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DNA, RNA and Protein Synthesis By the end of this lesson, I can Relate how Griffith’s bacterial experiments showed that a hereditary factor was involved in transformation. Summarize how Avery’s experiments led his group to conclude that DNA is responsible for transformation in bacteria. Describe how Hershey and Chase’s experiment lead to the conclusion that DNA, not proteins, is the hereditary molecule in viruses. Evaluate the contributions of Franklin and Wilkins in helping Watson and Crick discover DNA’s double helix structure. Describe the three parts of a nucleotide Summarize the role of the covalent and hydrogen bonds in the structure of DNA. Relate the role of the base-pairing to the structure of DNA. Summarize the process of DNA replication. Identify the role of enzymes in the replication of DNA Describe how complimentary base pairing guides DNA replication. Compare the number of replication forms in prokaryotic and eukaryotic cells during DNA replication. Describe how errors are corrected during DNA replication. Outline the flow of genetic information in cells from DNA to protein. Compare the structure of RNA with that of DNA. Describe the importance of the genetic code. Compare the role of mRNA, rRNA, and tRNA in translation. Identify the importance of learning the human genome. Vocabulary Virulent Transformation Bacteriophage Nucleotide Deoxyribose Nitrogenous base Purine Pyrimidine Base-pairing rules Complementary base pair Base sequence DNA replication Helicase Replication fork DNA polymerase Semi-conservative replication Mutation Ribonucleic acid (RNA) Transcription Translation Protein synthesis Ribose Messanger RNA (mRNA) Ribosomal RNA (rRNA) Transfer RNA (tRNA) RNA polymerase Promoter Termination signal Genetic code Codon Anticodon genome 1. Discovery of DNA a. Griffith’s Experiments Fredrick Griffith was studying Streptococcus pnuemoniae (S. pnuemoniae) Some types or strains of this bacterium cause the lung disease pneumonia in mammals. 1 DNA, RNA and Protein Synthesis Griffith was trying to develop a vaccine against a disease causing or virulent strain of the bacterium Bacteria are surrounded by a polysaccharide that protects it from an organism’s defense mechanisms The virulent bacteria grow as smooth edged colonies. This bacterium does cause pneumonia. The second strain of S. pneumonia does not cause pneumonia and lacks a capsule. It is called R strain because it grows in rough edged colonies. Transformation is the transfer of genetic material from one cell to another cell or from one organism to another organism. 1. This is the basis of Griffith’s experiment below b. Avery’s Experiments Oswald Avery set out to test whether the transforming agent in Griffith’s experiment was protein, RNA, or DNA. 1. Scientists used enzymes to separately destroy each of the 3 molecules in heat killed S cells. 2. Used a protease enzyme to destroy protein in heat-killed cells in the first experiment. 3. An enzyme called RNase to destroy the RNA in the second experiment 4. And an enzyme called DNase to destroy the DNA in the third experiment. 5. They then separately mixed the three experimental batches of heatkilled S cells with live R cells and injected mice with the mixtures. Avery found that the cells missing protein and RNA were able to transform R cells into S cells and kill the mice. But cells missing DNA did not transform R cells into S cells and the mouse survived. Avery concluded that DNA is responsible for transformation in bacteria. 2 DNA, RNA and Protein Synthesis c. Hershey-Chase Experiment Martha Chase and Alfred Hershey set out to test whether DNA or proteins was the hereditary material viruses transfer when viruses enter a bacterium These viruses are called bacteriophages or just plain phages. 1. Hershey and Chase used radioactive sulfur (35S) to label the protein and radioactive phosphor (32P) to label DNA in the phage. 2. They then allowed protein labeled and DNA labeled phage to separately infect Escherichia coli (E. coli) bacteria. 3. They then removed the phage coats from the cells in a blender 4. They then used a centrifuge they were able to separate the phage from the E. coli. a. Found that all of the viral DNA and little of the protein had entered the E. coli cells. b. Concluded that DNA is the hereditary molecule in viruses. 