(Lost in) TranslationSlide 2The cell-free systemTranslation Machinery in Prokaryotes (for comparing with Eukaryotes)How is right AUG selected for translation in Prokaryotes?Translational Initiation in EukaryotesScanning Model of InitiationSlide 8Apparent Exceptions to the Scanning Model?Slide 10Slide 11ConclusionsIs the first “good” AUG really favored? Effect of Repeated Initiation Sequences (replicas)Effect of RNA Secondary Structure in the 5’ UTR (Leader)Slide 15Slide 16Initiation Factors (except eIF-4)eIF4 (eIF4F)eIF4A and eIF4BSlide 20Slide 21eIF1 & eIF1ASlide 23Slide 24(Lost in) Translation 1. Briefly review prokaryotic machinery2. Initiation in Eukaryotes3. Where in the world is Peptidyl Transferase?4. tRNA charging: The second code1968 Nobel Prize in Physiology & Medicine (for deciphering the genetic code) “Triumph of the Chemists” H.G. Khorana R. Holley M. Nirenberg Used a cell-free protein synthesis system from E. coli, programmed it with natural and synthetic RNAs of defined sequence, and determined the sequence of the peptides produced.The cell-free system1. S-30 fraction–ribosomes, tRNAs, tRNA synthetases, other soluble protein factors2. 20 amino acids3. GTP & ATP4. Energy generating system–to keep producing ATP and limit [ADP] –PEP + pyruvate kinase PEP + ADP + Pi  pyruvate + ATP5. Mg2+ and K+ (NH4+)Translation Machinery in Prokaryotes (for comparing with Eukaryotes)•Ribosomes:-70S (composed of L (50S) and S (30S) subunits)-contain 23S (L), 16S (S), and 5S (L) rRNAs-each subunit (L and S) contains ~30 proteins•Initiation factors: if1, if2, if3•Elongation factors: ef-Tu, ef-Ts, and G•Termination (release) factor(s): Rf1 and Rf2 •Translation is initiated with fmet (N-formylated methionine).How is right AUG selected for translation in Prokaryotes?1. Many mRNAs contain a sequence preceding the start codon that base-pairs with the 3'-end of 16S rRNA (Shine-Dalgarno sequence) start5'----GGAGG-------AUG-----3’ mRNA 3'----CCUCC--------5' 16S rRNAFunction: helps position mRNA in ribosome.2. The AUG itself is also very important3. There is a S-D independent mode of translation initiation in E. coli4. Translate internal ORFs of polycistronic mRNAsS-DTranslational Initiation in Eukaryotes•Begins with methionine that is not formylated•tRNA (tRNAiMet) different from the one that is used for internal methionine codons•Translation start determined by the AUG and surrounding sequence•Translation start site also affected by RNA structure at the 5’ end of the mRNAScanning Model of Initiation•Proposed by M. Kozak •Small subunit of ribosome (+ initiation factors, GTP and tRNAiMet) binds to the 5’ Cap, and scans along the mRNA until the first AUG•Translation starts at the first AUG•Model seems to work for most mRNAsScanning (or Kozak) Model for Translation Initiation in EukaryotesFig. 17.16ATPApparent Exceptions to the Scanning Model?•Translation of some mRNAs (5-10%) doesn’t start at first AUG (ribosome skips one or more AUGs)•Comparative sequence analysis of these mRNAs revealed the following consensus sequence at the AUG that is used: -5 -4 -3 -2 -1 +1 +2 +3 +4C C R C C A U G G R=purine•Positions -3 and +4 are particularly important, based on mutagenesis studiesConclusion: When the upstream AUG was in a weak context (like F9), then the downstream one is used. Or, put another way, the first AUG in the right context is used.Fig. 17.18Effect of the context of an upstream “barrier” ATG on initiation of preproinsulin mRNA. proinsulinFig. 17.232nd ed.Upstream ATG is an ineffective barrier if followed by a Stop codon.In some mRNAs, the first ATG is in a favorable context, but is still not used. Kozak noted that there was usually a Stop codon in between the start codons in these mRNAs. So she engineered such a situation in the preproinsulin mRNA and tested its affect on translation.Result: Translation was good at the downstream ATG as long as it was in a good context.Stop codonConclusions•An upstream AUG does not interfere if it’s context (-3,+4) is poor, or if it is followed quickly by an in-frame Stop codon.-In the latter case, it may be that the ribosomes don’t fall off the mRNA after translating such a short ORF.-In natural mRNAs, upstream ORFs are very short, unless they have a regulatory role.Is the first “good” AUG really favored? Effect of Repeated Initiation Sequences (replicas)AUG AUG AUGTranslation started mainly at the first AUG.Fig. 17.19Effect of RNA Secondary Structure in the 5’ UTR (Leader)Poorly translatedTranslated wellTrans. wellNot translatedAdapted from Fig. 17.20Conclusions•Secondary structure (hairpin) at very 5’ end of RNA can prevent 40S subunit from binding •Scanning ribosomes can melt out some hairpins ( ΔG= -30 kcal/mole), but not highly stable ones ( ΔG= -62 kcal/mole)•Initiator tRNA (tRNAiMet) also important in recognizing AUG–(yeast) Anticodon of tRNAiMet changed to UCC, translation started at first “good” AGG in his4 mRNA (Fig. 17.21).Fig. 17.22Summary of translation initiation in Eukaryotes.Resists binding to 60S subunitInitiation Factors (except eIF-4)eIF-1(and 1A): promotes scanning *eIF-2: binds tRNAiMet to 40S subunit, requires GTP (which gets hydrolyzed to GDP) eIF-2B: catalyzes exchange of GTP for GDP on eIF-2*eIF-3: binds to 40S subunit, prevents 60S subunit from binding to iteIF-5: stimulates 60S subunit binding to the 48S pre-initiation complex*eIF-6: binds to 60S subunit, helps prevent 40S subunit from binding to it* prokaryotic counterparteIF4 (eIF4F)eIF4F Originally isolated based on its ability to bind the Cap-nucleotide 7MeGTP. Composed of 3 subunits, a 24-kDa protein that binds the Cap, and 2 others that stabilize the complex and have other roles: 1. eIF4G - versatile adaptor2. eIF4A - RNA helicase3. eIF4E - binds the CapFig. 17.25eIF4A and eIF4BeIF4A•also exists outside of the eIF4F complex•contains a DEAD motif (aspartate-glutamate-alanine-aspartate) characteristic of RNA helicases•RNA helicase activity was demonstrated (right panel) and found to require ATP and to be stimulated by another protein, eIF4BeIF4B •binds RNA, stimulates eIF-4ARole in translation: Unwind hairpins in the 5’ UTRs17.26eIF4G – helps recruit 40S subunit to mRNA; can interact with eIF4E, eIF4A, eIF3, and poly-A binding protein (Pab1); may be responsible for the synergistic effect of Cap and polyA-tail on