORI is 246 bp with several repeat sequences that are AT rich (9-and 13-mers)
        
        DnaA binding bends DNA; torsional strain opens the DNA and allows helicase access and the holoenzyme can now begin replicating DNA
        
        Regulation of initiation: competition between Dam and SeqA proteins
        
        Initiating replication depends on methylation status – and DnaA requires fully methylated DNA to bind
        
        1) DNA adenine methylase (Dam) methylates “A” in GATC (11 sites in 245-bp ORI, way more than expected by chance)
        
        2) DNA is hemi-methylated for a time following initiation, because high concentration of SeqA protein outcompetes Dam for GATC site binding
        
        3) As Dam outcompetes SeqA, it can re-methylate, initiation can happen again
        
        4) Dam begins to outcompete SeqA when the latter’s concentration is lowered at the origin, as it will bind hemimethylatedDNA all over the chromosome during the replication process
    - - Prokaryotes: enzymatically unlink two new circular chromosomes directly opposite the ori
      - Eukaryotes have to deal with problem of DNA shortening at ends of chromosomes
        
        Telomeres: sequences at ends of chromosomes that are repeated hundreds or thousands of times
        
        shorten upon each round of replication, since the terminal RNA primer, once removed, can’t be filled in with DNA (no primed DNA there to do DNA synthesis!)
        
        Telomerase: has RNA (part of which is reverse complement of telomere repeat sequence) and reverse transcriptase component
        
        Mechanism of action
        
        1) RNA template binds to repeat segment on telomere
        
        2) Telomerase extends 3’ end on the shortened chromosome end
        
        3) Extending the 3’ end gives sufficient DNA for addition of RNA primer and then DNA pol to synthesize missing strand; chromosome length thus preserved
        
        Remember that telomerase does not fill in the shortened DNA gap!
    - - DNA synthesis requires primed template; proceeds by extending the 3’ end
      - Chemistry (please be able to draw the following
        
        5’ triphosphate on incoming nucleotide added to 3’ hydroxyl; proceeds 5’ >>3’
        
        3’ oxygen on last nucleotide on 3’ end attacks phosphate on incoming free nucleotide with di-phosphate leaving group (SN2 reaction)
    - - Catalysis
        
        Very high processivity: template moves through “palm” portion of enzyme at ~1000 nt polymerized/second
        
        Positioning of proper nucleotide in active site
        
        Responds to proper nucleotide geometry, by: 1) forming proper H-bonds with complementary template nucleotide; and 2) not allowing RNA nucleotides to react
- - - - Requires special attention for replication because of linearity, size, and multiple # of chrom
  - - - In humans, large portion of the chromosome is repetitive DNA; very small amount is actual coding sequence for genes
    - - Some plants have multiple copies of many genes, increasing overall genome size
  - - - structure
        
        146 bp of DNA wrapped around histone protein core “octet” – two H2a/H2b heterodimers and two H3/H4 heterodimers
        
        20-60bp of linker DNA between each nucleosome
      - histone proteins
        
        Histone fold - 3 alpha helices in each histone allows dimerization with partner histone
        
        N-terminal tails have dual function: structural (serve as “threads” upon which the DNA coils) and regulatory (can be covalently modified epigenetically)
        
        Composed of significant amount of (+) lys and arg residues; this is electrostatically favorable for DNA association
        
        Highly conserved across all eukaryotes, except in N-terminal tail region
    - - regulated use of compacted DNA
        
        Early discoveries of how DNA compaction influences its use
        
        Grosveldet al (1987) use Southern blotting with HEL cells expressing epsilon hemoglobin gene (embryonic gene in embryonic cells)
        
        15kb region of epsilon gene promoter evaluated using Southern blot: if no nucleosomes (as predicted), will be accessible to micrococcal nuclease digestion
        
        Regulatory region is sensitive to nuclease; same region in adult cell line is not
        
        Conclusions: nucleosomes are present when DNA not accessed; absent when actively transcribed
        
        Hypothesis: nucleosomes should not be present where DNA is actively utilized (eg, where regulatory proteins need to bind at a promoter)
        
        Method used: Southern blotting – identify a specific DNA fragment
        
        Whole genome cut with restriction enzyme all fragments run on a gel
        
        Must know at least a short amount of nucleotide sequence of fragment of interest in order to make a “probe” – a radioactively-labeled short single stranded DNA
        
