Introduction to Molecular Biology
Introduction to Molecular Biology LIFESCI 3
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This 4 page Class Notes was uploaded by Nellie Botsford on Friday September 4, 2015. The Class Notes belongs to LIFESCI 3 at University of California - Los Angeles taught by Staff in Fall. Since its upload, it has received 105 views. For similar materials see /class/177799/lifesci-3-university-of-california-los-angeles in Life Sciences at University of California - Los Angeles.
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Date Created: 09/04/15
Discussion Week 7 Ch 14 7 Translation Initiation prokaryotic 3 initiation factors IFs are required pg 435 IFl 7 binds to Asite assists binding of IF2 IF2 7 binds to IFl in Asite a G protein GTP is hydrolyzed when it recognizes tRNA in the Psite IF3 7 binds to Esite on 308 ribosome prevents binding of SOS subunit wo mRNA displaced by mRNA Elongation 3 sites exist in the rRNA A P and E A 7 Amino acyl charged tRNA binding site charged or amino acyl tRNA is bound to the mRNA via a codonanticodon interaction EF Tu 7 an escort for charged tRNAs masking the aa from peptide formation until the right moment EFTu forms a complex with an aa and a GTPEFG complex to bind to the Asite When GTP is hydrolyzed the escort leaves and peptide bonds are free to form P 7 Eeptide bond formation site the next peptide bond bt amino acids occurs here E 7 Exit site the last aa that has just left the rRNA complex its codonanticodon are no longer bound Each have parts in both the large and small subunit of the ribosome These sites are defined relative to the rRNA complex not the amino acid or peptide Ie they will be dynamic with respect to the amino acids being bound Ex see pg 445 t 0 aal7aa2 aa3 aa4 W W mRNA E P A t 1 aal7aa27aa3 aa4 aa5 W W mRNA E P A Termination There is no complementary tRNA to the stop codons rather there are release factors which bind to the site in order to terminate translation Release factors 7 RFs recognize stop codons in mRNA and terminate translation by hydrolyzing peptide RFs mimic a tRNA functionally but not structurally GGQ motif acts as an anticodon Ch 15 7 The Genetic Code Reading frames 3 possible for ssRNA 6 possible for dsDNA For the following sequence of DNA several mutations are observed GATTACAGATTACA Which of the following mutations would be most detrimental to an offspring with the following DNA Hunk in terms of ORFs and get eXcited about LS4 Normal sequence GATTACAGATTACA GATTA GATTACA Deletion of 2 base pairs GAGTACAGATTACA Mutation of one base pair GATTACAG ACA Deletion of 3 base pairs Think of the Central Dogma of Biology wlien would a mutation be tlie most detrimental tlie least detrimental 4 types of RNA listed chronologically are needed to translate the message from DNA to protein 0 hnRNA 7 heteronuclear refers to the primary transcript in the nucleus before modifications 1 mRNA 7 messenger transfers DNA into RNA 2 tRNA 7 transfer or translating transfers RNA into amino acids 3 rRNA 7 ribosomal transfers amino acids into proteins Protein is written in a degenerate triplet code Degenerate here means redundant Why 20 amino acids and a stop signal are required for translation but a triplet code means that 64 amino acids are possible Base pairs per amino acid Possible amino acids 4 2 16 3 64 4 256 Wliat would be an advantage oflia vmg e base pair per amino acid One disadvantage Wliat would be an advantage oflia vmg four base pairs per amino acid One disadvantage Our triplet code allows for 63 amino acids and a stop signal Wli y do we onlylia ve 20 Although there are 64 possible codes present in DNA significantly less than 64 tRNAs exist This should help answer the above question Why The wobble position Codons are present in mRNA and complemented by an anticodon in tRNA Codon def 7 3 bases on mRNA that code for a specific amino acid Anti codon def 7 3 bases on tRNA that code for the amino acid on tRNA Wobble position def 7 5 end of an anticodon 3 end ofa codon a place in which unique base pairing can exist and a new more versatile base Inosine can be found A new nucleotide code is needed to transcribe this reglon Wobble position For example UUU and UUC both code for phe What does this mean It means that the we will need to synthesize signi cantly fewer than 64 tRNA molecules to code for the all 20 amino acids At the wobble position in tRNA a unique base eXists to make translation more efficient Inosine deaminated Guanine The unique code for tRNA at the wobble position is this Codon Anticodon U or C G G C U A A or G U A U or C I EX Alanine in your reader Codons Anticodons GCU CGI GCC CGI GCA CGI GCG CGC This means that we only need two tRNA molecules to code for all possible alanine codons What s the minimum number of tRNA molecules needed to code for every amino acid You know it has to be greater than twenty because alanine and others need at least two but less than 64 because of this redundancy Ch 20 7 Techniques of Molecular Biology Gels SDSPAGE We ve been doing this in lab 7 hopefully it sounds familiar SDS 7 sodium dodecyl sulfate a negativelycharged molecule which coats proteins on a gel creating a uniform charge mass ratio and denaturing the protein along with heat Reducing agent 7 mercaptoethanol or BME breaks disulfide linkages Adding or omitting a reducing agent will give information about the quaternary interactions between subunits Running a gel 1 Samples are loaded into wells 2 An electrochemical gradient is set up to push the negativelycoated molecules to the positive anode 3 Smaller proteins travel farther compare to a ladder of known protein masses 2D gels Run in two dimensions p1 and mass pI pH at which compound is neutrally charged run on a pH gradient mass is run similar to the SDSPAGE by electrophoresis All samples loaded in same well pH gradient set up to separate based on p1 xcoord Neutralize gel to uniformly reset charge of all proteins before adding SDS to set mz constant electrophoresis separates samples based on mass ycoord bP N Antibodies used in the mol bio laboratory not in ch 20 An antigen is injected into an animal who subsequently produces antibodies to the foreign antigen these antibodies are puri ed and can be used to nd the antigen of interest Primary 7 1 recognize antigen directly by recognition of the epitope or binding site antiantigen Secondary 7 2 recognize constant region of primary antibodies contain uorescent or radioactive marker antiantibody Think beyond the scope of the course why might we use both antibodies in the lab Why don t we just include the uorescent or radioactive on the primary antibody There are lots of reasons for this think of a couple To determine if the antigen of interest is present e g on a gel 1 Blot the gel with a piece of nitrocellulose to adsorb proteins from the gel 2 wash nitrocellulose with a solution of 10 antibodies Antibodies stick to the epitope 3 wash nitrocellulose with a solution of 20 antibodies Antibodies stick to the primary antibody and show us where they are hv or radioactive decay This process for proteins is known as a Western Blot F or fun look up Southern and Northern blots in the LS dictionary Biologists do have a sense of humor This is how a pregnancy test works but with stationary antibodies recognizing hCG beyond course scope httpwwwscienceprojectscomCCPregyancyhtml Chromatography Ionexchange Column beads are coated with an ionic substance positive or negative Samples will stick to beads magnitude of interaction depends on polarity Samples eluted with a solution of increasing salt concentration creating an equilibrium bt bound samples and dissolved samples Often coupled with enzymatic activity to detect sample of interest Cation exchange negativelycharged beads a cation exchange column catches cations Anion exchange positivelycharged beads binds anions Affinity most specific beads recognize an epitope on molecules of interest ligands often antibodies ex a polyT column is used to purify mRNA from all RNA only mRNA has a polyA tail Other methods Mass Spectrometry Time of ight TOF tandem MSMS discussed gives mass of fragments whole molecule Xray crystallography With a pure crystal of sample it gives the 3D structure of the molecule when applicable practical arguably the single best piece of molecular imaging
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