1. Chapter 32 The Genetic Code Including a review of tRNA structure from Chapter 12 section 7

2. Review of Steps in Gene Expression From Access Excellence: http://www.accessexcellence.org/AB/GG/steps_to_Prot.html RNA = nucleotide sequence protein = amino acid sequence Adaptor molecule

3. Translating the Message How does the sequence of mRNA translate into the sequence of a protein? What is the genetic code? How do you translate the "four-letter code" of mRNA into the "20-letter code" of proteins? And what are the mechanics like? There is no obvious chemical affinity between the purine and pyrimidine bases and the amino acids that make protein. Three major advances gave the clues to solving this dilemma

4. Clue #1 The Discovery that Proteins are made on ribosomes By Paul Zamecnik (early 1950s) He asked where in the cell are proteins synthesized? Injected rats with radioactive amino acids A short time after injection (when the amino acids should be incorporated into newly-synthesized proteins) he killed the rats, harvested their livers, ground them up and divided the cell components into “subcellular fractions” by centrifugation

5. Results Radioactivity was found in small ribonucleoprotein particles visible by electron microscopy. These were later characterized and called “ribosomes” (since they had RNA as a major component) From Lehninger “Principles of Biochemistry” p 1021

6. Clue #2 The Discovery that amino acids are “Activated” By Hoagland and Zamecnik They incubated amino acids with the cytosolic fraction of liver cells, and with ATP They found the amino acids became “activated” during the incubation Activation consists of attaching the amino acids to a heat-stable soluble RNA (which we now know is tRNA) Activated amino acids are called aminoacyl- tRNAs The enzymes that do the activation are called aminoacyl-tRNA synthetases (next class!)

7. Clue #3 Crick’s Adaptor Hypothesis Francis Crick thought about the problem He reasoned that a small nucleic acid could serve as an adaptor between RNA and protein synthesis if it could bind both RNA and an amino acid His idea was that one end of the adaptor would bind a specific amino acid and the other would bind to a specific sequence in the RNA that coded for that amino acid

8. Crick’s Adaptor Hypothesis From Lehninger “Principles of Biochemistry” p 1021 Must have one adaptor per amino acid Therefore there must be a family of adaptors: These are the tRNAs each tRNA can recognize specific sequences in the RNA transcript Each is “charged” with the amino acid that is specified by that sequence

9. Review of tRNA Structure tRNAs are the “adaptors” in protein synthesis There are many different tRNAs, each has a distinct sequence However, all tRNA have several conserved features 1) small (73-93 nucleotides long) 2) they have a conserved secondary structure - 4 stems and 4 loops with important functions 3) they contain many unusual bases Inosine (I), pseudouridine (  ), dihydrouridine (D), ribothymidine (T), and methylated bases ( m G, m I)

10. Unusual bases in tRNAs

11. Invariant bases Amino acid addition site Varies in size Interacts with the ribosome Base pairs with the codon in the mRNA transcript

12. Example of a specific tRNA The yeast alanyl-tRNA

13. The cloverleaf tRNA folds into an L-shape by interactions between conserved bases in the stems and loops

14. tRNA tertiary structure: banana or L-shape Non- canonical base pairs stabilize the 3 o structure

15. Animation of tRNA secondary and tertiary structure

16. Elucidation of the Genetic Code 4 major advances helped figure out the code 1) The demonstration of colinearity between genes and protein 2) The idea of triplet codons 3) Deciphering the first word (UUU= Phe) 4) Deciphering the rest of the code

17. The Colinearity of Gene and Protein Structures Yanofsky provided evidence in 1964: he showed that the relative distances between mutations in DNA were proportional to the distances between amino acid substitutions in E. coli tryptophan synthase

18. Charles Yanofsky - 1964 (trp operon) Had a collection of mutants in the trpA gene (coding for tryptophan synthase) He made 2 determinations using these mutants: 1) determined the position of the mutation in the gene 2) determined the position of the mutant amino acid in the protein encoded by each mutant gene gene protein Mutant 1 Mutant 2 Mutant 3 Therefore gene sequence is colinear with protein sequence.