2. DNA Structure a. DNA Double Helix In 1950s a James Watson teamed up with Francis Crick to try and determine the structure of DNA. By 1953 they had put together a model for the structure of DNA. They proposed that DNA is made up of 2 chains that wrap around each other in the shape of a double helix, similar to that of a winding spiral staircase. 1. Their final model was correct and was remarkable because it explained how DNA could replicate. 3 DNA, RNA and Protein Synthesis They relied on other scientists’ work to develop their DNA model. 1. Part of that work was X-ray diffraction photographs of DNA crystals 2. These photographs and crystals were produced by Rosalind Franklin and Maurice Wilkins. 3. In 1962 Watson, Crick and Wilkins received the Nobel Prize in Medicine for their work on DNA. Franklin died in 1958 and could not be named on the award. b. DNA Nucleotides DNA is a nucleic acid made of 2 long chains or strands of repeating subunits called nucleotides. Each nucleotide consists of 3 parts 1. A 5 carbon sugar, a phosphate group, and a nitrogenous base. 4 DNA, RNA and Protein Synthesis a. 5 carbon ring is called deoxyribose b. The Phosphate group consists of a phosphorus atom bonded to 4 oxygen atoms. c. Nitrogenous base contains nitrogen atoms and carbon atoms and is a base (accepted hydrogen ions) Bonds hold DNA together 1. The nitrogenous bases on one strand of DNA face and form bonds called hydrogen bonds with the bases on the other strand. 2. Nitrogen bases are bonded together by 2 or 3 hydrogen bonds. a. They for the steps of the staircase. 3. The base pairs are uniform in width because in each pair one base has a 2 ring structure and the other has a single ring structure. 5 DNA, RNA and Protein Synthesis Nitrogenous Bases 1. Purines a. Ademine i. Nitrogenous bases that have a double ring of carbon and nitrogen atoms 1. Adenine and Guanine b. Pyrimidines i. Nitrogenous bases that have a single ring of carbon and nitrogen atoms 1. Cytosine and Thymine 6 DNA, RNA and Protein Synthesis c. Complementary Bases In 1949, Erwin Chargaff observed that the percentage of adenine equals the percentage of thymine and the percentage of cytosine and guanine are also equal to each other in the DNA of a variety of organisms. 1. This observation was key to understanding the structure of DNA because it meant that bases pair by base-pairing rules. a. In DNA cytosine on one strand pairs with guanine on the opposite strand. b. Thymine and Adenine pair up together. c. These are known as complementary base pairs. i. Each complementary base pair consists of one double ringed purine and a single ringed pyrimidine. 2. Due to base pairing rules, the order of nitrogenous base pairs on one strand is complementary to the order of bases on the other strand. a. For example: ATTC on one strand would have a complementary sequence of TAAG i. This is known as base sequence. 7 DNA, RNA and Protein Synthesis 3. DNA Replication a. How DNA Replication Occurs DNA Replication is the process by which DNA is copied in a cell before a cell divides by mitosis, meiosis, or binary fission. During replication the nucleotides strands of the original double helix separate along the strands. Strands are complementary and each strand serves as a template to make a new complementary strand. After replication the 2 identical stranded DNA molecule separates and move to the new cells forming during cell division. 1. Steps of DNA Replication. a. Step 1 i. Enzymes called helicases separate the DNA Strand. 1. Helicase moves along the DNA strand and breaks the hydrogen bonds between complimentary bases. 2. This action allows the 2 DNA strands of the double helix to separate from each other. 3. The Y-shaped region that results when the two strands separates is called a replication fork. b. Step 2 i. DNA polymerases add complementary nucleotides 1. DNA polymerases adds complementary nucleotides that are floating freely inside the nucleus. 2. As the nucleotides on the newly forming strands are added, covalent bonds form between the adjacent nucleotides. 3. Covalent bonds form between the deoxyrobose sugar on the nucleotide and the phosphate group of the next nucleotide. 4. Hydrogen bonds form between the complementary bases on the original and the new strand. c. Step 3 i. DNA polymerase finishes 1. DNA polymerase finishes replication of the DNA and fall off. 2. Results in 2 separate and identical DNA strand that are ready to move to new cells during cell division. 