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David Sadava, David M. Hillis, H. Craig Heller, May R. Berenbaum La nuova biologia.blu Genetica, biologia molecolare ed evoluzione S DNA and Gene Expression What Is the Evidence that the Gene Is DNA? By the 1920s it was known that chromosomes consisted of DNA and proteins. A new dye that stained DNA provided evidence that DNA is the genetic material. • It was in the right place • It varied among species • It was present in the right amounts What Is the Evidence that the Gene Is DNA? Experimental evidence came from work on two strains of Streptococcus pneumoniae. A substance from cells of one strain (even when dead) could produce a heritable change in the other strain. What Is the Evidence that the Gene Is DNA? To identify this substance, Oswald Avery treated samples to destroy different molecules. If DNA was destroyed, the transforming activity was lost. There was no loss of activity with destruction of proteins or RNA. What Is the Evidence that the Gene Is DNA? Hershey-Chase experiment: used bacteriophage T2 virus to determine whether DNA, or protein, is the genetic material. Part of the virus enters E. coli cells and converts the cell into a virus replication machine. What Is the Evidence that the Gene Is DNA? Bacteriophage were grown with either 35S to label the proteins, or with 32P to label the DNA. After infection, bacterial cells and viral remains were separated—the bacteria cells were labeled with 32P, indicating that DNA had entered the cells. What Is the Structure of DNA? The structure of DNA was determined using many lines of evidence. One crucial piece came from X-ray diffraction. Rosalind Franklin prepared crystallographs from DNA samples. Her images suggested a double-stranded helix with 10 nucleotides in each full turn. What Is the Structure of DNA? Chemical composition: Biochemists knew that DNA is a polymer of nucleotides. Each nucleotide consists of deoxyribose, a phosphate group, and a nitrogen-containing base. The four different nucleotides differed only in the bases: •Purines: adenine (A), guanine (G) •Pyrimidines: cytosine (C), thymine (T) What Is the Structure of DNA? Erwin Chargaff noticed that in all DNA, the amount of purines is the same as the amount of pyrimidines. Chargaff’s rule What Is the Structure of DNA? Francis Crick and James Watson used model building, plus the physical and chemical evidence to solve the structure of DNA. They published their results in 1953. What Is the Structure of DNA? Four key features of DNA structure: • It is a double-stranded helix • It is right-handed • It is antiparallel • The outer edges of the bases are exposed in major and minor grooves What Is the Structure of DNA? Antiparallel strands: direction of strand is determined by the sugar–phosphate bonds. Phosphate groups connect to the 3′ C of one sugar, and the 5′ C of the next sugar. Results in one chain with a free 5′ phosphate group—the 5′ end; The other chain has is a free 3′ hydroxyl group—the 3′ end. What Is the Structure of DNA? The two chains are held together by: 1. Hydrogen bonding between bases – complementary base pairing: One purine (A or G) with one pyrimidine (T or C) 2. Van der Waals forces between adjacent bases on the same strand. What Is the Structure of DNA? The double-helix structure is essential to DNA function: • Stores genetic information: with millions of nucleotides, the base sequences store a huge amount of information • Susceptible to mutations • Precisely replicated in cell division by complementary base pairing • Genetic information is expressed as the phenotype—nucleotide sequence determines sequence of amino acids in proteins How Is DNA Replicated? The mechanism of DNA replication is semiconservative (each parent strand is a template; new molecules have one old and one new strand). Ingredients needed: • Deoxyribonucleoside triphosphates dATP, dCTP, dGTP, and dTTP (dNTPs, the monomers of DNA) • DNA molecules to serve as template • DNA polymerase enzyme How Is DNA Replicated? Two steps in DNA replication: • Double helix is unwound, making two template strands • New nucleotides form complementary base pairs with template DNA and are linked by phosphodiester bonds How Is DNA Replicated? Nucleotides are added to the new strand at the 3′ end. Formation of the phosphodiester linkage is a condensation reaction. Bonds linking the phosphate groups of the triphosphate nucleosides are broken, releasing energy that drives the reaction. How Is DNA Replicated? DNA polymerase requires a primer, a short starter strand—usually RNA. The primer is complementary to the DNA template and is synthesized by an enzyme called a primase. DNA polymerase then adds nucleotides to the 3′ end until that section is complete, and the primer is degraded. How Is DNA Replicated? At the replication fork DNA opens up like a zipper in one direction. The leading strand grows at its 3′ end as the fork opens. In the lagging strand the exposed 3′ end gets farther from the fork, and an unreplicated gap forms. How Is DNA Replicated? Synthesis of the lagging strand occurs in small, discontinuous stretches called Okazaki fragments. The final phosphodiester linkage between fragments is catalyzed by DNA ligase. How Is DNA Replicated? Eukaryote chromosomes have repetitive sequences at the ends called telomeres. In humans the sequence is TTAGGG, repeated about 2,500 times. Continuously dividing cells have telomerase, which catalyzes addition of lost telomeres. Telomerase is expressed in most cancer cells, and is important in their ability to keep dividing. It is a target for anti-cancer drugs. How Are Errors in DNA Repaired? DNA polymerases initially make many mistakes, and