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1. Chapter 15 : The Genetic Code 柳兴凤 生物科学班 200431060185

2. This chapter mainly describes the classic experiments that led to the elucidation of the genetic code , and lays out the rules by which the code is translated . The nucleotide sequence information is based on a three letter code , while the protein sequence information is based on twenty different amino acids . The code is degenerate with two or more codons (in most cases ) specifying the same amino acid . There are also specific codons that indicate where translation should start and where it should stop .

3. This chapter contains four parts : Topic one : The code is degenerate Topic two : Three rules govern the genetic code Topic three : Suppressor mutations can reside in the same or a different gene Topic four : The code is nearly universal

4. The outline : 1.At the very heart of the Central Dogma is the concept of information transfer from the linear sequence of the four letter alphabet of the polynucleotide chain into the 20-amino acid language of the polypeptide chain . 2.Translation takes place on ribosomes . 3.Translation is mediated by special adaptor molecules known as tRNAs .

5. The total number of permutations of the triplets is 64,a value well in excess of the number of amino acids .Which of these triplet codons are responsible for specifying which amino acids , and what are the rules that govern their use ? In this chapter , we discuss the nature and underlying logic of the genetic code , how the code was “cracked ,”and the effect of mutations on the coding capacity of messenger RNA .

6. Topic one : THE CODON IS DEGENERATE

7. Degeneracy (密码子的简并性）： The phenomenon of many amino acids are specified by more than one codon . Synonyms (同义密码子）： Codons specified the same amino acid . Table 15-1 The Genetic Code

8. The features of the code : 1 . 61 of the 64 possible triplets specify an amino acid , with the remaining three triplets being chain-terminating signals . 2 . When the first two nucleotides are identical , the third nucleotide can be either cytosine or uracil and the codon will still code for the same amino acid . Often , adenine and guanine are similarly interchangeable . 3 . Not all degeneracy is based on equivalence of the first two nucleotides . ( Figure 15-1 )

9. CUC CUG Figure 15-1 Codon-anticodon pairing of two tRNA Leu molecules

10. How there can be great variation in the AT/GC ratios in the DNA of various organisms without correspondingly large changes in the relative proportion of amino acids in their proteins ? This can be explained by codon degeneracy , especially the frequent third-place equivalence of cytosine and uracil or guanine and adenine .

11. §1-1 Perceiving Order in the makeup of the Code 1 . The code evolved in such a way as to minimize the deleterious effects of mutations . 1 ) . Mutations in the first position of a codon will often give a similar ( if not the same ) amino acid . 2 ) . Codons 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 . 3 ) . If a codon suffers a transition mutation in the third position , rarely will a different amino acid be specified .

12. 2 . Whenever the first two positions of a codon are both occupied by G or C , each of the four nucleotides in the third position specifies the same amino acid . 3 . Whenever the first two positions of the codon are both occupied by A or U , the identity of the third nucleotide does make a difference .

13. §1-2 Wobble in the Anticodon Why the opinion of that a specific tRNA anticodon would exist for every codon is wrong ? 1 ) . Highly purified tRNA species of known sequence could recognize several different codons . 2 ) . An anticodon base was not one of the 4 regular ones , but a fifth base , inosine .

14. Wobble concept ( 摆动理论 ) : 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 any of several bases located at the 3’ end of a codon . (This is devised by Francis Crick in1966)

15. The wobble rules : 1 . Not all combinations are possible , with pairing restricted to those shown in Table 15-2 . 2 . The pairings permitted by the wobble rules are those that give ribose-ribose distances close to that of the standard A:U or G:C base pairs . 3 . The wobble rules do not permit any single tRNA molecule to recognize four different codons .