8 DNA, RNA and Protein Synthesis 3. Each strand contains 1 old DNA strand and 1 new DNA strand. a. This is known as semi-conservative replication because each new DNA has CONSERVED one of the 2 original DNA strands. 2. Action at the Replication Fork i. DNA synthesis occurs in different directions on each strand. ii. As the replication fork moves along the original DNA, synthesis of the one strand follows the movement of the replication fork. iii. Synthesis on the other strand moves in the opposite direction, away from the replication fork, leaving gaps in the newly synthesized DNA strand. iv. These gaps are joined together by an enzyme called DNA ligase. 3. Prokaryotic and Eukarytoic replication a. Prokaryotic cells i. These have circular chromosome. ii. Replication begins at one pace along the chromosome. iii. 2 replication forks are formed and proceed in opposite directions (like 2 zippers opening in opposite directions. Replication continues until they meet and the entire DNA is copied. b. Eukaryotic cells i. Chromosomes long but not circular. 9 DNA, RNA and Protein Synthesis ii. DNA polymerase adds 50 nucleotides per second. At this rate it would take 53 days to replicate the largest human chromosome. 1. Replication begins in many points or origins along the DNA. 2. 2 replication forks move in opposite directions. 3. Only simultaneous replication along chromosomes could allow for rapid replication of DNA. b. Replication Errors DNA replication usually occurs with great accuracy. Only about one error occurs for every billion paired nucleotides added. DNA polymerase has repair functions that “proofread” DNA When a mistake occurs in replication the base sequence of the newly formed DNA differs from the base sequence of the original DNA. A change in nucleotide sequence is called a mutation. 1. Mutations can have serious effects on the function of important genes and can disrupt cell function. 4. PROTEIN SYNTHESIS a. FLOW OF GENETIC INFORMATION A gene is a segment of DNA that is located on an autosome (chromosome) and it codes for a hereditary character. 1. Gene determines hair color a. Gene directs the making of the protein called melanin (a pigment) in hair follicle cells through an intermediate – the nucleic acid called ribonucleic acid or RNA b. Below summarizes the flow of genetic information in a eukaryotic cell. i. During transcription DNA acts as a template for the synthesis of RNA. ii. RNA directs the assembly of proteins iii. Forming protein based on information in DNA and carried out by RNA is protein synthesis or gene expression. iv. This central concept is symbolized as: 1. DNA => RNA => Protein v. Proteins do important work in cells: 1. Protect the body against infections 2. Carry oxygen in red blood cells 10 DNA, RNA and Protein Synthesis b. RNA STRUCTURE AND FUNCTION Like DNA, RNA is a nucleic acid made of nucleotides However as shown below RNA differs from DNA in four basic ways a. RNA contains the sugar ribose, not deoxyribose found in DNA. b. RNA contains the nitrogenous base URACIL instead of Thymine found in DNA. c. RNA is usually single stranded rather that a double helix i. However, within in a single stranded RNA molecule some regions fold to form short double stranded sections. ii. In the double stranded regions guanine forms base pairs with cytosine and URACIL forms base pair with adenine d. RNA is usually much shorter than DNA 11 DNA, RNA and Protein Synthesis Types of RNA 1. mRNA a. messenger RNA i. a single stranded RNA molecule that carries the instructions from a gene to make a protein. ii. In eukaryotic cells, mRNA carries the genetic “message” from DNA in the nucleus to the ribosomes in the cytosol. 2. rRNA a. ribosomal RNA i. is part of the structure of ribosomes. ii. Ribosomes are organelles that carry out protein synthesis. iii. Ribosomes are mad of mRNA and other proteins. 12 DNA, RNA and Protein Synthesis 3. tRNA a. transfer RNA i. transfers amino acids to the ribosome to make a protein c. TRANSCRIPTION The process by which the genetic instructions in a specific gene are transcribed or “rewritten” into an RNA molecule. Takes place in the nucleus of eukayriotic cells and in the DNA containing region in the cytoplasm of prokaryotic cells. 1. Step 1 13 DNA, RNA and Protein Synthesis a. RNA polymerase, an enzyme that catalyzes the formation of RNA on a DNA template, binds to a promoter. i. A promoter is a specific nucleotide sequence of DNA where RNA polymerase binds and initiates transcription. ii. After RNA polymerase binds to the promoter, the DNA strands unwind and separate. 