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From DNA to Protein: (Central Dogma). From DNA to Protein: Genotype to Phenotype. One Gene, One Polypeptide DNA, RNA, and the Flow of Information Transcription: DNA-Directed RNA Synthesis The Genetic Code Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes

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From DNA to Protein: (Central Dogma)

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    1. From DNA to Protein:(Central Dogma)

    2. From DNA to Protein: Genotype to Phenotype • One Gene, One Polypeptide • DNA, RNA, and the Flow of Information • Transcription: DNA-Directed RNA Synthesis • The Genetic Code • Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • Translation: RNA-Directed Polypeptide Synthesis • Regulation of Translation • Posttranslational Events • Mutations: Heritable Changes in Genes

    3. One Gene, One Polypeptide • A gene is defined as a DNA sequence. • There are many steps between genotype and phenotype; genes cannot by themselves produce a phenotype.

    4. One Gene, One Polypeptide • In the 1940s, Beadle and Tatum showed that when an altered gene resulted in an altered phenotype, that altered phenotype always showed up as an altered enzyme. • They experimented with strains of the bread mold Neurospora: a wild-type, and several mutant strains. • Their results suggested that mutations cause a defect in only one enzyme in a metabolic pathway. • This lead to the one-gene, one-enzyme hypothesis.

    5. Figure 12.1 One Gene, One Enzyme (Part 1)

    6. Figure 12.1 One Gene, One Enzyme (Part 2)

    7. One Gene, One Polypeptide • The gene–enzyme connection has undergone several modifications. Some enzymes are composed of different subunits coded for by separate genes. • This suggests, instead of the one-gene, one enzyme hypothesis, a one-gene, one-polypeptide relationship.

    8. DNA, RNA, and the Flow of Information • The expression of a gene takes place in two steps: • Transcription makes a single-stranded RNA copy of a segment of the DNA. • Translation uses information encoded in the RNA to make a polypeptide.

    9. DNA, RNA, and the Flow of Information • RNA (ribonucleic acid) differs from DNA in three ways: • RNA consists of only one polynucleotide strand. • The sugar in RNA is ribose, not deoxyribose. • RNA has uracil instead of thymine. • RNA can base-pair with single-stranded DNA (adenine pairs with uracil instead of thymine) and also can fold over and base-pair with itself.

    10. DNA, RNA, and the Flow of Information • Francis Crick’s central dogma stated that DNA codes for RNA, and RNA codes for protein. • How does information get from the nucleus to the cytoplasm? • What is the relationship between a specific nucleotide sequence in DNA and a specific amino acid sequence in protein?

    11. Figure 12.2 The Central Dogma

    12. DNA, RNA, and the Flow of Information • Messenger RNA, or mRNA moves from the nucleus of eukaryotic cells into the cytoplasm, where it serves as a template for protein synthesis. • Transfer RNA, or tRNA, is the link between the code of the mRNA and the amino acids of the polypeptide, specifying the correct amino acid sequence in a protein.

    13. Figure 12.3 From Gene to Protein

    14. DNA, RNA, and the Flow of Information • Certain viruses use RNA rather than DNA as their information molecule during transmission. • These viruses transcribe from RNA to RNA; they make a complementary RNA strand and then use this “opposite” strand to make multiple copies of the viral genome by transcription. • HIV and certain tumor viruses (called retroviruses) have RNA as their infectious information molecule; they convert it to a DNA copy inside the host cell and then use it to make more RNA.

    15. Transcription: DNA-Directed RNA Synthesis • In normal prokaryotic and eukaryotic cells, transcription requires the following: • A DNA template for complementary base pairing • The appropriate ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) to act as substrates • The enzyme RNA polymerase

    16. Transcription: DNA-Directed RNA Synthesis • Just one DNA strand (the template strand) is used to make the RNA. • For different genes in the same DNA molecule, the roles of these strands may be reversed. • The DNA double helix partly unwinds to serve as template. • As the RNA transcript forms, it peels away, allowing the already transcribed DNA to be rewound into the double helix.

    17. Transcription: DNA-Directed RNA Synthesis • The first step of transcription, initiation, begins at a promoter, a special sequence of DNA. • There is at least one promoter for each gene to be transcribed. • The RNA polymerase binds to the promoter region when conditions allow. • The promoter sequence directs the RNA polymerase as to which of the double strands is the template and in what direction the RNA polymerase should move.

    18. Figure 12.4 (Part 1) DNA is Transcribed in RNA

    19. Transcription: DNA-Directed RNA Synthesis • After binding, RNA polymerase unwinds the DNA about 20 base pairs at a time and reads the template in the 3¢-to-5¢ direction (elongation). • The new RNA elongates from its 5¢ end to its 3¢ end; thus the RNA transcript is antiparallel to the DNA template strand. • Transcription errors for RNA polymerases are high relative to DNA polymerases.

    20. Figure 12.4 (Part 2) DNA is Transcribed in RNA

    21. Transcription: DNA-Directed RNA Synthesis • Particular base sequences in the DNA specify termination. • Gene mechanisms for termination vary: • For some, the newly formed transcript simply falls away from the DNA template. • For other genes, a helper protein pulls the transcript away. • In prokaryotes, translation of the mRNA often begins before transcription is complete.

    22. Figure 12.4 (Part 3) DNA is Transcribed in RNA

    23. The Genetic Code • A genetic code relates genes (DNA) to mRNA and mRNA to the amino acids of proteins. • mRNA is read in three-base segments called codons. • The number of different codons possible is 64 (43), because each position in the codon can be occupied by one of four different bases. • The 64 possible codons code for only 20 amino acids and the start and stop signals.

