1. ©1998 Timothy G. Standish Transcription and Translation Timothy G. Standish, Ph. D.

2. ©1998 Timothy G. Standish Outline: The genetic code: A brief introduction The genetic code helps control the impact of point mutations The genetic code is improbable and does not look random The genetic code is very unlikely to change The genetic code is not completely universal Summary

3. ©1998 Timothy G. Standish The Genetic Code: A Brief Introduction

4. DNA mRNA Transcription Introduction The Central Dogma of Molecular Biology Cell Polypeptide (protein) Translation Ribosome ©1998 Timothy G. Standish

5. Information Only Goes One Way The central dogma states that once “information” has passed into protein it cannot get out again. The transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein, may be possible, but transfer from protein to protein, or from protein to nucleic acid, is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein. Francis Crick, 1958

6. ©1998 Timothy G. Standish The Genetic Language The genetic code is a written language not unlike English or German. While English uses 26 letters to spell out words, genetic languages use only 4 nucleotide “letters”. The nucleotide language of DNA is transcribed into the nucleotide language of RNA.

7. ©1998 Timothy G. Standish The Nucleotide Language DNA- ATGCATGCATGC RNA- AUGCAUGCAUGC It is not unlike different versions of English. Me thinks it is a weasel I think it is a weasel

8. OH O CH 2 Sugar H OH A Nucleotide NH 2 N N N N Base P O OH HO O Phosphate ©1998 Timothy G. Standish

9. Pyrimidines NH 2 O N N NH N Guanine N N Adenine N N NH 2 N O N O N Cytosine Uracil (RNA) CH 3 N O N O NH N O N O Thymine (DNA) Purines ©1998 Timothy G. Standish Two Families of Bases

10. ©1998 Timothy G. Standish Nucleotide Words Words in the nucleotide language are all 3 letters or bases long. These three base “words” are called codons This means that there can only be 4 3 = 64 unique words.

11. SUGAR-PHOSPHATE BACKBONE B A S E S H P O O HO O O CH 2 NH 2 N NH N N HOH P O O HO O O CH 2 NH 2 N N N N H P O OH HO O O CH 2 NH 2 N N N N O A Codon Guanine Adenine Arginine ©1998 Timothy G. Standish

12. The Genetic Code Helps To Control The Impact Of Point Mutations

13. ©1998 Timothy G. Standish Redundancy in the Code Codons code for only 20 words, or amino acids. In addition to the amino acids, the start and stop of a protein need to be coded for There are thus a total of 22 unique meanings for the 64 codons, so many codons are synonyms. The fact that many amino acids are coded for by several codons is called degeneracy

14. ©1998 Timothy G. Standish Why Not Use Shorter Codons? If each codon was only 2 bases long, there would be 4 2 = 16 possible unique codons This would not provide enough unique meanings to code for the 22 things (20 amino acids plus start and stop) that have to be coded for.

15. ©1998 Timothy G. StandishSentences Sentences in the nucleic acid language are called genes. Each gene contains a sequence of codons that describe the primary structure (amino acid sequence) of a polypeptide (protein). At the beginning of each gene is a start codon In the middle is a sequence of codons for amino acids At the end is a stop codon

16. ©1998 Timothy G. Standish The Protein Language The protein language is very different from the nucleotide language Sentences are called polypeptides or proteins It is analogous to pictographic languages like Chinese or Egyptian Hieroglyphics. Each symbol has a meaning in pictographic languages and in proteins, each amino acid has a unique meaning or specific effect. Words are not a sequence of nucleotides, but each AA in the primary structure

17. ©1998 Timothy G. Standish Comparison of Languages English - Dog Chinese - DNA - CGT RNA - CGU Amino Acid - Arginine

18. ©1998 Timothy G. Standish Redundancy: Synonyms and Codon Degeneracy English - Synonyms for dog: Canine Hound Mutt Cur Pooch Nucleic acids - Synonyms for Arginine: CGU CGC CGA CGG AGA AGG

19. ©1998 Timothy G. Standish S E C O N D B A S E A GGU GGC GGA GGG Gly* AGU AGC AGA AGG Arg G CGU CGC CGA CGG Arg G UGU UGC UGA UGG C GAU GAC GAA GAG AAU AAC AAA AAG Glu CAU CAC CAA CAG A UAU UAC UAA UAG Stop Tyr GUU GUC GUA GUG Val AUU AUC AUA AUG start Ile CUU CUC CUA CUG Leu U UUU UUC UUA UUG Leu Phe Met/ GCU GCC GCA GCG Ala ACU ACC ACA ACG Thr CCU CCC CCA CCG Pro C UCU UCC UCA UCG Ser UCAGUCAG U UCAGUCAG UCAGUCAG UCAGUCAG Gln † His Trp Cys THIRDBASETHIRDBASE FIRSTBASEFIRSTBASE The Genetic Code Asp Lys Asn † Stop Ser Neutral Non-polar Polar Basic Acidic †Have amine groups *Listed as non-polar by some texts