2. Step 2 a. RNA polymerase adds free RNA nucleotides that are complementary to the nucleotides on one of the DNA strands. b. As in replication complementary base pairing determines the nucleotide sequence in the newly formed RNA. i. If the bases on the DNA strand was ATCGAC, the bases on the RNA strand would be UAGCUG ii. Unlike DNA replication transcription uses only a specific region (a gene) on one of the two DNA strands to serve as the template. c. As RNA polymerase moves past the separated DNA strand rewinds. 3. Step 3 a. During this step RNA polymerase reaches a terminal signal i. Terminal signals are a specific sequence of nucleotides that marks the end of a gene. b. Upon reaching this stop signal, RNA polymerase releases both the DNA and the newly formed RNA. c. The RNA made during this transcription can be one of many types including mRNA, tRNA, or rRNA. d. This newly formed RNA can now do its job within the cell and the RNA polymerase can begin transcribing another gene. 14 DNA, RNA and Protein Synthesis 15 DNA, RNA and Protein Synthesis d. THE GENETIC CODE During the next process of gene expression , amino acids are assembled based on instructions encoded in the sequence of nucleotides in the mRNA 1. The genetic code is the term for the rules that relate how a sequence of nitrogenous bases in nucleotides corresponds to a particular amino acid. 2. In the code 3 adjacent nucleotides (“letters”) in mRNA specify an amino acid (“word”) in a polypeptide. a. Each 3-nucleotide sequence in mRNA that encodes an amino acid or signals a start or stop signal is called a codon. b. The figure below lists 64 mRNA codons and the corresponding amino acid they encode for in most organisms. i. For example the codon GCU specifies the amino acid alanine in the genetic code. ii. This ode is nearly universal to all life forms on Earth and supports the notion that all organisms share an ancient common ancestor. c. Some amino acids are encoded by 2, 3, or more different codons. These codon often differ from one another by just one nucleotide. 16 DNA, RNA and Protein Synthesis e. TRANSLATION Although the instructions for creating a protein are copied from DNA to mRNA, all three major types of RNA are involved in translation Translation is the making of proteins 1. Protein structure a. Every protein is composed of one or more polypeptides. b. Polypeptides are chains of amino acids linked together by peptide bonds. c. 20 different amino acids found in the peptides of living things. d. Each polypeptide chain may consist of hundreds or thousands of the 20 different amino acids. i. They are arranged in a sequence specific to each protein. ii. The amino acid sequence determines how the polypeptides will twist and fold into 3D structure of the protein iii. The shape of the protein is critical to the function of the protein. 2. Steps of translation a. Step 1 i. 2 ribosomal RNA subunits, tRNA and mRNA join together 1. Enzymes first attach a specific amino acid to the end of each tRNA according to the genetic code. 17 DNA, RNA and Protein Synthesis 2. The other end of each tRNA contains the anticodon, 3 nuleotides on the RNA that are complementary to the sequence of a codon I mRNA. 3. A tRNA carring the amino acid methionine at one end and the anticdon UAC at other end pairs with the start codon AUG on the mRNA. 4. The first amino acid in nearly all polypeptides is methionine, but this amino acid maybe a removed later b. Step 2 i. The polypeptide chain is put together ii. tRNA carrying the appropriate amino acid pairs its anticodon with the second codon in the mRNA iii. the ribosome then detaches methionine from the first tRNA, and a peptide bond forms between methionine and the second amino acid. iv. The first tRNA then exits the ribosome. The ribosome then moves a distance of one codon along the nRNA c. Step 3 i. The polypeptide chain continues to grow as the mRNA moves along the ribosome ii. A new tRNA moves in, carrying an amino acid for the mRNA codon iii. The growing polypeptide chain moves from one tRNA to the amino acid attached to the next tRNA. iv. Chain grows one step a time d. Step 4 i. The ribosome reaches the stop codon e. Step 5 i. The components of translation come apart. ii. The tRNA leaves the ribosome and the ribosome moves away from the nRNA. iii. The process starts all over again made 18 DNA, RNA and Protein Synthesis f. Comparing and Contrasting Translation and Trancrption. 19