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Content Chapter 1 Introduction Chapter 2 The Structures of DNA and RNA Chapter 10 R egulation in Eukaryotes Chapter 4 DNA Mutation and Repair Chapter 5 RNA Transcription Chapter 6 RNA Splicing Chapter 7 Translation Chapter 8 The Genetic code  Chapter 9 R egulation in prokaryotes Chapter 3 DNA Replication

HOW TO LEARN THIS COURSE WELL ？  To learn effectively  To preview and review  Problem-base learning  Making use of class time effectively  Active participation  Bi-directional question in class  Group discussion  Concept map  Tutorship  To call for reading, thingking and discussing of investigative learning

E VALUATION ( GRADING ) SYSTEM  Question in-class and attendance : 10 points  Group study and attendance: 20 points  Final exam: 70 points  Bonus

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 3/22/05

Ch 5 : Transcription Ch 6 : RNA Splicing Ch 7 : Translation Ch 8 : The Genetic code 4/3/05 EXPRESSION OF THE GENOME

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

Topic 1: THE CODE IS DEGENERATE CHAPTER 8 The Genetic Code Codon: degenerate Anticodon: wobble Codon: degenerate Anticodon: wobble

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 ). 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 8-1 The Genetic Code TABLE 8-1 The Genetic Code

C ODING ROLE #1 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. 2.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. 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. 2.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.

Figure 8-1 Codon-anticodon pairing of two tRNA Leu moleculars Figure 8-1 Codon-anticodon pairing of two tRNA Leu moleculars CUG CUC

AT/GC ratios proportion of amino acids 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.

Perceiving Order in the Makeup of the Code 1.The genetic code evolved in such a way as to minimize the deleterious effects of mutations. 2.Code degeneracy may serve as a safety mechanism to minimize errors in the reading of codons. The Code Is Degenerate

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.

Wobble in the Anticodon ( 反密码子具有摇摆性 ) 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 The Code Is Degenerate

Inosine inosine adenine Inosine arises through enzymatic modification of adenine

W OBBLE C ONCEPT 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.

Table 8-2 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 G U or C C G A U U A or G I A, U, or C

T HE W OBBLE R ULES 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

Figure 8-2 Wobble base pairing Figure 8-2 Wobble base pairing The ribose-ribose distances for the wobble pairs are close to those of A:U or G:C base pairs

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

W HY 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.

Figure 8-3 Structure of yeast tRNA(Phe) The adjacent base The adjacent base is always a bulky modified purine residue.

Three Codons Direct Chain Termination  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). The Code Is Degenerate

How the Code Was Cracked ( 解开 )  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 The Code Is Degenerate

Stimulation of Amino Acid Incorporation by Synthetic mRNAs 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 The Code Is Degenerate

Figure 8-4 Polynucleotide phosphorylase reaction How the RNA is synthesized? [XMP]n + XDP = [XMP]n+1 + P How the RNA is synthesized? [XMP]n + XDP = [XMP]n+1 + P

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.

Mixed 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 The Code Is Degenerate

Such experiment can determine the composition of the codons, but not the order of the three nucleotides. See Table 8-3 on Page 467

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.  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.

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

Figure 8-5 Preparing oligo-ribonucleotides

Table 8-5 copolymer Codons Recognized Amino Acids Incorporated or Polypeptide Made Codon Assignment (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’ (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’

Topic 2: THREE RULES GOVERN THE GENETIC CODE CHAPTER 8 The Genetic Code

T HREE R ULES  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.

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. 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.

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

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.  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.

Topic 3: SUPPRESSOR MUTATIONS CAN RESIDE IN THE SAME OR A DIFFERENT GENE CHAPTER 8 The Genetic Code 4/22/05

 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 Reverse the harmful mutations by a second genetic change

 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.

Figure 8-6 Suppression of frameshift mutations

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.  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.

Figure 15-7 a Figure 8-7 a

Figure 8-7 b

Nonsense Suppressors also Read Normal Termination Signals (OOPs) Suppressor mutations  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??.  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??.

Topic 4: THE CODE IS NEARLY UNIVERSAL Topic 4: THE CODE IS NEARLY UNIVERSAL CHAPTER 8 The Genetic Code 4/22/05

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 ecpression in bacteria)

 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.

Table 8-6 Genetic Code of Mammalian Mitochondria

1.“The genetic code is degenerate” What does it mean? What are the benefits? 2.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? 3.What are the three roles governing the genetic code? What are the mutations altering genetic code? 4.What are suppressor mutations? ( 种类 ) 5.What are the benefits of the code universality? Key points of the chapter