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Download Topic # 7: Nucleic Acids
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Topic # 7: Nucleic Acids I. DNA Structure and replication A. Semi-conservative replication of DNA 1. The replication of DNA is semiconservative and depends on the complementary bases pairing 2. When a cell prepares to divide, the two strands of the double helix separate 3. Each old strand serves as a template to build a new strand two new molecules form 4. The new strands form by adding nucleotides, one by one and linking them together I. DNA Structure and replication 5. Each new DNA strand is made up of half a new strand and half of an old strand semi-conservative 6. complementary base pairing a. When the strands are open, nucleotides are exposed b. Adenine will attract free Thymine Cytosine will attract free Guanine Thymine will attract free Adenine Guaning will attract free Cytosine c. Enzymes direct this process II. The Hershey Chase Experiment A. Alfred Hershey and Martha Chase 1. Wanted to determine if DNA or proteins were the genetic material 2. Used viruses to perform the experiments 3. Viruses are made up of proteins on the outside and DNA on the inside 4. The T4 bacteriophage was used for the research 5. They added a radioactive marker to the protein on the T4bacteriophage 6. No radioactivity in the resulting culture 7. They then added a radioactive marker to II. The Hershey Chase Experiment DNA of the T4 bacteriophage 8. The resulting culture contained the radioactivity III. DNA Structure and replication A. The Watson and Crick model suggested semi-conservative replication 1. DNA structure suggested a mechanism for DNA replication 2. Several lines of evidence came together to lead to the knowledge of the structure of DNA III. DNA structure and replication a. Linus Pauling: molecular modeling b. Rosalind Franklin: X-ray diffraction and careful examination of her photographs c. Erwin Chargaff: base composition studies 3. Watson and Crick a. First models had sugar-phosphate strands wrapped around each other b. nitrogen bases were facing outward c. Franklin countered that the nitrogen bases would face the center of the helix III. DNA structure and replication 4. Franklin’s X-Ray diffraction showed that the DNA helix was tightly packed 5. The tight packing could only occur if a purine paired with a pyrimidine and if their bases were upside down a. A-T, T-A b. C-G, G-C 6. This pairing suggests the mechanism by which DNA replication could occur B. The role of nucleosomes in DNA packing 1. Nucleosomes help to supercoil DNA 2. DNA is associated with proteins - histone III. DNA structure and replication 3. Histones package the DNA into structures called nucleosomes a. Central core of 8 histone molecules b. DNA wrapped around it c. An additional histone (the H1 histone) holds the DNA in place III. DNA structure and replication B. The leading strand and the lagging strand 1. DNA replication is continuous on the leading strand and discontinuous on the lagging strand 2. The two strands are antiparallel 3. The leading strand is synthesized continuously 4. The lagging strand is made in fragments moving away from the replication fork 5. These fragments are called Okazaki fragments III. DNA structure and replication C. Proteins involved in replication 1. DNA replication is carried out by a complex system of enzymes 2. DNA helicase unwinds the DNA at the replication fork 3. Topoisomerase releases the strain that develops ahead of the helicase 4. Single-stranded binding proteins keep the strands apart long enough to allow the template strand to be copied 5. RNA primer a. Many RNA primers are on the lagging III. DNA structure and replication strand, but only one is needed on the leading strand b. RNA primers are formed by RNA primase c. They are 10-12 nucleotides long d. The primers are sequenced according to the part of the strand where they are placed e. RNA primers are necessary to initiate the activity of DNA polymerase C. DNA polymerase 1. Covalently links the nucleotide to the 3’ III. DNA structure and replication a. Proof-reading b. Polymerization c. Removal of RNA primers as they are no longer needed D. The direction of replication 1. DNA polymerases can only add nucleotides to the 3’end of a primer 2. Prokaryotes have only one origin of replication 3. Eukaryotes have many different origins 4. Replication occurs in both directions away from the origin III. DNA structure and replication 5. The PO4-3 of the new DNA nucleotide is added to the 3’ carbon of the deoxyribose of the nucleotide at the end of the chain 6. Therefore, DNA replication occurs in a 5’3’ direction III. DNA structure and replication E. Non-coding regions of DNA have important functions 1. Some regions of DNA do not code for proteins but have other important functions 2. DNA is used as a guide for the production of polypeptides using the genetic code 3. not all of DNA codes for polypeptides 4. some code for production of tRNA or rRNA 