Protein Synthesis 2 Major topics covered: •Translation: initiation, elongation and termination •Comparison of eukaryotic translation to prokaryotic •Medical relevance of translation: two points related text: Biochemistry Garret and Grisham, 4th ed. Chapter 30 contact info: David A. Schneider, Ph.D. Department of Biochemistry and Molecular Genetics [email protected] office #: 934-4781 Reminder of Friday’s lecture: • Translation is the process of making protein from an RNA template • Fidelity is affected by several steps • tRNAs are the “adapters” that translate the 4-nucleotide language of DNA/RNA into the 20-amino acid language of proteins. • Aminoacyl-tRNA synthetases are ancient, but accurate enzymes. • Ribosomes are large, complicated “machines”. I will start with a general overview of translation (the example is eukaryotic) The ribosome is the ribozyme that catalyzes peptide bond formation. What other factors participate in translation, and how is the whole process orchestrated? The ribosome is the ribozyme that catalyzes peptide bond formation. What other factors participate in translation, and how is the whole process orchestrated? Translation consists of three steps: 1) Initiation 2) Elongation 3) Termination A general cartoon of the translation process So, where does this start? In bacteria, the first codon in the mRNA (AUG) leads to initiation and recruitment of the formyl-methionyl tRNA How does the ribosome find the first codon? The “Shine-Delgarno” sequence in the mRNA: Base pairing between the Shine Dalgarno sequence and the 3´ end of 16S rRNA facilitates translation initiation. Consequently, the efficiency of translation initiation is determined by: 1) How well the S.D. sequence conforms to the consensus sequence that is complementary to the 3´ end of 16S rRNA. 2) The distance between the S.D. sequence and the start codon (a 7 base spacer is optimal). Three translation initiation factors are required (in addition to the ribosome and aa-tRNA) The process of translation initiation in prokaryotic cells High translation initiation rates lead to multiple ribosomes per message (“polysomes”) Electron micrograph of polysomal mRNA Note of interest: ribosome occupancy on mRNA plays a major role in determining mRNA decay rate The translation elongation cycle The chemistry of peptide bond formation Translation terminates when a stop codon (UAA, UAG, UGA) enters the A-site Translation termination factors: RF-1 = recognizes UAA and UAG RF-2 = recognizes UAA and UGA RF-3 = G-protein; helps trigger hydrolysis (by the 23S rRNA) RRF = liberates ribosome/release factors Important term = molecular mimicry Translation factors use molecular mimicry to utilize common binding sites on the ribosome From Ramakrishnan, Cell 108: 557 (2002) Translation is a cycle (final overview) A more detailed animation of translation, including factors Translation is a highly conserved process among all living things… However, important differences exist between bacteria and eukaryotes (e.g. you!) Important difference #1: Ribosomes are substantially different Important difference #2: mRNA is very different in prokaryotes versus eukaryotes Bacterial mRNA: lacks 5’ cap, poly-A tail not required, multiple orfs per transcript, SD sequence eukaryotic mRNA: 7-MeG cap, poly-A tail, one orf per transcript, no sequence specific binding Consequence: translation mechanisms are different, primarily at the initiation step The structural arrangement and required factors for translation initiation are substantially different in eukaryotes, compared to bacteria Overview of eukaryotic translation initiation Step 1: eIF1, 1A, 3 and 5 bind to 40S (not shown) tRNAiMet-eIF2:GTP is recruited Step 2: eIF4 proteins associate with mRNA (cap and tail) and bind 43S preinitiation complex Scanning Step 3: eIF5-mediated ejection of IFs and 60S binds Translation elongation is very similar in eukaryotes and prokaryotes eEF1a eEF1b eEF2 Translation termination in eukaryotes is mechanistically similar to prokaryotes… Important difference: only one release factor is required What have we learned (lectures 1&2)? •tRNAs “adapt” the 4-base nucleotide code to a 20 amino acid protein code. •Charging of tRNAs and codon:anticodon interactions are critical for fidelity. •Ribsomes are big-big-big ribozymes… that we can now visualize in some detail. •Translation is a complicated process that is geared to be efficient and accurate! •Eukaryotic translation varies from prokaryotic translation most significantly at the initiation step. We know that translation and ribosome composition varies between bacteria and eukaryotic cells Why does this matter? We know that translation and ribosome composition varies between bacteria and eukaryotic cells Why does this matter? Fungi and bacteria often occupy the same environment and battle for the same resources. Thus, they try to kill each other We benefit! Several common antibiotics with mode of action and molecular target (of some) mapped Note: your mitochondrial ribosomes are similar to those of bacteria, thus some toxicity occurs Puromycin is a charged tRNA (tRNATyr) analog: as expected it inhibits translation in all organisms Many human genetic disorders originate from nonsense mutations Nonsense mutations: premature stop codons in orf leading to termination of translation and incompletely synthesized protein Many human genetic disorders originate from nonsense mutations Nonsense mutations: premature stop codons in orf leading to termination of translation and incompletely synthesized protein PTC124 has progressed effectively through Phase 2 clinical trials and can rescue CFTR mRNA levels -Kerem, et al. The Lancet (2008) THE END -any questions?