DNA

1. The Central Dogma Transcription Transcription Protein DNA RNA Translation Translation Replication 1. Genetic information transfer from polynucleotide chain into polypeptide chain. 2. Take place in ribosomes. 3. tRNAs recognize codons.

2. Chapter 15 The Genetic Code

3. With four possible nucleotide at each position, the total number of permutations of these triplets is 64,a value well in excess of the number of amino acids, so what are the rules that govern their use? • In this chapter, we will discuss the nature and underlying logic of the genetic code, how the code was “cracked”, and the effect of mutation on the coding capacity of messenger RNA.

4. Outline • The genetic code is degenerate • Three rules goven the genetic code • Suppressor mutation can reside in the same or a different gene • The code is nearly universal

5. Section 1The code is degenerate

7. Many amino acids are specified by more than one codon-degeneracy (简并性). Codons specifying the same amino acid are called synonyms (同义密码子)

8. Degeneracy • Often, when the first two nucleotides are identical, the third nucleotide can be either C or U without changing the code. A and G at the third position are interchangeable as well. • Transition in the third position of a codon specifies a same amino acid. Transversion in this position changes the amino acid about half the time.

9. Code degeneracy explains how there can be great variation in the AT/GCratios in the DNA of various organisms without large changes in the proportionof amino acids in their proteins. organisms without large changes in the proportion of amino acids in their proteins.

10. Figure 15-1 Codon-anticodon • pairing of two tRNA Leu moleculars CUG CUC

11. Detail1Perceiving order in the Makeup of the Code The genetic code evolved in such a way as to minimize the deleterious effects of mutations. Code degeneracy may serve as a safety mechanism to minimize errors in the reading of codons.

12. For instance, mutations in the first position of a codon will often give a similar amino acid. Furthermore, codon with pyrimidines in the second position specify mostly hydrophobic amino acids, whereas those with purines in the second position correspond mostly to polar amino acids. • Hence, because transition are the most common type of point mutations, a change in the second position. • Another in the code is that whenever the first two position of a codon are both occupied by G or C, each of the four nucleotides in the third position specifies the same amino acid. As G:C base pair are stronger than A:U base pair, mismatchs in pairing the third codon base are often tolerated if the first two positions make strong G:C base pairs.

13. Detail 2Wobble in the Anticodon • Some tRNA could recognize several different codons • Inosine is present in the anticodon loop as a fifth base

14. The wobble rules do not permit any single tRNA molecule to recognize four different codons. Three codons can be recognized only when inosine occupie the position of the anticodon.

15. Inosine inosine adenine Inosine arises through enzymatic modification of adenine

16. Wobble Concept In 1966, Francis Crick devised the wobble concept. It states that the base at the 5’ end of the anticodon is not as spatially confined as the other two, allowing it to form hydrogen bonds with more than one bases located at the 3’ end of a codon.

17. Table 15-2 Pairing Combinations with the Wobble Concept Base in 5’ Anticodon Base in 3’ Codon G U or C C G A U U A or G I A, U, or C

18. The Wobble Rules • The pairings permitted are those give ribose-ribose distancesclose to that of the standard A:U or G:C base pairs. • The ribose-ribose distances: Purine-purine: too long Pyrimidine-pyrimidine: too short

19. The ribose-ribose distances for the wobble pairs are close to those of A:U or G:C base pairs Figure 15-2 Wobble base pairing

20. The 3-D structure of tRNA shows that the stacking interactions between the flat surfaces of the 3 anticodon bases + 2 followed bases position the first (5’) anticodon base at the end of the stack, thus less restricted in its movements. The 3’ base appears in the middle of the stack, resulting in the restriction of its movements.

21. The adjacent base The adjacent base is always a bulky modified purine residue. Figure 15-3 Structure of yeast tRNA(Phe)

22. Details 3Three Codons Direct Chain Termination • Three codons do not correspond to any acid. Instead, the signify chain termination. As discussed, UAA, UAG, and UGA, are read by specific proteins known as release factors:RF1 and RF2 in bacteria and eRF1 in eukaryotes. Release factor enter the A site of the ribosome and trigger hydrolysis of the peptidyl-tRNA occupying the P site, resulting in the release of the newly synthsized protein.

23. Details 4How the Code Was Cracked In the 1961, one year after the general outline of how messenger RNA participate in protein synthesis had been established, the use of artificial mRNAs and the availability of cell-free systems for carrying out protein synthesis began to make it possible to crack the code (see Chapter 2).

24. Details 5Stimulation of amino acid incorporation by synthetic mRNA Extracts from E. coli cells can incorporate amino acids into proteins. After several minutes the synthesis came to a stop because the degradation of mRNA. The addition of fresh mRNA to extracts caused an immediate resumption of synthesis This led the scientist an opportunity to elucidate the nature of the code using synthetic RNA

25. How the RNA is synthesized? [XMP]n + XDP = [XMP]n+1 + P

26. And scientists found: • Poly-U codes for Polyphenylalanine • CCC was assigned as a proline codon and AAA as a lysine codon • The guanine residues in poly-G firmly hydrogen bond to each other and form mutistranded trip helices that do not bind to riboomes.

