1. ©2001 Timothy G. Standish John 1:1-3 1In the beginning was the Word, and the Word was with God, and the Word was God. 2The same was in the beginning with God. 3All things were made by him; and without him was not any thing made that was made.

2. ©2001 Timothy G. Standish The Genetic Code: The Language of Life Timothy G. Standish, Ph. D.

3. ©2001 Timothy G. Standish 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

4. ©2001 Timothy G. Standish The Universal Language “[The universe] is written in the language of mathematics, and its characters are triangles, circles, and other geometrical figures, without which it is humanly impossible to understand a single word of it....” Galileo Galilei, The Controversy on the Comets of 1618

5. ©2001 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 DNA nucleotide language is transcribed into the RNA nucleotide language The nucleotide language must be translated into the amino acid language to make proteins

6. ©2001 Timothy G. Standish The Nucleotide Language DNA - ATGCATGCATGC RNA - AUGCAUGCAUGC It is not unlike different Bible versions. Psalm 139:14 KJV I will praise thee; for I am fearfully and wonderfully made: marvelous are thy works; and that my soul knoweth right well. NIV I praise you because I am fearfully and wonderfully made; your works are wonderful, I know that full well.

7. ©2001 Timothy G. Standish Nucleotide Words Words in the nucleotide language are all 3 letters or bases long This means that there can only be 4 3 = 64 unique words 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.

8. 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

9. ©2001 Timothy G. Standish The Genetic Code Helps To Control The Impact Of Point Mutations

10. ©2001 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 Because all codons are used many act as synonyms, having different sequences, but the same meaning The fact that many amino acids are coded for by several codons is called degeneracy

11. ©2001 Timothy G. StandishSentences Genes can be thought of as sentences in the nucleic acid language 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, acting like a capital letter is the start codon In the middle is a sequence of codons for amino acids The period at the end is a stop codon

12. ©2001 Timothy G. Standish The Protein Language The protein language is very different from the nucleotide language Polypeptides are the sentences 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

13. ©2001 Timothy G. Standish Comparison of Languages English - God Chinese - Hieroglyphics - DNA - CGT RNA - CGU Amino Acid - Arginine

14. ©2001 Timothy G. Standish Redundancy: Synonyms and Codon Degeneracy English - Synonyms for God: Lord Father Deity the Almighty Jehovah Nucleic acids - Synonyms for Arginine: CGU CGC CGA CGG AGA AGG

15. ©2001 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 Stop Codon Names - Ochre - Amber -Not named UAA UAG UGA UAA UAG UGA

16. ©2001 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

17. ©2001 Timothy G. Standish Codon Assignment Is Fortuitous Because of wobble base pairing, 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

18. ©2001 Timothy G. Standish The Genetic Code Is Improbable And Does Not Look Random

19. ©2001 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

20. ©2001 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 amino acids and “stop” must be assigned one codon, only 64 - 21 = 43 codons could be truly randomly assigned Net probability is the likelihood of initial assignment times probability of random assignment of remaining codons

21. ©2001 Timothy G. Standish Initial Codon Assignment Theory would indicate initial codon assignment must have been random Lewin in Genes VI, p 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

22. ©2001 Timothy G. Standish Codon Assignment does not look random The genetic code does not like uneven numbers.

23. ©2001 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

24. ©2001 Timothy G. Standish Codon Assignment Is Not Strongly Correlated to 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

25. ©2001 Timothy G. Standish The Genetic Code Is Not Completely Universal

26. ©2001 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

27. ©2001 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

28. ©2001 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: 1.Replacement of former stop codons in genes vital for life, and whose activity will be destroyed, with still functional stop codons 2.Production of new tRNAs with anticodons that recognize the codon as an amino acid and not stop anymore 3.Modification of the release factor (eRF) to restrict its binding specificity so that it no longer binds the former stop codon All changes “appear to have occurred independently in specific lines of evolution” (Lewin, Genes VI)