19. What is the nature of the Code 1:1 correspondence can’t work Therefore nucleotides must be read in combinations Is 2 enough? 4X4 = 16 different combinations possible - not enough But 3 would give 4X4X4 = 64 combinations This would be enough to code for 20 amino acids Therefore the concept of the triplet codon was born mRNA (nucleotides) protein (amino acids) 4 different nucleotides 20 different amino acids

20. What is the nature of the Code Is the code overlapping or non-overlapping? Is the code punctuated or non-punctuated? These details were worked out by Crick Crick used mutagens to introduce changes into a coding sequence Used mutagens that either induced point mutations in the DNA or insertions. After generating mutants he checked the proteins coded by the mutant sequences

21. X Changes 3 amino acids X Changes 1 amino acid Changes all following amino acids following amino acids are unchanged

22. The Nature of the Genetic Code summary of results A group of three bases codes for one amino acid The code is not overlapping The base sequence is read from a fixed starting point, with no punctuation The code is degenerate (in most cases, each amino acid can be designated by any of several triplets)

23. Biochemists Break the Code Assignment of "codons" to their respective amino acids Marshall Nirenberg and Heinrich Matthaei Worked with an in vitro translation system from E. coli Cell-free extract Ribosomes tRNAs Amino acids Enzymes ATP, GTP + mRNA = protein

24. They generated RNAs that are homopolymers using the enzyme polynucleotide phosphorylase (catalyzes random synthesis of RNA chains) Deciphering the first word i.e. nNDPs polynucleotide phosphorylase (NMP)n + nPi Using this system they made a polyU mRNA by programming their reaction with UDP When this was put into the cell-free extract it should be translated into a protein made up of amino acids coded by the codon UUU

25. Experiment: They set up 20 different test tube reactions Each one was spiked with a different radioactive amino acid They programmed each with the polyU RNA Then recovered the proteins by acid precipitation Under these conditions the proteins precipitate but the free amino acids do not Then they asked which reaction (out of the 20) has radioactivity in the protein pellet? Deciphering the first word (continued)

26. Biochemists Break the Code Results Marshall Nirenberg and Heinrich Matthaei showed that poly-U produced polyphenylalanine in a cell- free solution from E. coli. In other words, only the test tube reaction spiked with radioactive Phe generated a radioactive pellet They repeated the experiment with other synthetic homopolymer RNAs Poly-A gave polylysine (AAA = Lys) Poly-C gave polyproline (CCC = Pro) Poly-G gave polyglycine (GGG = Gly)

27. Getting at the Rest of the Code Work with nucleotide copolymers (poly (A,C), etc.), revealed some of the codes Gobind Khorana (organic chemist) -synthesized DNA composed of alternating copolymers eg: ACACACACACAC….. Then used RNAP to make RNA from the DNA template eg: UGUGUGUGUGUGU…… This RNA transcript has two possible alternating codons: UGU GUG UGU GUG In a translation extract you should get a protein with 2 alternating amino acids

28. Nirenberg and Leder Took a cell-free translation extract (ribosomes and tRNAs charged with their specific amino acid) Added a synthetic triplet RNA (a codon) eg UUU They found that addition of that simple triplet RNA to the cell-free extract could stimulate the binding of the tRNA that recognized that codon to a ribosome Since the tRNA is covalently linked to the amino acid that is coded for by the codon, therefore that amino acid gets localized to the ribosome If they collect the ribosomes from the experiment they can identify which amino acid was brought to the ribosome by that triplet codon

29. Nirenberg and Leder UUU AAA Phe Ribosome Ternary complex Very large Can be captured on a filter Experiment: for each triplet RNA set up 20 reactions, each one spiked with a different radioactive amino acid. Ask which reaction generates radioactivity on the filter. That’s the amino acid coded for by the triplet codon! Triplet RNA

31. Getting at the Rest of the Code Finally Marshall Nirenberg and Philip Leder cracked the entire code in 1964 They showed that trinucleotides bound to ribosomes could direct the binding of specific aminoacyl-tRNAs (See Figure 31.6) By using C-14 labeled amino acids with all the possible trinucleotide codes, they elucidated all 64 correspondences in the code Found that all the codons (except the 3 stop codons) specified an amino acid There are 64 codons and 20 amino acids Therefore amino acids can be encoded by >1 codon

32. Features of the Genetic Code All the codons have meaning: 61 specify amino acids, and the other 3 are "nonsense" or "stop" codons The code is unambiguous - only one amino acid is indicated by each of the 61 codons The code is degenerate - except for Trp and Met, each amino acid is coded by two or more codons Codons representing the same or similar amino acids are similar in sequence 2nd base pyrimidine: usually nonpolar amino acid 2nd base purine: usually polar or charged aa