27. Detail 6Mixed Copolymers Allowed Additional Codon Assignments • Poly-AC contain 8 codons: CCC, CCA, CAC, ACC, CAA, ACA, AAC, and AAA. They code for Asp, Glu, His, Thr & Pro (CCC), Lys (AAA).The proportions of the 8 codons incorporated into polypeptide products depend on the A/C ratio

28. As there is no way of knowing from random copolymer . Such experiment can determine the composition of the codons, but not the order of the three nucleotides.

29. Detail 7Transfer RNA binding to defined trinucleotide codons In 1964, the discovery of a method utilized the fact that specific aminoacyl-tRNA molecules can bind to ribosome-mRNA complexes which does not demand the presence of long mRNA provided a relatively easy way of determining the order of nucleotides within many codons.

30. Detail 8Codon assignments from repeating copolymers • At the same time that the trinucleotide binding technique became available, organic chemical and enzymatic technique were being used to prepare synthetic polyribonucleotides with known repeating sequences. (Ribosomes tart protein synthesis at random points along these regular copolymers)

31. Figure 15-5 Preparing oligo-ribonucleotides

32. Table 15-5 Amino Acids Incorporated or Polypeptide Made Codons Recognized Codon Assignment copolymer (CU)” CUC|UCU|CUC… Leucine 5’-CUC-3’ Serine UCU (UG)” UGU|GUG|UGU… Cystine UGU Valine GUG (AC)” ACA|CAC|ACA… Threonine ACA Histidine CAC (AG)” AGA|GAG|AGA… Arginine AGA Glutamine GAG (AUC)”AUC|AUC|AUC… Polyisoleucine 5’-AUC-3’

33. Section 2Three rules govern the genetic code

34. Three Rules 1.Codons are read in a 5’ to 3’ direction. 2.Codons are nonoverlapping and the message contains no gaps. 3.The message is translated in a fixed reading frame which is set by the initiation codon.

35. Three Kinds of Point Mutations Alter the Genetic Code • 1. Missense mutation: An alternation that changes a codon specific for one amino acid to a codon specific for another amino acid.

36. 2. Nonsenseorstop mutation:An alternation causing a change to a chain-termination codon. • 3.Frameshift mutation: Insertions or deletions of one or a small number of base pairs that alter the reading frame.

37. The insertion of a single base drastically alters the coding capacity of the message not only at site the insertion but for the remainder of the messengers well. Likewise, the insertion (or deletion) of two base would have the effect of throwing the entire coding sequence, at and downstream of the insertions, into a different reading frame

38. illustration Ala Ala Ala Ala Ala Ala Ala Ala 5’-GCU GCU GCU GCU GCU GCU GCU GCU Ala Ala SerCys Cys Cys Cys Cys 5’-GCUGCU AGC UGC UGC UGC UGC UGC Ala Ala Ser Cys Met Leu HIS Ala 5’-GCU GCU AGC UGC AUG CUG CAU GCU

39. Genetic proof that the code is read in units of three • A classic experiment involving bacteriophage T4 • Because the gene could tolerate three insertions but not one or two, the genetic code must be read in units of three.

40. Section 3Suppressor mutations can reside in the same or a different

41. Key points 1.Reverse (back) mutations: change an altered nucleotide sequence back to its original arrangement. 2.Suppressor mutations: suppress the change due to mutation at site A by producing an additional genetic change at site B. (1) Intragenic suppression (2) Intergenic suppression

42. Key points 3.Suppressor genes: genes that cause suppression of mutations in other genes. Suppressor mutations work by producinggood (or partially good) copies of the protein that are made inactive by the original harmful mutation.

43. Key points • 4.Intergenic suppresion involes mutant tRNAs . Mutant tRNA genes suppress the effects of nonsense mutations in protein-coding genes. They act by reading a stop codon as if it were a signal for a specific amino acid.

44. Figure 15-7 a Figure 15-7 a

45. Figure 15-7 b

46. Key points 5.Nonsense suppressors also read normal termination signals. The act of nonsense suppression is a competition between the suppressor tRNA and the release factor.

47. Key points In the presence of the suppressor tRNA, more than half of the chain-terminating signals are read as specific amino acid codons. E.coli can tolerate this misleading of the UAG stop codon.

48. Section 4The code is nearly universal

49. The results of large-scale sequencing of genomes have confirmed the universality of the genetic code. Benefits of the universal codes 1.Allow us to directly compare the protein coding sequences among all organisms. 2.Make it possible to express cloned copies of genes encoding useful protein in different host organism. Example: Human insulin expression in bacteria)

50. In certain subcellular organelles, the genetic code is slightly different from the standard code. • Mitochondrial tRNAs are unusual in the way that they decode mitochondrial messages. • Only 22 tRNAs are present in mammalian mitochondria. The U in the 5’ wobble position of a tRNA is capable of recognizing all four bases in the 3’ of the codon.