1. Welcome Each of You to My Molecular Biology Class

2. Molecular Biology of the Gene, 5/E --- Watson et al. (2004)
Part I: Chemistry and Genetics
Part II: Maintenance of the Genome
Part III: Expression of the Genome
Part IV: Regulation
Part V: Methods
3/22/05

3. Part III: Expression of the Genome
Ch 12: Mechanisms of Transcription
Ch 13: RNA Splicing
Ch 14: Translation
Ch 15: The Genetic Code

4. CHAPTER 15 The Genetic Code
Molecular Biology Course
CHAPTER 15
The Genetic Code

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

6. Topic 1: THE CODE IS DEGENERATE
CHAPTER 15 The Genetic Code
Topic 1: THE CODE IS DEGENERATE
Codon: degenerate
Anticodon: wobble

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).
TABLE 15-1
The Genetic Code

8. Coding role #1
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. pairing of two tRNA Leu moleculars
CUC
CUG
Figure 15-1 Codon-anticodon
pairing of two tRNA Leu moleculars

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

11. Perceiving Order in the Makeup of the Code
The Code Is Degenerate
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. Coding role #2 1.The second position of a codon:
Pyrimidines-hydrophobic amino acids
Purines-polar amino acids
2.If the first two positions are both occupied by G or C, each of the four nucleotides in the third position specifies the same amino acid.

13. Wobble in the Anticodon
(反密码子具有摇摆性)
The Code Is Degenerate
Question: Is there a specific tRNA for every codon? (If it was true, at least 61 different tRNAs would exist.)
The answer is NO
Some tRNA could recognize several different codons
Inosine is present in the anticodon loop as a fifth base

14. Inosine arises through enzymatic modification of adenine

15. 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.

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

18. 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

19. Critical Thinking
The wobble concept predicted that at least three tRNAs exist for the six serine codons (UCU, UCC, UCA, UCG, AGU, and AGC). Why?

20. Why wobble is allowed at the 5’ anticodon
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 is always a bulky modified purine residue.
Figure 15-3 Structure of yeast tRNA(Phe)

22. Three Codons Direct Chain Termination
The Code Is Degenerate
Three codons, UAA, UAG, and UGA signify chain termination.
They are not read by tRNAs but by proteins called release factors (RF1 and RF2 in bacteria and eRF1 in eukaryotes).

23. How the Code Was Cracked (解开)
The Code Is Degenerate
See Chapter 2, Page 35:
Establishing the Genetic Code
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

24. Stimulation of Amino Acid Incorporation by Synthetic mRNAs
The Code Is Degenerate
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?
Figure 15-4 Polynucleotide phosphorylase reaction
How the RNA is synthesized?
[XMP]n + XDP = [XMP]n+1 + P

26. Experimental Results:
UUU codes for phenylalanine.
CCC codes for proline.
AAA codes for lysine.
The guanine residues in poly-G firmly hydrogen bond to each other and form multistranded triple helices that do not bind to ribosomes.

27. Mixed Copolymers Allowed Additional Codon Assignments
The Code Is Degenerate
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. Such experiment can determine the composition of the codons, but not the order of the three nucleotides.
See Table 15-3 on Page 467

29. Transfer RNA Binding to Defined Trinucleotide Codons (1964)
The Code Is Degenerate
A method to order the nucleotides within some of the codons.
Specific amino-acyl-tRNA can bind to ribosome-mRNA complexes.
The addition of trinucleotide results in corresponding amino-acyl-tRNA attachment.

30. Codon Assignments from Repeating Copolymers
The Code Is Degenerate
Organic chemical and enzymatic techniques were used to prepare synthetic polyribonucleotides with known repeating sequences.

31. Figure 15-5 Preparing oligo-ribonucleotides

32. Amino Acids Incorporated or Polypeptide Made
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. Topic 2: THREE RULES GOVERN THE GENETIC CODE
CHAPTER 15 The Genetic Code
Topic 2: THREE RULES GOVERN THE GENETIC CODE
4/22/05

34. Three Rules Codons are read in a 5’ to 3’ direction.
Codons are nonoverlapping and the message contains no gaps.
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
Three Rules Govern 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.
2. Nonsense or stop mutation: An alternation causing a change to a chain-termination codon.

36. 3. Frameshift mutation: Insertions or deletions of one or a small number of base pairs that alter the reading frame.

37. Genetic Proof that the Code Is Read in Units of Three
Three Rules Govern the Genetic Code
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.

38. CHAPTER 15 The Genetic Code
Topic 3: SUPPRESSOR MUTATIONS CAN RESIDE IN THE SAME OR A DIFFERENT GENE
4/22/05

39. Reverse the harmful mutations by a second genetic change
Reverse (back) mutations: change an altered nucleotide sequence back to its original arrangement.
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

40. Suppressor genes: genes that cause suppression of mutations in other genes.
Suppressor mutations work by producing good (or partially good) copies of the protein that are made inactive by the original harmful mutation.

41. Figure 15-6 Suppression of frameshift mutations

42. Intergenic Suppression Involves Mutant tRNAs
Suppressor mutations
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.

43. Figure 15-7 a
Figure 15-7 a

44. Figure 15-7 b

45. Nonsense Suppressors also Read Normal Termination Signals (OOPs)
The act of nonsense suppression is a competition between the suppressor tRNA and the release factor.
In E. coli, Suppression of UAG codons is efficient, and suppression of UAA codon average is inefficient. Why??.
Suppressor mutations

46. THE CODE IS NEARLY UNIVERSAL
CHAPTER 15 The Genetic Code
Topic 4:
THE CODE IS NEARLY UNIVERSAL
4/22/05

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

48. However, 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.

49. Table 15-6 Genetic Code of Mammalian Mitochondria

50. Key points of the chapter
“The genetic code is degenerate” What does it mean? What are the benefits?
What is the wobble concept? Is there structural evidence? How the wobble in the anticodon affect the number of tRNAs to recognize the 61 codons?
What are the three roles governing the genetic code? What are the mutations altering genetic code?
What are suppressor mutations? (种类)
What are the benefits of the code universality?