    24. Figure 12.5 The Universal Genetic Code

    25. The Genetic Code • AUG, which codes for methionine, is called the start codon, the initiation signal for translation. • Three codons (UAA, UAG, and UGA) are stop codons, which direct the ribosomes to end translation.

    26. The Genetic Code • After subtracting start and stop codons, the remaining 60 codons code for 19 different amino acids. • This means that many amino acids have more than one codon. Thus the code is redundant. • However, the code is not ambiguous. Each codon is assigned only one amino acid.

    27. The Genetic Code • In the early 1960s, molecular biologists broke the genetic code. • Nirenberg prepared an artificial mRNA in which all bases were uracil (poly U). • When incubated with additional components, the poly U mRNA led to synthesis of a polypeptide chain consisting only of phenylalanine amino acids. • UUU appeared to be the codon for phenylalanine. • Other codons were deciphered from this starting point.

    28. Figure 12.6 Deciphering the Genetic Code

    29. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The molecule tRNA is required to assure specificity in the translation of mRNA into proteins. • The tRNAs must read mRNA correctly. • The tRNAs must carry the correct amino acids.

    30. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The codon in mRNA and the amino acid in a protein are related by way of an adapter—a specific tRNA molecule. • tRNA has three functions: • It carries an amino acid. • It associates with mRNA molecules. • It interacts with ribosomes.

    31. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • A tRNA molecule has 75 to 80 nucleotides and a three-dimensional shape (conformation). • The shape is maintained by complementary base pairing and hydrogen bonding. • The three-dimensional shape of the tRNAs allows them to combine with the binding sites of the ribosome.

    32. Figure 12.7 Transfer RNA

    33. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • At the 3¢ end of every tRNA molecule is a site to which its specific amino acid binds covalently. • Midpoint in the sequence are three bases called the anticodon. • The anticodon is the contact point between the tRNA and the mRNA. • The anticodon is complementary (and antiparallel) to the mRNA codon. • The codon and anticodon unite by complementary base pairing.

    34. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • Each ribosome has two subunits: a large one and a small one. • In eukaryotes the large one has three different associated rRNA molecules and 45 different proteins. • The small subunit has one rRNA and 33 different protein molecules. • When they are not translating, the two subunits are separate.

    35. Figure 12.9 Ribosome Structure

    36. Preparation for Translation: Linking RNAs, Amino Acids, and Ribosomes • The small ribosomal subunit plays a role in validating the three-base-pair match between the mRNA and the tRNA. • If hydrogen bonds have not formed between all three base pairs, the tRNA is ejected from the ribosome.

    37. Translation: RNA-Directed Polypeptide Synthesis • Translation begins with an initiation complex: a charged tRNA with its amino acid and a small subunit, both bound to the mRNA. • This complex is bound to a region upstream of where the actual reading of the mRNA begins. • The start codon (AUG) designates the first amino acid in all proteins. • The large subunit then joins the complex. • The process is directed by proteins called initiation factors.

    38. Figure 12.10 The Initiation of Translation

    39. Translation: RNA-Directed Polypeptide Synthesis • Ribosomes move in the 5¢-to-3¢direction on the mRNA. • The peptide forms in the N–to–C direction. • The large subunit catalyzes two reactions: • Breaking the bond between the tRNA in the P site and its amino acid • Peptide bond formation between this amino acid and the one attached to the tRNA in the A site • This is called peptidyl transferase activity.

    40. Figure 12.11 Translation: The Elongation Stage

    41. Translation: RNA-Directed Polypeptide Synthesis • After the first tRNA releases methionine, it dissociates from the ribosome and returns to the cytosol. • The second tRNA, now bearing a dipeptide, moves to the P site. • The next charged tRNA enters the open A site. • The peptide chain is then transferred to the P site. • These steps are assisted by proteins called elongation factors.

    42. Translation: RNA-Directed Polypeptide Synthesis • When a stop codon—UAA, UAG, or UGA—enters the A site, a release factor and a water molecule enter the A site, instead of an amino acid. • The newly completed protein then separates from the ribosome.

    43. Figure 12.12 The Termination of Translation

    44. Regulation of Translation • Antibiotics are defensive molecules produced by some fungi and bacteria, which often destroy other microbes. • Some antibiotics work by blocking the synthesis of the bacterial cell walls, others by inhibiting protein synthesis at various points. • Because of differences between prokaryotic and eukaryotic ribosomes, the human ribosomes are unaffected.

    45. Regulation of Translation • Polysomes are mRNA molecules with more than one ribosome attached. • These make protein more rapidly, producing multiple copies of protein simultaneously.

    46. Figure 12.13 A Polysome (Part 1)

    47. Figure 12.13 A Polysome (Part 2)

    48. Posttranslational Events • Two posttranslational events can occur after the polypeptide has been synthesized: • The polypeptide may be moved to another location in the cell, or secreted. • The polypeptide may be modified by the addition of chemical groups, folding, or trimming.

    49. Figure 12.14 Destinations for Newly Translated Polypeptides in a Eukaryotic Cell

    50. Posttranslational Events • As the polypeptide chain forms, it folds into its 3-D shape. • The amino acid sequence also contains an “address label” indicating where in the cell the polypeptide belongs. It gives one of two sets of instructions: • Finish translation and be released to the cytoplasm. • Stall translation, go to the ER, and finish synthesis at the ER surface.