29. ©2001 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

30. ©2001 Timothy G. Standish Wobble Base Pairing

31. ©2001 Timothy G. Standish The Rules of Codon Anticodon Base Pairing Three things affect the way in which base pairing occurs between codons on mRNA and anticodons on tRNA: 1 How the two molecules “twist” when annealing - They are not free to form a perfect A helix 2 The environment of the Ribosome A site 3 Chemical modification of bases These three factors change the usual base pairing seen in DNA and RNA, particularly at the first base of anticodons/third base of codons

32. ©2001 Timothy G. Standish D Arm - Contains dihydrouridine Acceptor Arm - A specific amino acid is attached to the 3’ end T  C arm -  stands for pseudouridine Extra Arm - May vary in size Anticodon Transfer RNA (tRNA)

33. ©2001 Timothy G. Standish D Arm - Contains dihydrouridine T  C arm -  stands for pseudouridine Anticodon Transfer RNA (tRNA) Dihydro- uridine O O NH H H H H N Pseudo- uridine O O HNNH C

34. N O H N O N N H Cytosine H O N N N N N H H Guanine - + + + - - Base Pairing Guanine And Cytosine

35. N O N O N H + - Uracil N N N N H N H - + Adenine Base Pairing Adenine And Uracil

36. Base Pairing Adenine And Cytosine N O H N O N N H Cytosine - + - N N N N H N H - + Adenine

37. N O N O N H + - Uracil Base Pairing Guanine And Uracil H O N N N N N H H Guanine + + -

38. N O N O N H Uracil + - Wobble Base Pairing Guanine And Uracil H O N N N N N H H Guanine + + -

39. N S N O N H + - 2 Thio- uracil N N N N H N H - + Adenine Base Pairing Adenine And 2-Thiouracil

40. S N O N H Wobble Base Pairing Guanine And 2-Thiouracil H O N N N N N H H Guanine + + - 2-Thiouracil forms only one hydrogen bond with guanine which is not enough to form a stable pair in the environment of the ribosome A site + 2 Thio- uracil

41. N O H N O N N H Cytosine O N N N N H H Inosine - + - + - Wobble Base Pairing Inosine And Cytosine

42. O N N N N H H Inosine - + N O N O N H Uracil + - Wobble Base Pairing Inosine And Uracil

43. N N N N H N H - + Adenine O N N N N H H Inosine + - Wobble Base Pairing Inosine And Adenine

44. ©2001 Timothy G. Standish The Wacky Rules of Wobble Base Pairing -------------A or G -------A -------------G -------------U -------------C or U -------------C U or G First anticodon base: U 2-S-U C A G I Third codon base:

45. ©2001 Timothy G. Standish Modified RNA Bases: Uridine H O C C N C C N Ribothymidine (T) O H C H H H H H O C C N C C N H Dihydrouridine (D) O H H H S C C N C C N H 4-thiouridine (S 4 U) O H H O C C C H O H NN C Pseudouridine (  ) H O C C N C C N H Uridine O H

46. ©2001 Timothy G. Standish Wobbling and tRNA Numbers The net effect of wobble base pairing is to reduce the number of tRNAs that must be produced by a cell In reality cells do not make 61 different tRNAs, one for each codon Many tRNAs have anticodons that anneal to several different codons Codons are known for which there are more than one tRNA, although each tRNA carries the same amino acid (i.e., methionine)

47. ©2001 Timothy G. Standish Summary: Are Codons The Language of God? The genetic code appears to be non-random in nature and designed with 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 Taken together, this evidence indicates the hand of a Designer in the genetic code and does not support the theory that life originated due to random processes or that all organisms share a common ancestor

48. ©2001 Timothy G. Standish

49. Modified RNA Bases: Uridine H O C C N C C N Ribothymidine O H C H H H H H O C C N C C N H Dihydrouridine O H H H O C C N C C N H Uridine O H H O C C C H O H NN C Pseudouridine

50. ©2001 Timothy G. Standish H Other Modified RNA Bases: Cytidine O H C N C N C C N H H Cytidine O

51. ©2001 Timothy G. Standish Other Modified RNA Bases: Adenosine OO H C N N C C N N C C N H H H Adenosine