5. some regulate gene expression 6. the majority of the eukaryotic genome is non-coding 7. repetitive sequences can be common III. DNA structure and replication 8. telomeres a. Repetitive sequences at the end of a chromosome b. Serves a protective function for the chromosome i. The enzymes that replicate DNA cannot continue replication all the way to the end of the chromosome ii. The DNA sacrifices the repetitive telomere portion each time the cell divides protects the genes IV. Transcription and gene expression A. Regulation of gene expression by proteins 1. Gene expression is regulated by proteins that bind to specific base sequences in DNA 2. Some proteins are always necessary for the survival of an organism 3. These are expressed in an unregulated fashion 4. Other proteins need to be produced at certain times and in certain amounts so their expression needs to be regulated 5. In prokaryotes, it happens as a result of IV. Transcription and gene expression environmental factors Ex. E. coli can only produce the enzyme needed to break down lactose after the disaccharide has been absorbed 6. In eukaryotes, genes are also regulated in response to environmental conditions as well 7. There are proteins that regulate transcription when they bind to DNA a. Enhancers b. Silencers c. Promoter-proximal elements nearer to the promoter and binding of proteins IV. Transcription and gene expression B. The impact of the environment on gene expression 1. The environment of a cell and of an organism has an impact on gene expression 2. “nature vs nuture” debate 3. Twin studies have been done to see what phenotypes are expressed and if they can be attributed to environment or to heredity 4. For some traits, the influence of the environment is unequivocal a. Skin pigmentation during sun exposure 5. embryonic development IV. Transcription and gene expression a. Embryos contain uneven distribution of chemicals called morphogens b. Concentration of morphogens affect gene expression c. Produce different fates for the embryonic cells i. Dependent on their position in the embryo d. Coat color in cats i. “C” gene codes for the enzyme tyrosinase – the 1st step in pigment production IV. Transcription and gene expression ii. “c” is a mutant allele that allows for normal pigment production at temps below body temperature C. Nucleosomes regulate transcription 1. Nucleosomes help to regulate transcription in eukaryotes 2. eukaryotic DNA is associated with histones 3. chemical modification of the tails of histones help to determine if a gene will be expressed or not a. Acetyl group c. Phosphate group b. Methyl group IV. Transcription and gene expression D. The direction of transcription 1. Transcription occurs in a 5’ to 3’ direction 2. Production of mRNA occurs in three stages a. Initiation b. Elongation c. Termination 3. Begins near a site in DNA called the promoter 4. Once binding of RNA polymerase occurs, the DNA is unwound by the RNA polymerase forming an open complex 5. RNA polymerase slides along the DNA IV. Transcription and gene expression 6. synthesizes a single strand of RNA An animation of the process… E. Post-transcriptional modification 1. Eukaryotic cells modify mRNA after transcription 2. Transcription occurs in the nucleus and modification afterward also occurs there before it leaves the nucleus 3. Introns are removed from the RNA introns intervening sequences…they intervene with the genetic sequence IV. Transcription and gene expression 4. Exons are left behind the expressed portion of the gene F. mRNA splicing 1. Splicing of mRNA increases the number of different proteins an organism can produce 2. alternative splicing is a process during gene expression whereby a single gene codes for multiple proteins 3. a particular exon may or may not be included in the final mRNA 4. So proteins translated from alternatively spliced mRNAs will differ in their IV. Transcription and gene expression 4. Exons are left behind the expressed portion of the gene F. mRNA splicing 1. Splicing of mRNA increases the number of different proteins an organism can produce 2. alternative splicing is a process during gene expression whereby a single gene codes for multiple proteins 3. a particular exon may or may not be included in the final mRNA 4. So proteins translated from alternatively spliced mRNAs will differ in their V. Translation A. The structure of the ribosome 1. The use of molecular visualization software to analyze the structure of eukaryotic ribosomes and a tRNA molecule 2. Ribosome structure includes a. proteins and ribosomal molecules (rRNA) b. two sub-units, one large and one small c. Three binding sites for tRNA on the surface of the ribosome – two tRNA molecules can bind at the same time to the same ribosome V. Translation surface of the ribosome e. Each ribosome has three tRNA binding sites i. E – exit site ii. P – peptidyl site iii. A – amino acyl site 3. All tRNA molecules have a. Sections that become double-stranded by base pairing, creating loops b. A triplet