16. 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

17. 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

18. Why wobble is not seen in the first (5’) position of the code ? Restriction of bases movements: 1 ) . In the three-dimensional structure of tRNA , the three anticodon bases—as well as the two following (3’) bases in the anticodon loop—all point in roughly the same direction (figure 15-3) . 2) . The first (5’) anticodon base is at the end of the stack and is perhaps less restricted in its movements than the other two anticodon bases—hence , wobble in the third (3’) position of codon . 3 ) . Not only does the third (3’) anticodon base appear in the middle of the stack , but the adjacent base is always a bulky modified purine residue .

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

20. §1-3 Three Codons Direct Chain Termination The chain-termination codons , UAA , UAG , and UGA ,are read not by special tRNAs but by specific proteins known as release factors. Release factors 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 synthesized protein .

21. §1-4 How the Code Was Cracked The identification of the codons for a given amino acid would require : 1 . Exact knowledge of the nucleotide sequences of a gene .(while the then-current methods were very primitive . ) 2 . The corresponding amino acid order in its protein product . (At that time , the elucidation ,although a laborious process , was already a very practical one . )

22. §1-5 Stimulation of Amino Acid Incorporation by Synthetic mRNAs The extracts prepared from dells of E. coli , that were actively engaged in protein synthesis ,were capable of incorporating radioactively-labeled amino acids into proteins .

23. 1) . Protein synthesis in these extracts proceeded rapidly for several minutes and then gradually came to a stop . 2) . There was a corresponding loss of mRNA owing to the action of degradative enzymes present in the extract . However , the addition of fresh mRNA to extracts that had stopped making protein caused an immediate resumption of synthesis .

24. How the RNA is synthesized? [XMP]n + XDP = [XMP]n+1 + P Polynucleotide phosphorylase is normally responsible for breaking down RNA , and no template DNA or RNA is required for RNA synthesis with this enzyme ; the product depends entirely on the ratio of the various ribonucleoside diphosphates added to the reaction mixture .

25. 1.Under physiological conditions favors the degradation of RNA into nucleoside diphosphates . 2.By use of high nucleoside diphosphate concentrations , this enzyme can be made to catalyze the formation of internucleotide 3’ 5’ phosphodiester bonds and thus make RNA molecules (figure 15-4) . 3.In all these mixed polymers ,the base sequences are approximately random ,with the nearest-neighbor frequencies determined solely by the relative concentrations of the reactants .

26. Figure 15-4 Polynucleotide phosphorylase reaction

27. §1-6 Poly-U Codes for Polyphenylalanine 1.Under the right conditions in vitro , almost all synthetic polymers will attach to ribosomes and functions as templates . 2.A high magnesium concentration can circumvent the need for initiation factors and the special initiator fMet-tRNA , allowing chain initiation to take place without the proper signals in the mRNA . 3.Pol-U selects phenylalanyl tRNA molecules exclusively , thereby forming a polypeptide chain containing only phenylalanine (polyphenylalanine) .

28. §1-7 Mixed Copolymers Allowed Additional Codon Assignments We can ratiocinate the codon assignments by the proportions of different codons .(the proportions of the codons vary with the copolymer base ratio , eg: table 15-3 in page 468 . ) But there is no way of knowing the order from random copolymers .

29. §1-8 Transfer RNA Binding to Defined Trinucleotide Codons Trinucleotide effect : (三核苷酸效应） 1.Even in the absence of all the factors required for protein synthesis , specific aminoacyl-tRNA molecules can bind to ribosome-mRNA complexes . 2.This specific binding does not demand the presence of long mRNA molecules . In fact , the binding of a trinucleotide to a ribosome is sufficient .

30. The function of the discovery of thes trinucleotide effect : It provided a relatively easy way of determining the order of nucleotides within many codons .

31. §1-9 Codon Assignments from Repeating Copolymers Organic chemical and enzymatictechniques were being used to prepare synthetic polyribonucleotides with known repeating sequences (Figure 15-5) .

32. Figure 15-5 Preparing oligo-ribonucleotides

33. Ribosomes start protein synthesis at random points along these regular copolymers ; yet they incorporate specific amino acids into polypeptides . (Table 15-5) The sum of all these observations : they permitted the assignments of specific amino acids to 61 out of the possible 64 codons ,with the remaining three chain-terminating codons , UAG , UAA , AND UGA , not specifying any amino acid .

