MCB 150 Exam II Outline-Eukaryotes.docx

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University of Illinois
Molecular and Cell Biology
MCB 150
Brad Mehrtens

Eukaryotes DNA Organization Linear chromosomenucleus  HOWEVER due to the Endosymbiotic theory  chloroplasts + mitochondria come from ancestral bacterial cells o cells had their own genomes o circular double stranded DNA also found in these SEMIAUTONOMOUS ORGANELLES  stroma of chloroplasts/matrix of mitochondria PACKING DNA  to get things out of the way AND to be able to access DNA  Eukaryotes: 6 billion base pairs of DNA in diploid genomes (half from mom half from dad) o Chopped into chromosomes (segmented) o All the chromosomes > 2 meters in length  Must cram it together to be 1-10 micro meters (nucleus/nucleoid region) EUKARYOTIC DNA ORGANIZATION  Roger Kornberg o Crack open cell--> take out nucleuscrack nucleustake out chromatin  DNA complexed with Protein (chromatin)  Stretch out chromatin o “beads on a string”  Beads on a String o “beads”=protein o “string”=DNA  Kornberg isolated  Nuclease: a family of enzymes capable of hydrolyzing phosphodiester linkages (degrading nucleic acid)  Gentle nuclease activity o Just long enough to cut the string in between the beads (not the string wrapped around the beads) linker DNA  Separated spots: NUCLEOSOMES o Salt preparation: string falls off the beads  String=Double stranded DNA w/ 146 base pairs that were touching the beads  Beads= proteins: 8 different polypeptide chains in one “bead”  DNA wrapped around 8 polypeptides with 146 base pairs touching it o Proteins beads:  HISTONES: have DNA wrapped around it and can be changed when it has to  Basic  Small  H1, H2A, H2B, HB, H4  + charged proteins interacting with DNA o Phosphate groups (negative) interact with + charged Protein to hold it in place in an organized fashion  Phosphates are negative, so positive charged histones can stabilize it (must be basic)  Highly conserved: o Primary sequence are highly similar across different species (like RNA)  H4 (cat): 102 A.A. and comare with H4 (pea plant) 100 A.A. are exactly the same o Don’t change the sequenceDIE  BACTERIA DON’T HAVE HISTONES o Have proteins that interact o ARCHAE HAVE histones (but no nucleus)  NUCLEOSOME:  2 molecules of each histone o EXCEPT H1  146 base pairs of DNA  10 nm diameter chromatin fiber  CHROMATOSOME: more accurate  2 molecules of each histone (touching 147 base pairs)  H1 histone o Inside outer boundary of H1, not touching core  Base pairs that are not bound to Histone (~20 bp) o 10 on each side o 166 bp total  interact with one anothermore organization  TAILS:  “Random coil region”  at amino terminus  A.A. of tails can be chemically modified in a covalent but reversible way (add stuff with enzymes) o A) add phosphate (Kinase)/remove phosphate (Phosphatase) o B) add acetyl group (acetylase) /remove acetyl group (deacetylase) o C) Add methyl group (methylase) /remove methyl group (demethylase)  Why remove: they act as signals  Does the cell need to wind DNA or unwind DNA? Does something need to be worked on? REGULATIONallow the cell to condense or relax DNA as the needs of that region see fit  they come in combos  histone code: combos of chains and modifications refer to certain signals HIGHER LEVELS OF ORGANIZATION  10 nm organization isn’t enough  Chromatosomes intearact with one another o 1) telephone chord  coil nucleosome 6 per turn and pack them together  Flexibile zig-zag: H1 holds zig zag in place o 30 nm Fiber (more organized to get more DNA into a smaller space)  o must pack 2 meters of DNA into 1-10 MICRONS (nm) space in the nucleus/nucleoid region Lecture 14 DNA Organization  Is Chromatin always organized to the same extent during the cell’s life? o Eukaryotes: Phases  Interphase: growing/ replicating  M-Phase: nuclear division (mitosis) + cytoplasmic division (cytokinesis)  2 daughter cells (cytokenesis) o must pack DNA tightly when separating  1 way to organize DNA during Interphase and another during M Phase  Interphase: in Eukaryotes o Chromatin tight or unwound (working on it-->accessibility)  Loosely Organized: euchromatin  Distributed throughout the cell evenly  Mostly in 30 nm fibers (or less)  Tightly/More condensed: heterochromatin  Subsequent levels of organization  Anything more organized than 30 nm o pockets around nucleus o Periphery of nucleus (mostly) o Looks darker on an electron micograph  M-Phase : chromatin condensed o Nucleolus disappears o Chromatin condenses further into “chromosomes”  More heterochromatin  Tighter/organized o Must take ½ DNA and put each half on one side of the cell  Easier to break the tighter organized structures, so that’s what you want in order to split chromosomes in half for cell division  Minimize the risk of breaking the DNA  o Heterochromatin Organization:  Loop 30 nm fibers into 300 nm fibers  Take looped fibers and coil loops for 700 nm structures (half a mitotic chromosome)  2 replicated double helical modelseach sister chromatid  TOTAL 1400 nm (two 700 nm structures together)  