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Visualizing the triplet code Reading Frame: 1 2 3 THE FAT CAT ATE THE BIG RAT HEF ATC ATA TET HEB IGR AT EFA TCA TAT ETH EBI GRA T add or delete one base ===> frameshift THE FAN TCA TAT ETH EBI GRA T THE FAC ATA TET HEB IGR AT Visualizing the triplet code Implications of the code from the rII experiment If you add (or delete) 3 nucleotides you get back to the same reading frame But…….the sequence will encode additional, fewer or different amino acids But………what is the code? How does each 3 base codon translate into the 20 amino acids? Key innovations 1. In vitro translation 2. Synthetic nucleic acids RNA protein cell extract ribosomes amino acids tRNA 5’-AAAAAAAAAAAAAAAAAAAAAAAA-3’ 5’-CCCCCCCCCCCCCCCCCCCCCCCC-3’ 5’-GGGGGGGGGGGGGGGGGGGGGGGG-3’ 5’-UUUUUUUUUUUUUUUUUUUUUUUU-3’ But………what is the code? Nirenberg and Matthei 5’-UUUUUUUUUUUUUUUUUUUUUUUU-3’ RNA (polyU) protein only 14C phe resulted in labeled protein 14C radiolabeled amino acids Nobel Prize, 1968 UUU AAA CCC GGG – – – - phenylalanine lysine proline glycine But………what is the code? Other key experiments/findings: 1. Synthetic RNA made with dinucleotides, trinucleotides etc. - (e.g. 5’-UCUCUCUCUCUCUCU-3’ encodes poly Leu-Ser) - H. Gobind Khorana – Nobel Prize, 1968 2. Matching trinucleotides with amino acids 3. Point mutations leading to single amino acid substitutions - Charles Yanofsky, trp operon in E. coli 4. Point mutation leading to a single amino acid substitution in sickle cell anemia 1. Translation of synthetic RNA 5’-UCUCUCUCUCUCUCUCUCUCUCUCUCUCUCU-3’ N-SerLeuSerLeuSerLeuSerLeuSerLeu -C N-LeuSerLeuSerLeuSerLeuSerLeuSer -C H. Gobind Khorana Nobel Prize, 1968 Combinations of alternating dinucleotides, trinucleotides could be used to work out the code 2. Matching trinucleotides with amino acids Nirenberg and Leder experiment 14C-Tyr 5’-UAC-3’ labeled ribosome cell extract ribosomes 14C-tRNATyr 3’ AUG5’ 5’-UAC-3’ Ribosome-bound codon only binds to the corresponding tRNA 3. Point mutations leading to single amino acid substitutions Charles Yanofsky, trp operon in E. coli Single nucleotide change altered a single amino acid Confirmed the code and showed it is not overlapping 4. Point mutation leading to a single amino acid substitution in sickle cell anemia Demonstrated the universality of the code and showed for the first time that a genetic disease resulted from a change in a specific protein (gene) The genetic code Nearly universal across organisms – evolutionarily early Degenerate – multiple codons for some amino acids (wobble) Punctuation – start (AUG, Met) and stop (UAA, UGA, UAG) Practice problem Mix uracil and guanine to make mRNA U:G=3:1 Synthesize protein in vitro Analyze amino acid frequencies Codon probabilities at U:G=3:1 codon probability ratio UUU P (UUU) = 3/4 X 3/4 X 3/4 = 27/64 P (UUG) = 3/4 X 3/4 X 1/4 = 9/64 P (UGU) = 3/4 X 1/4 X 3/4 = 9/64 P (GUU) = 1/4 X 3/4 X 3/4 = 9/64 P (UGG) = 3/4 X 1/4 X 1/4 = 3/64 P (GGU) = 1/4 X 1/4 X 3/4 = 3/64 P (GUG) = 1/4 X 3/4 X 1/4 = 3/64 P (GGG) = 1/4 X 1/4 X 1/4 = 1/64 0.42 UUG UGU GUU UGG GGU GUG GGG 0.14 0.14 0.14 0.05 0.05 0.05 0.01 total 1.00 Amino acid abundances at U:G=3:1 (UUU):(UUG):(UGU):(GUU):(UGG):(GGU):(GUG):(GGG) 0.42: 0.14: 0.14: 0.14: 0.05: 0.05: 0.05: 0.01 Phe : Leu : Cys : Val : Trp : Gly 0.42: 0.14 : 0.14: 0.19: 0.05: 0.06 Conclusions: Identify which codons specify which amino acids Prove that the code is degenerate tRNA – the final piece of the basic puzzle Predicted by Francis Crick in 1955, published in 1958 “I cannot conceive of any structure (for either nucleic acid) acting as a direct template for amino acids, or at least as a specific template………In its simplest form there would be 20 different kinds of adaptor molecule, one for each amino acid, and 20 different enzymes to join the amino acid to their adaptors.” Identifed by Hoagland and Zamecnik in 1958 tRNAs specify the amino acid inserted tRNATyr Tyr anticodon 3’ AUG5’ 5’ NNNUACNNN3’ Tyr codon amino acyl tRNA synthetases load (“charge”) the tRNA with the amino acid tRNAs and the suppressor concept tRNATyr WT AUG GUG UAC GAC AAG AGA UAA Met Val Tyr Asp Lys Arg Stop tRNATyr SuTyr Tyr Mut 1 (Nonsense Mutant) AUG GUG UAG GAC AAG AGA UAA Met Val Stop - Mut 1 Su AUG GUG UAG GAC AAG AGA UAA Met Val Tyr Asp Lys Arg Stop 3’ AUG5’ 5’ GUGUACGAC3’ Tyr AUC5’ 5’ GUGUAGGAC3’ Stop Can you think of other types of suppressors? 3’ Practice problem A protein found in E. coli has the following amino acid sequence: Met-Leu-Trp-Ala-Ile-Ile-Cys-Asp In a mutant strain of E. coli, the anticodon of a tRNA has been altered from 5’-ACA-3’ to 5’-CCA-3’, resulting in a new amino acid sequence for the protein. Predict the amino acid sequence of this new polypeptide. Assume that the anticodon mutation alters the codon that the tRNA reads but does not alter the amino acid that the tRNA is charged with. cys cys anticodon (tRNA) 3’ACA 5’ 3’ACC 5’ 5’UGU 3’ 5’UGG 3’ Cys codon (mRNA) Trp Cys 3’ 5’ ACA 5’ NNNUGUNNN3’ Cys Met-Leu-Cys-Ala-Ile-Ile-Cys-Asp 3’ 5’ ACC 5’ CUCUGGGCC3’ Cys Lakota Sioux Native Americans believe the Goddess of Peace once appeared in the form of a white buffalo calf. As legend goes, chances are one in 10 million that a white buffalo will ever enter this world. Midterm 1. Happy to consider regrades if written in ink and submitted by 11/16/12 2. Exams have been photocopied 3. Final scores to grades for prior years (out of 405 total points) A- or better B- or better C- or better ~350 ~300 ~200 4. We don’t know how the curve will turn out this year