BIO 344 1st Edition Lecture 16 Outline of Last Lecture I. Breaking the genetic codea. Punctuationb. Degeneracy of the codec. tRNAs and the genetic coded. Conservation of the DNA sequencee. Conservation of the protein sequencef. Translationi. Amino acyl tRNA synthetaseOutline of Current Lecture I. Translation: RibosomesII. Finding the Start sitea. Shine delgarnob. 5’ capc. Ternary complexd. Effect of 5’ cap on translatione. Effect of 3’ poly A tail on translationf. PolioIII. Elongationa. Ef-Tu/GTPb. Ef-Ts/GDPIV. Fidelitya. Rejecting tRNAV. TranslocationCurrent LectureTranslation—Ribosomes- Composed of RNA and protein- Large subunit = Assembles the polypeptideo 50s= prokaryoteso 60s= eukaryotes S= Svedberg units-- sedimentationThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Small subunit= decodes, binds the mRNAo 30s= prokaryoteso 40s= eukaryotes- Functional sites= tRNA binds hereo A= amino acyl tRNA siteo P= pepitdyl tRNA siteo E= exit siteFinding the Start site- Bacteriao 5’_UTR____SD__AUG-lacZ__SD__AUG-lacY__SD__AUG-lacA___UTR__3’o SD for each, start/stop codons for each, polypeptide for eacho Shine Delgarno Conserved sequence 8-10 nucleotides upstream of translation start site- Mostly purines- Binds to 3’ of small ribosomal subunit and scans to find AUG start site- Base pairs and forms a helix with each other- Most consensus SD, drives translation most efficiently- Eukaryoteso 5’-cap__UTR__AUG______orf_______STOP____-3’o 5’ cap recognition scan until find AUGo 5’ cap effect on translation Firefly luciferase has luminescent gene- Can be used as a reporter for protein expression- RNA+ cell extract luciferase translation assayed by measuring light Results: 5’ capped vs uncapped is 21 fold more efficient How?- eIF4E protein binds cap - eIF4G bound to eIF4Eo eIF4G recruits small subunit to the 5’ cap endo 4G is large and serves as a scaffold (landing platform) for other proteins to bind- mRNA____cap-4E-4G- 40s- ribosome finds AUG with Met charged tRNA anticodono ternary complex with 40s to find AUG =eIF2-met-tRNA-MetGTP- If AUG and anticodon but wrong tRNA or uncharged, it will not be recognized—MUST be ternary complex (will come back to this)- Bacteria also utilize ternary complexo 3’ poly A tail effect on translation 50% increase in translation with than without—1.5 fold PABP= polyA binding protein It should be the last thing the ribosome sees, right? So why does it effect translation? o 5’ and 3’ together If effects are additive, translation will increase ~30 fold, BUT together, translation increases 300 fold- Effects are synergistic; greater than additiveo They must be working cooperatively- RECALL: repressor-operator in transcriptiono Repressor binding to O1 enhances binding to O3 and enhanced repression- 3’ and 5’ end come togethero PABP bound to poly A caused 4E to bind to capo Tethering increases local concentration, increases cap binding, increases ribosome finding and binding the sitePolio and Viral Hijacking of translation machinery- Polio targets eIF4G- Experiment:o infect culture Bands show 4G is getting degraded as time passeso Add 36S radioactively labeled Met at various times, make extract, run SDS PAGE, do Western blot with eIF4G antibodies After time, only viral proteins are being made, no host proteins being made Virus cuts 4G loses interaction with 4E but not the 40s Now blind to 5’ end can’t translate its own message, host protein liberated to be attacked by virusElongation (similar in eukaryotes and bacteria)- After locating the start site, ternary complex dissociates with GTP hydrolysis upon recognition of the start codono eIF2 and initiation factors will recycle with GDPo large subunit clamps ono initiator tRNA is in P site, but from this point forward, all tRNA start at the A site- Elongation begins with aminoacyl tRNA bindingo mRNA samples the tRNA at the A site until it finds the right oneo next, amino acyl tRNA binds to A site and a peptide bond is formed with the amino acid in the P siteo the previous tRNA moves to the E site for exit and the tRNA bound to the growing polypeptide chain moves to the P site to make the A site available for the next tRNAo GTPase Ef-Tu delivers the tRNA to the ribosome After recognition of correct tRNA, Ef-Tu hydrolyzes GTP-GDP and peptidyl transferase reaction can then occur For next peptide to be added and elongation to continue, GDP must be exchanged for GTP in order to recruit the tRNAs- Done with Ef-Ts GTPase- GEF= GTP exchange factorFidelity in Translation- Is it the right tRNA?o If no base pairing (wrong anticodon), no stability, tRNA leaves with Ef-Tu-GTP (before hydrolysis)- Is it a near cognate? (=2 bp and 1 in wobble)o Ef-Tu-GTP hydrolysis occurs but tRNA may still be ejectedTranslocation- Ef-G GTPase drives translocation to reset register for a vacant A
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