sister chromatids run LENGTHWISE o each has 2 arms (4 arms total)  Homologous Chromosome: copy of chromosome 7 from mom and dad o Genes in same place but are NOT the same sequences  Each gets replicated so you have 2 pairs of sister chromatids which represent chromosome 7  Telomeres: identifies the end of chromosome arm o Centromeric region + telomeric regionproteins there that recognize key sequences of bases in centromeres (telomeres) and proteins bind for different reasons o 1) Identifies the end of a linear DNA molecule  ends of linear DNA molecuels indicate that something got broken  if a repair mechanism encounters the end of a linear molecule, repair machine assumes something is broken and tries to fix it o this does not want to happen (linking two different chromosomes together) o must go out of way to say that it is not brokensome telomere proteins do this o 2) recognizes the end of a Linear Chromosome  check out Telomeres in TEXT BOOK  Levels of DNA organization o o Euchromatin:  2nm30nm o Heterochromatin:  30 nm + = 1400 nm  only have to unpack certain regions to work on them, not the whole thing DNA Base Pairs  4 base pairs o Adenine, Guanine, Cytosine, Thymine o Sequence of bases looked at as a unit of information  4 possibilities of base pair sequence combos  n=base pairs  250 million base pairs in HUMAN DNA  “sequences” are recognized as “shapes” Eukaryotic Transcription Eukaryotic o RNA POLYMERASE I o Transcription of rRNA genes o RNA POLYMERASE II o mRNA genes o RNA POLYMERASE III o tRNA genes o leftover rRNA work o in the absence of any helper proteins, if you only look at polymerase subunits, it does a very bad job recognizing its own promoter region because it has NO sigma factor o needs a helper coenzyme o Transcription Factors: accessory protein to help in eukaryotic transcription  Recruit RNA POL to sit down on strands to start transcription once it finds the hot spots  Regulatory purposes  o each one recognizes its own promoter since each RNA has its own promoter scheme o POL I o POL II o POL III o Transcription FactorsStart Transcription in Eukaryotes o Eukaryotic o Protein coding gene  TATA BOX: -30 bases upstream  Written in T and A in sequence  Easier to unwind AT base pairs  Load in first TF  Then RNA POLYMERASE can sit down after the assembled TF are set in place  TF naming: ie) TFIIDeverything you need to know about that molecule  TF: transcription factor  II: which type of RNA pol it is working for  D: order it was discovered  All TF left behind once transcription begins (must like sigma factor being taken away but TF is not part of the polymerase Differences in genes b/t bacteria and eukaryotes  Length of the resulting transcript in bacteria is approx. the same length of the gene in which it was transcribed (ALL INFO USED)  In Eukaryotes: there is a lot of extra DNA (repeated sequences, etc…)NON CODING DNA that doesn’t get coded for protein o SPACER DNA: in between gene x + y (a lot of DNA) o INTRON: within gene x  Intervening sequences  Must be removed  EXONS: expressed sequences that are used for a continuous coding regionprotein  DNA sequence is much longer than the resulting mRNA that participates in protein synthesis  RNA POLYMERASES don’t know introns from exons o They transcribe everything (until told to stop) o Introns are cut out AFTER transcription through RNA splicing o Resulting mRNA=MATURE  1) genes transcribed into large precurser mRNAs  2) called primary or initial transcripts or pre-RNA  3) RNA processed into continuous coding sequences SPLICING: intron removal (join two ends together)  introns can vary in length, but all of the base pairs must be removed  occurs: post-transcriptonally o not RNA pol’s job o not translated o does not mean you have to wait until you get all the way from promoter region to terminator region and THEN start taking out introns  it actually just happens when it’s allowed to happen and once the machine recognizes an intron/exon junction, it’ll start taking out the introns even while RNA polymerase is working on the rest of the chain o must happen before mRNA exported into cytoplasm because ribosome’s cannot recognize the difference between introns and exons  will make the wrong protein otherwise  not the case in bacteria because there are no introns o All introns have something in common  Conserved sequences at both endssplice point (rest of intron not important)  Complex of protein + RNA: ribonucleoprotein complex  Molecules that recognize intron/exon junctions: ribonucleoprotein complex o Special kind of RNA in it o Small nuclear RNA: snRNA  Ribonucleoprotein complex + snRNA: snRNPs  Remove intron + join ends of exon o SPLICING st  1 thing bound: Boundary between exon 1 + intron: 5’ splice site (on the 3’ of exon) bound by snrnp  2 thing bound: sequence of bases with a very important Adenine  invariant A not at splice site  first cut: take snrnps and bring togheter into a splisosome  cuts the 5’ splice site  take 5’ intron end that was just cut and l
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