20. ©1998 Timothy G. Standish Codon Assignment Is Fortuitous Effect of mutations is minimized in the genetic code: Mutation of the third base in a codon changes the codon meaning only 1/3 of the time In AAs with only two codons, the mutation always has to be purine to pyrimidine or vice versa to change the AA coded for. This is much harder than purine to purine or pyrimidine to pyrimidine mutation

21. ©1998 Timothy G. Standish Codon Assignment Is Fortuitous Because of wobble base pairing, this arrangement means less than 61 tRNAs have to be made 53% of purine to purine or pyrimidine to pyrimidine mutations in the second position result in codons with either the same meaning (i.e. UAA to UGA both = stop) or coding for chemically related amino acids

22. ©1998 Timothy G. Standish The Genetic Code Is Improbable And Does Not Look Random

23. ©1998 Timothy G. Standish Possible Codon Assignments The probability of getting the assignment of codons to amino acids we have can be calculated as follows: –There are 21 meanings for codons: 20 amino acids 1 stop 1 start, which doesn’t count because it also is assigned to methionine –64 Codons If we say that each codon has an equal probability of being assigned to an amino acid, then the probability of getting any particular set of 64 assignments is: 0.0000000000000000000000000 0000000000000000000000000 00000000024 or

24. ©1998 Timothy G. Standish Problems With Codon Assignment Under Miller-Urey type conditions, more than the 20 amino acids would have been available To estimate probability, we assume only 20, but this changes the odds As all 20 amion acids and “stop” must be assigned one codon, only 64 - 21 = 43 codons could be truely randomly assigned Net probability is the likelyhood of initial assignment times probability of random assignment of remaining codons

25. ©1998 Timothy G. Standish Initial Codon Assignment Theory would indicate initial codon assignment must have been random Lewin in Genes VI pp 214, 215 suggests the following scenario: 1 A small number of codons randomly get meanings representing a few amino acids or possibly one codon representing a “group” of amino acids 2 More precise codon meaning evolves perhaps with only the first two bases having meaning with discrimination at the third position evolving later 3 The code becomes “frozen” when the system becomes so complex that changes in codon meaning would disrupt existing vital proteins

26. ©1998 Timothy G. Standish Codon Assignment Does not look random The genetic code does not like uneven numbers.

27. ©1998 Timothy G. Standish Initial Codon Assignment If natural selection worked on codons, the most commonly used amino acids might be expected to have the most codons If there was some sort of random assignment, the same thing might be expected This is not the case

28. ©1998 Timothy G. Standish Codon Assignment Is Not Strongly Correlated With Use Met Trp Cys His Tyr Phe Thr Arg Ser Leu Pro Ile Gln Asp Lys Glu Asn Val Gly Ala 1 2 3 4 5 6 Number of Codons 10 8 6 4 2 % In Proteins

29. ©1998 Timothy G. Standish The Genetic Code Is Very Unlikely To Change

30. ©1998 Timothy G. StandishInitiation The small ribosome subunit binds to the 5’ untranslated region of mRNA The small ribosomal subunit slides along the mRNA 5’ to 3’ until it finds a start codon (AUG) The initiator tRNA with methionine binds to the start codon The large ribosomal subunit binds with the initiator tRNA in the P site

31. ©1998 Timothy G. Standish How Codons Work: tRNA the Translators tRNA - Transfer RNA Relatively small RNA molecules that fold in a complex way to produce a 3 dimensional shape with A helices Associate a given amino acid with the codon on the mRNA that codes for it

32. ©1998 Timothy G. Standish A E Large subunit P Small subunit Translation - Initiation fMet UAC GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

33. ©1998 Timothy G. Standish A E Ribosome P UCU Arg Aminoacyl tRNA Phe Leu Met Ser Gly Polypeptide CCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

34. ©1998 Timothy G. Standish A E Ribosome P Phe Leu Met Ser Gly Polypeptide Arg Aminoacyl tRNA UCUCCA Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

35. ©1998 Timothy G. Standish ANYTHING ACID AMINE Protein Synthesis C O OHCN H H H C HOH C H O CN H H H C HH C H O OHCN H H H C HOH Serine C H O OHCN H H H C HH Alanine H C O OHC R N H H Amino Acid H2OH2O

36. ©1998 Timothy G. Standish A E Ribosome P CCA Arg UCU Phe Leu Met Ser Gly Polypeptide Translation - Elongation GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

37. ©1998 Timothy G. Standish A E Ribosome P Translation - Elongation Aminoacyl tRNA CGA Ala CCA Arg UCU Phe Leu Met Ser Gly Polypeptide GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