DNA can be damaged by chemicals, UV radiation, and other threats. Cells have three repair mechanisms: proofreading, mismatch repair, excision repair. How Does Information Flow from Genes to Proteins? Gene expression occurs in two steps: •Transcription: DNA sequence is copied to a complementary RNA sequence •Translation: RNA sequence is template for an amino acid sequence This model was proposed by Crick and Watson, and called “The central dogma of molecular biology.” How Does Information Flow from Genes to Proteins? The central dogma suggested that information flows from DNA to RNA to protein, which raised two questions: • How does genetic information get from the nucleus to the cytoplasm? • What is the relationship between a DNA sequence and an amino acid sequence? How Does Information Flow from Genes to Proteins? Three kinds of RNA are involved in gene expression: 1.Messenger RNA (mRNA) and transcription— one strand of DNA is copied to a complementary mRNA strand. In eukaryotes, the mRNA moves to the cytoplasm. 2.Ribosomal RNA (rRNA) and translation— ribosomes are protein synthesis factories made up of proteins and rRNA 3.Transfer RNA (tRNA)— can bind a specific amino acid, and recognize specific sequences in mRNA. How Is the Information Content in DNA Transcribed to Produce RNA? Transcription occurs in three phases: 1. Initiation: RNA polymerase binds to a DNA sequence called a promoter. Promoters tell the enzyme where to start and which strand of DNA to transcribe. The promoter has an initiation site where transcription begins. How Is the Information Content in DNA Transcribed to Produce RNA? 2. Elongation: RNA polymerase unwinds DNA about 10 base pairs at a time; reads template in 3′ to 5′ direction. The transcript is antiparallel to the DNA template strand. RNA polymerases do not proofread and correct mistakes. How Is the Information Content in DNA Transcribed to Produce RNA? 3. Termination: Specified by a specific DNA sequence. Mechanism in eukaryotes is not well understood. In bacteria, the transcript forms a loop and falls away from the DNA; or a helper protein binds to the transcript and causes it to detach from the DNA. How Is the Information Content in DNA Transcribed to Produce RNA? The genetic code specifies which amino acids will be used to build a protein. Codon: a sequence of three bases, something like a three-letter “word.” Each codon specifies a particular amino acid. How Is the Information Content in DNA Transcribed to Produce RNA? AUG is the start codon. Stop codons— UAA, UAG, and UGA. The genetic code is nearly universal, redundant (more codons than amino acids) but not ambiguous. How Is RNA Translated into Proteins? Transfer RNA (tRNA) links mRNA codons with specific amino acids. Each tRNA has three functions: • It binds to a specific enzyme that attaches it to only one amino acid: it is then “charged” • Binds to mRNA at a triplet called the anticodon, which is complementary to an mRNA codon • Interacts with ribosomes How Is RNA Translated into Proteins? tRNAs are charged by aminoacyl-tRNA synthetases. Each enzyme is specific for one amino acid and its corresponding tRNA. Translation occurs at a ribosome. It holds mRNA and charged tRNAs in the correct position to allow assembly of the polypeptide. Ribosomes can make any type of protein, they can be used over and over. Most cells have thousands of them. How Is RNA Translated into Proteins? Ribosomes have two subunits, large and small, held together non-covalently. In eukaryotes, the large subunit has three different molecules of ribosomal RNA (rRNA) and 49 different proteins in a precise pattern. The small subunit has one rRNA and 33 proteins. How Is RNA Translated into Proteins? A large subunit has three tRNA binding sites: • A (aminoacyl tRNA) site binds with anticodon of charged tRNA • P (peptidyl tRNA) site where tRNA adds its amino acid to the growing chain • E (exit) site where tRNA sits before being released from the ribosome How Is RNA Translated into Proteins? Translation occurs in three steps: 1. Initiation An initiation complex forms—a charged tRNA and small ribosomal subunit, both bound to mRNA. How Is RNA Translated into Proteins? 2. Elongation Another charged tRNA enters A site and the large subunit catalyzes two reactions: • Bond between tRNA in P site and its amino acid is broken • Peptide bond forms between that amino acid and the amino acid on tRNA in the A site How Is RNA Translated into Proteins? 3. Termination Translation ends when a stop codon enters the A site. Stop codons bind a protein release factor which hydrolyzes bond between the polypeptide and the tRNA in the P site. The polypeptide then separates from the ribosome. What Happens to Polypeptides after Translation? After translation, polypeptides may move into an organelle, or out of the cell. They are often modified by the addition of new chemical groups that affect their function. Polypeptide emerges from the ribosome and folds into its 3-D shape. It may contain a signal sequence indicating where in the cell it belongs. Example: a nuclear localization signal (NLS) -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val- What Happens to Polypeptides after Translation? What Happens to Polypeptides after Translation? Most polypeptides are modified after translation: • Proteolysis: Polypeptide is cut by proteases, (e.g., signal sequence is removed) • Glycosylation: Addition of sugars to form glycoproteins. The sugars can act as signals; others form membrane receptors • Phosphorylation: Addition of phosphate groups catalyzed by protein kinases Adapted from Life: The Science of Biology, Tenth Edition, Sinauer Associates, Sunderland, MA, 2014 Inc. All rights reserved