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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

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

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

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.

Topic 1: THE CODE IS DEGENERATE CHAPTER 15 The Genetic Code Topic 1: THE CODE IS DEGENERATE 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). TABLE 15-1 The Genetic Code

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.

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

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 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.

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) 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

Inosine arises through enzymatic modification of adenine

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.

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

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

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

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?

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.

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

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).

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

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

How the RNA is synthesized? Figure 15-4 Polynucleotide phosphorylase reaction 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 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

Such experiment can determine the composition of the codons, but not the order of the three nucleotides. See Table 15-3 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.

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 15-5 Preparing oligo-ribonucleotides

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’

Topic 2: THREE RULES GOVERN THE GENETIC CODE CHAPTER 15 The Genetic Code Topic 2: THREE RULES GOVERN THE GENETIC CODE 4/22/05

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.

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.

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.

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

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

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 15-6 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.

Figure 15-7 a Figure 15-7 a

Figure 15-7 b

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

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

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)

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 15-6 Genetic Code of Mammalian Mitochondria

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?