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30 Cards in this Set
- Front
- Back
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Chemists in 1968 that got nobel prize for deciphering the genetic code
THey used cell free protein synthesis system to detereine peptides produced |
-H.G. Khorana
-R. Holley -M. Nirenberg |
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The Cell-Free System consists of?
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-S-30 fraction - ribosomes, tRNA's, tRNA synthetases, other soluble protein factors
-20 amino acids -GTP and ATP -Energy generating system to keep producing ATP and PEP + pyruvate kinase -Mg2+ and K+ (NH4+) |
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Ribosomes in translation machinery in Prokaryotes
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-70S(composed of L (50S) and S (30S) subunits)
-contain 23S (L), 16S (S) and 5S (L) rRNA's -each subunit (L and S) contains ~30 proteins |
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Initiation and Elongation factors in translation machinery in prokaryotes. Also termination (release) factors
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-if1, if2, if3
-ef-Tu, ef-Ts, and G -Rf1 and Rf2 Translation initiated by fmet (N-formylated methionine) |
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How right AUG selected for translation in prokaryotes?
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-S-D sequence about 7nt upstream of start codon base pairs with the 3' end of 16S rRNA (AGGAGG)
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How is translation initiated in Eukaryotes?
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-Begins with methionine that is NOT FORMYLATED
-tRNA (tRNAiMet) different from the one that is used for internal methionine codons -Start determined by the AUG and surrounding sequence -start site also affected by RNA structure at the 5' end of the mRNA |
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Scanning (or Kozak) model for translation initiation in Eukaryotes
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-40S subunit along with IF's, Met-tRNA, and GTP recognize the m7G cap at 5' end of mRNA so the subunit can bind.
-40S scans down seaching for AUG and melts stem loop in way. -once AUG found, the 60S can join so initiation can occur -ATP required for scanning energy |
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Exceptions to the Scanning model?
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-Translation of some mRNAs (5-10%) does not start at first AUG
-Positions -3 and +4 are important, based on mutagenesis studies. -Purine at -3 -Guanine at +4 |
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Initiation factors (except eIF4)
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-eIF-1(and iA) - promotes scanning
-eIF-2 - binds tRNAiMet to 40S, requires GTP -eIF-2B - catalyzes exchange of GTP for GDP on eIF-2 -eIF-3: binds to 40S, prevents 60S from binding to it -eIF-5: stimulates 60S binding to 48S pre initiation complex -eIf-6: binds to 60S, helps prevent 40S from binding to it |
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eIF-4
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-eIF4F: ability to bind cap 7MeGTP
-F contains E,A,G -eIF4E:binds the cap -eIF4A:RNA helicase; laso oustide complex; contains DEAD motif; requires ATP and stimulated by eIF4B -eIF4G:versatile adaptor; helps recruit 40S to mRNA;responsible for synergistic effect of cap and polyAtail on translation -eIF4B:binds RNA and stimulates eIF4A; together they unwind hairpins in 5' UTR |
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IRES are not capped but translated. How is this done?
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Viral protease clips off N-terminus of eiF4G so it cant bind iEF4E. eIF4G binds viral protein that binds the IRES, promoting translation of uncapped viral mRNAs
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eIF2, interferons, and viruses
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Interferons are anti viral proteins induced by viral infection
-they repress translation by triggering phosphorylation of eIF2alpha -kinase is DAI (dsRNA) which triggers the same pathway of virus. inhibits protein synthesis -overall they block reproduction of the virus |
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Translation elongation in Prokaryotes
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1. EF-Tu brings in second tRNA to the A site. The P site is already occupied by fMettRNA.