34. 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’

35. Topic two : THREE RULES GOVERN THE GENETIC CODE

36. The genetic code is subject to three rules that govern the arrangement and use of codons in messenger RNA : 1 . Codons are read in a 5’ to 3’ direction . (eg : The base order of the dipeptide NH2-Thr-Arg-COOH is 5’- ACGCGA-3’ ,but not 3’-GCAAGC-5’) 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 .

37. §2-1 Three Kinds of Point Mutations Alter the Genetic Code 1 . Missense mutation (错义突变） An alteration that changes a codon specific for one amino acid to a codon specific for another amino acid .

38. 2 . Nonsense (无义突变) or Stop mutation (终止突变) An alteration causing a change to a chain-termination codon . (a more drastic effect ) 3 . Frameshift mutation (移码突变）: Insertions or deletions of one or a small number of base pairs that alter the reading frame .

39. 1 ) . The insertion (or for that matter the deletion ) of a single base or two bases drastically alters the coding capacity of the message not only at the site of the insertion but for the remainder of the messenger as well . 2 ) . An insertion of three extra bases at nearby positions in a message will drastically alter the stretch of message , at and between the three insertions . But mRNA downstream of the three inserted bases will be in its proper reading frame and hence , completely unaltered .

40. eg : Ala Ala Ala Ala Ala Ala Ala Ala 5’-GCU GCU GCU GCU GCU GCU GCU GCU-3’ Insertion of an A in the message : Ala Ala Ser Cys Cys Cys Cys Cys 5’-GCU GCU AGC UGC UGC UGC UGC UGC-3’ Insertion of a three extra bases at nearby positions in a message : Ala Ala Ser Cys Met Leu His Ala Ala Ala 5’-GCU GCU AGC UGC AUG CUG CAU GCU GCU GCU-3’

41. §2-2 Genetic Proof that the Code Is Read in Units of Three Francis Crick 、Sydney Brenner : a classic experiment involving bacteriophage T4 Conclusion : because the gene could tolerate three insertions but not one or two , the genetic code must be read in units of three . (This did so purely on the basis of a genetic argument . )

42. Topic three : SUPPRESSOR MUTATIONS CAN RESIDE IN THE SAME OR A DIFFERENT GENE

43. The effects of harmful mutations can be reversed by a second genetic change : 1 . Simplereverse (back) mutations(逆转突变) : Mutationschange an altered nucleotide sequence back to its original arrangement . 2 .Suppressor mutations ( 抑制突变) : The mutations occurring at different locations on the chromosome the suppress the change due to a mutation at site A by producing an additional genetic change at site B .

44. 1 ) . Intragenic suppressor (基因内抑制) : The suppressor mutations occurring within the same gene as the original mutation but at a different site in this gene . ( such as missense mutation and frameshift mutation . ) 2 ) . Intergenic suppressor (基因间抑制) : The suppressor mutations occurring in another gene .

45. Figure 15-6 Suppression of frameshift mutations

46. §3-1 Intergenic Suppression Involves Mutant tRNAs Suppressor genes do not act by changing the nucleotide sequence of a mutant gene . (eg : figure 15-7) Cell with nonsense suppressors contain mutationally-altered rRNAs Question : How their codons corresponding to these rRNAs could continue to be read normally ?

47. Figure 15-7 a Nosense suppression Figure 15-7 a

48. Figure 15-7 b Nosense suppression

49. §3-2 Nonsense Suppressors also Read Normal Termination Signals The act of nonsense suppression can be viewed as a competition between the suppressor tRNA and the release factor .

50. 1 .Suppression of UAG codons is efficient . In the presence of the suppressor tRNA , more than half of the chain-terminating signals are read as specific amino acid codons . 2 . Mutant cells producing UAA-suppressing tRNAs grow poorly .

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