        Denature DNA on gel; probe will bind to complementary sequence on fragment of interest, and can visualize this when exposing gel to film that detects radioactivity
      - modern discoveries: histone function allows DNA access
        
        the histone code
        
        Covalent modification of residues on N-terminal tails
        
        Conveys message for DNA use. Example: H3K14Ac (acetylate lysine 14 on histone H3) – transcribe this gene
        
        Acetylating neutralizes the positive charge on lys; DNA less able to associate
        
        Other types of modification and meanings
        
        N-terminal tails can be phosphorylated, methylated, etc
        
        Meanings such as transcribe/don’t transcribe, form heterochromatin, insert alternative histone protein (to repair DNA, for example)
        
        HATs and HDACs acetylalte and deactylate, respectively
        
        HDACs, epigenetic reg of DNA accessibility
        
        Example of “modern” HDAC importance: cancer biology (Harms & Chen paper, cont’d)
        
        Does the HDAC2 effect on MCF7 cell proliferation involve the tumor suppressor p53?
        
        Yes, since if they eliminate p53 activity in the context of HDAC2 knockdown, proliferation levels change compared to normal p53 activity
        
        Possible mechanisms: HDAC2 deacetylates p53 directly, or deacetylates histones important for p53 activity
        
        latter hypothesis favored, since the expression of a p53-regulated gene (p21) went up when p53 protein added AND HDAC2 inhibited
        
        Model: in MCF7 breast cancer cells, overactive HDAC2 is improperly deacetylating histones in the regulatory sequences of p53-regulated genes, so they are not expressed properly (ie, p53 can’t do its job)
        
        this would allow cells to enter S phase aberrantly, and could contribute to the cancer phenotype
        
        How do HATs and HDACs know where to act, since they’re non-specific?
        
        They need to access to specific DNA locations to promote specific gene expression, heterochromatin formation, etc
        
        HATs and HDACs are brought to histones as multi-protein complexes that accomplish common purpose
        
        Example: NuRD complex – has HDAC 1 and 2 AND proteins that direct them to specific DNA sequences so they can act on the neighboring histones
        
        Together, form the “acetylome,” – sum total of all acetylations in given cell at any one time (a staggering thought!)
        
        General protein acetylation (done by Class II HDAC, which themselves can be covalently modified to change activity)
        
        Example: shuttling HDAC between nucleus and cytoplasm
        
        Muscle cell differentiation: phosphorylated HDAC5 stays in cytoplasm; can’t get to nucleus to deacetylate MEF, which is required for MEF to act as a transcription factor and promote cell differentiation
        
        Specifically deacetylating histone proteins (done by Class I HDACs)
        
        Acetylation/deacetylation plays role in regulation of gene expression; one global area where this works functionally is progression of cell cycle (and thus is related to cancer state)
        
        Harms/Chen paper: MCF7 breast cancer cell line that overexpresses HDAC2; want to connect this activity to cancer phenotype
        
        showed that when HDAC2 expression is knocked down, cells stay in G1 more: suggests that when HDAC2 overexpressed, cells get to S phase easier
      - 30 nm fiber is an ordered, “zig-zag” coiled structure of nucleosomes
        
        This structure is facilitated by first compacting nucleosomes: Histone H1 brings adjacent nucleosomes close together
        
        Beyond that, what is known about compaction? Not a lot!
        
        DNA loops predominate (visible in frog lampbrush chromosomes). How they form and are maintained is under investigation
        
        One family of proteins identified that contributes to higher-order chromosome structure: SMC proteins (Structural Maintenance of Chromosomes)
        
        Two protein monomers join to form a “hinged” structure; it is thought that the ends attach to DNA and hinge region allows structural maintenance
        
        DNA binding motifs and apparent ATP use suggest mechanism for SMC protein use (bind to DNA loops; ATP hydrolysis allows compaction)
    - - To fit into typical eukaryotic cells, DNA must be compacted 104–105fold)
      - Replication, transcription must work through, and can be affected by, compacted DNA
      - Differential compaction of particular DNA can define cell type
        
        Euchromatin vs heterochromatin as a way to (sometimes) permanently shut off gene access