2. Peptidyl transferase forms a peptide bond b/w fMet and second tRNA. 3. in translocation step, EF-G shifts the message and the tRNAs one over. This moves the dipeptidyl tRNA to P site and deacyclated tRNA to E site and opens A. |
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Antibiotics that inhibit protein synthesis by binding to ribosomes
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-streptomycin: causes misreading
-chloramphenical: inhibits peptidyl transferase activity! -tetracycline: inhibits binding of tRNA -Erythromycin: inhibits translocation -cycloheximide: inhibits PT on 80S cytoplasmic ribosomes |
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Composition of e.coli ribosome
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-50S subunit:23S and 5S RNA and 34 L proteins
-30S subunit:16S RNA and 21 S proteins -together the 50S and 30S make up the 70S ribosome -Mg2+ keeps 30 and 50 together -Add urea to denature 50 and 30 into smaller components |
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Evidence for rRNA as the PT
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1. no ribosomal proteins have been modified that have PT activity
2. drugs that inhibit PT bind to the 23S rRNA, in the PT loop of Domain V 3. mutations that provide resistance to the drugs that inhibit PT map to the same loop 4. nearly all of the protein can be stripped from the 50S and still have PT activity 5. the xray crystal of the 50S shows that only RNA chains are close enough to catalyze a reaction |
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tRNA charging and structure
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-AA's are attached to the 3' terminal end of tRNA's (adenosine) via the 3' or 2' OH
-charging in 2 steps: AA+ATP->aminoacyl-AMP+PP and then Aminoacyl-AMP+tRNA->Aminoacyl-tRNA+AMP -catalyzed by Aminoacyl-tRNA synthetases -PP is an inhibitory product |
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Recognition of tRNAs by Aminoacyl-tRNA synthetases: the second genetic code
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-The synthetases recognize mainly the acceptor stem and anticodon. The CCA at very end is not as relevant.
-class I: aminoacylate the 2'OH -class II: aminoacylate the3' OH -diverse group of enzymes despite recognizing fairly similiar substrates. -classes bind different,but both bind the acceptor stem and anticodon loop |
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how is charging accuracy achieved given the structure of amino acids?
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-Isoleucine tRNA synthetase (ILeRS) discriminates very well
-accuracy by having 2 active sites: one that charges tRNA and one that hydrolyzes mischarged aminoacyl tRNAs(editing site) |
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Chloroplast DNA in green plants
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-circular, multi copy(20-100/organelle)
-~160,000bp; ~125 genes -Most genes of two types: photosynthesis and genetic functions (mostly translation) |
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# genes in certain organisms
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-Mycoplasma:517 genes smallest of all
-E coli:4300 -Cyanidioschyzon:4700 smallest eukaryote -Homo sapien:38,000 -Oryza(rice): 32000-56000 largest -Arabidopsis: 25000 smallest angiosperm genome |
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smallest genome for independent replicating organism?
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270-350 genes essential under lab conditions
-include, Glycolysis, ATP, ABC transporters, DNA pol., trxn and translation genes |
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what accounts for the wide differences in nuclear genome physical size?
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variation in:
-amounts of highly repeated DNA -AMounts of selfish DNA (transposons) -Frequency and sizes of introns -other intergenic DNA -Genetic redundancy -Humans have many large introns and plants have small introns |
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Genetic redundancy
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the sizes of many gene families has increased in some organisms more than others
-may account for much of the unexpectedly high genetic complexity of angiosperms relative to humans |
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Impact of horizontal transfer on genomes
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20% of the e coli genome was obtained by lateral transfer
-viral and bacterial pathogens can transfer DNA from host to host -Some nuclear genes came from organellar genomes -selfish DNAs occasionally transfer horizontally -HT is prevalent amont unicellular organisms (germ and vegetative cell lines the same) |
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What can you do with whole genomes and sequences?
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1. predict much about the functions of a poorly studied organism
2. can examine genome wide expression patterns with microarrays (like cancer vs. normal cells) -Hybridize slide to cDNA by RT from mRNAs. CAn slide with a laser to flouresce marked nucleotide. 3. Identify new drug targets (e.g. antibiotic target could be a gene unique to the pathogen) 4. more rapidly identify genes linked to a trait' 5. rapidly identify a gene for an identified protein by mass spectrometry (Proteomics) |
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Why would you want to make transgenic plants and animals?
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1. study gene function and regulation
2.generate new organismic tools for other fields of research 3. cure genetic diseases 4. improve agriculture and related raw materials 5. generate new systems or sources for bioengineered drugs (use plants instead of animals or bacteria) |
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Nobel prize in Physiology or medicine 2007 for principles for introducing specific gene modifications in mice by use or stem cells
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M. capecchi
Sir M. Evans O. Smithies -first KO mice in 1989 |
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Problem: DNA integration
DIfferent models? |
-Homologous or non homologous recombination
-Frequency of Non homo. is much greater than Homo. by at least 5000 fold in mammal cells -If you want Homo. integrants, which you need for knock-outs, you must have a selection scheme for those |
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How to Knock out a gene
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1. insert neo gene into the target gene
2. transform KO plasmid into embryonic stem cells 3. perform double selection to get cells with the homologous integration (neo and gangcyclovir resistant) 4. inject cells with the knocked out gene into a blastocyst. 5. Implant into pseudopregnant mouse 6. Identify offspring with transgene (chimeric mouse) |