38. ©1998 Timothy G. Standish A E Ribosome P Translation - Elongation CCA Arg UCU Phe Leu Met Ser Gly Polypeptide CGA Ala GAG...CU-AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA-AT GCA...TAAAAAA 5’ mRNA 3’

39. ©1998 Timothy G. Standish Aminoacyl-tRNA Synthetase Aminoacyl-tRNA Synthetase enzymes attach the correct amino acids to the correct tRNA This is an energy consuming process Aminoacyl-tRNA Synthetases recognize tRNAs on the basis of their looped structure, not by direct recognition of the anticodon

40. Gly Amino- acyl-tRNA Synthetase Gly CCA Amino- acyl-tRNA Synthetase Gly Amino- acyl-tRNA Synthetase A P Amino- acyl-tRNA Synthetase Making Aminoacyl- tRNA P P Pyrophosphate A P AMP Gly CCA Aminoacyl- tRNA Note that the amino acid is not paired with the tRNA on the basis of the anticodon. The correct tRNA for a given amino acid is recognized on the basis of other parts of the molecule. A P P P ATP ©1998 Timothy G. Standish

41. Requirements for Translation Ribosomes - rRNA and Protiens mRNA - Nucleotides tRNA –The RNA world theory might explain these three components Aminoacyl-tRNA Synthetase, –A protein, thus a product of translation and cannot be explained away by the RNA world theory L Amino Acids ATP - For energy This appears to be an irreducibly complex system

42. ©1998 Timothy G. Standish Reassignment of Stop Codons Changes in stop codon meaning must have occurred after meanings were “frozen” in other organisms, alternatively organisms that exhibit them must have evolved from organisms that never shared the universal genetic code All changes in stop codons must include three changes: –Replacement of stop codons that do not code for stop anymore with those that still do –Production of new tRNAs with anticodons that recognize the codon as not stop anymore –Modification of the release factor (eRF) to restrict its binding specificity further so that it no longer binds the stop codon with new meaning All changes “appear to have occurred independently in specific lines of evolution” (Lewin, Genes VI)

43. ©1998 Timothy G. Standish Changing Initial Codon Assignment Once codons have been assigned to an amino acid, changing their meaning would require: –Changing the tRNA anticodon or, much harder, changing the aminoacyl-tRNA synthetase –Changing all codons to be reassigned in at least the vital positions in those proteins needed for survival This seems unlikely The situation is complicated in cases where genes seem to have been swapped between the nucleus and mitochondria

44. ©1998 Timothy G. Standish The Genetic Code Is Not Completely Universal

45. ©1998 Timothy G. Standish Variation In Codon Meaning Lack of variation in codon meanings across almost all phyla is taken as an indicator that initial assignment must have occurred early during evolution and all organisms must have descended from just one individual with the current codon assignments Exceptions to the universal code are known in a few single celled eukaryotes and mitochondria and at least one prokaryote Most exceptions are modifications of the stop codons UAA, UAG and UGA serine Stop Common Meaning Stop Candida A yeast Euplotes octacarinatus A ciliate Paramecium A ciliate Organism Tetrahymena thermophila A ciliate leucine cysteine glutamine Modified Meaning CUG UGA UAA UAG Codon/s UAA UAG glutamine Stop Mycoplasma capricolum A bacteria tryptophanUGA Neutral Non-polar, Polar

46. ©1998 Timothy G. Standish Variation in Mitochondrial Codon Assignment UGA/G=Stop Universal Code Cytoplasm/ Nucleus Plants Yeast/ Molds Platyhelmiths Echinoderms Molluscs Insects Vertebrates UGA=Trp AGA/G=Ser AUA=Met CUN=Thr AUA=Ile AAA=Asn Nematodes NOTE - This would mean AUA changed from Ile to Met, then changed back to Ile in the Echinoderms UGA must have changed to Trp then back to stop Differences in mtDNA lower the number of tRNAs needed AAA must have changed from Lys to Asn twice

47. ©1998 Timothy G. Standish Summary: The genetic code appears to be Non-random in nature and incorporates considerable safeguards against harmful point mutations An evolutionary model suggests at least at some level of randomness in assignment of amino acids to codons No mechanism exists for genetic code evolution Thus variation in the genetic code suggests a polyphyletic origin for life

49. ©1998 Timothy G. Standish Question 1 How many bases are in a codon? A1A1 B2B2 C3C3 D4D4 E5E5

50. ©1998 Timothy G. Standish Question 2 True or False A)Mutating just one base in a codon may have a profound effect on the protein being coded for and consequently the organism B)Mutating the third base in a codon frequently has no effect on the protein being coded for C)Changing an amino acid in a protein will have less effect on a protein if the amino acid belongs to the same class as the original amino acid it is replacing

51. ©1998 Timothy G. Standish Question 3 Which of the following components of the translation process cannot be explained away by the RNA World theory? A)mRNA B)Ribosomes C)Aminoacyl-tRNA transferase D)tRNA