of bases called the anticodon which is part of a loop of seven unpaired bases V. Translation c. Two other loops d. The base sequence CCA at the 3’ end which forms a site for attaching an amino acid 4. tRNA-activating enzymes a. tRNA-activating enzymes illustrate enzyme-substrate specificity and the role of phosphorylation b. Each tRNA molecule is recognized by a tRNA-activating enzyme that attaches a specific amino acid to the tRNA, using ATP for energy V. Translation c. 20 different amino acids, so 20 different tRNA molecules (at least) d. The active site of the activating enzyme is specific to both the correct amino acid and the correct tRNA e. Energy from ATP is needed for the attachment of amino acids f. Once ATP and amino acid are attached to the active site, the amino acid is activated g. The activated amino acid is covalently attached to the tRNA V. Translation to the growing polypeptide B. Initiation of translation 1. Initiation of translation involves assembly of the components that carry out the process 2. to begin translation, mRNA binds to the small ribosomal subunit at an mRNA binding site 3. An initiator tRNA carrying methionine then binds at the start codon “AUG” 4. The large ribosomal subunit then bind to the small one 5. The initiator tRNA is in the P site V. Translation 6. The next codon signals another tRNA to bind 7. a peptide bond is formed between the amino acids in the P and the A site C. Elongation of the polypeptide 1. Synthesis of the polypeptide involves a repeated cycle of events 2. following initiation, elongation occurs through a series of repeated steps 3. the ribosome translocates three bases along the mRNA, moving the tRNA in the P site to the E site V. Translation 6. The next codon signals another tRNA to bind 7. a peptide bond is formed between the amino acids in the P and the A site C. Elongation of the polypeptide 1. Synthesis of the polypeptide involves a repeated cycle of events 2. following initiation, elongation occurs through a series of repeated steps 3. the ribosome translocates three bases along the mRNA, moving the tRNA in the P site to the E site V. Translation 4. this allows a tRNA with the appropriate anticodon to bind to the next codon 5. this vacates the A site 6. next amino acid can bind in the same way …on to the animations!!! D. Termination of translation 1. Disassembly of the components follows termination of translation 2. A stop codon is reached and the free polypeptide is released V. Translation 3. movement of translation is from the 5’ to 3’ end of the mRNA strand 4. Stop codons i. UAG, UAA, UGA ***SO!! Replication, Transcription and Translation ALL occur in a 5’ to 3’ direction” *** E. Free ribosomes 1. Free ribosomes synthesize proteins for use primarily in the cell 2. these are proteins destined for use in the cytoplasm, mitochondria and chloroplasts V. Translation F. Bound ribosomes 1. Bound ribosomes synthesize proteins primarily for secretion or for use in lysosomes 2. proteins must be sorted so they end up in their correct location 3. Proteins that are destined for use in the ER, the Golgi apparatus, lysosomes, the plasma membrane or outside the cell are synthesized by ribosomes bound to the ER G. The coupling of transcription and translation in prokaryotes 1. Translation can occur immediately after V. Translation transcription in prokaryotes due to the absence of a nuclear membrane 2. in eukaryotes, cellular functions are compartmentalized 3. in prokaryotes, they are NOT 4. once transcription is complete in eukaryotes, the transcript is modified before exiting the nucleus 5. In prokaryotes, as soon as the mRNA is transcribed, translation begins H. Primary Structure 1. The sequence and number of amino acids V. Translation the polypeptide is the primary structure 2. a chain of amino acids is called a polypeptide 3. The sequence is the primary structure I. Secondary structure 1. the secondary structure is the formation of alpha helices and beta pleated sheets stabilized by hydrogen bonding 2. yeah…there’s a picture on the next slide J. Tertiary structure 1. the tertiary structure is the further folding of the polypeptide stabilized by interactions Bonds form between the carboxyl and the amino group for both parts of the secondary structure V. Translation 2. three dimensional shape of the protein 3. it’s the result of the interaction of R-groups with one another 4. Several types a. Positively charged R-groups interact with negatively charged R-groups b. Hydrophobic amino acids will orient themselves toward the center of the polypeptide to avoid contact with water while hydrophilic amino acids will orient themselves outward c. Polar R groups will form hydrogen V. Translation with other polar R groups d. The R-group of the amino acid cysteine can form a covalent bond with the Rgroup of another cysteine forming what is called a disulfide bridge K. Quartenary structure 1. The quartenary structure exists in proteins with more than one polypeptide chain 2. Insulin is formed from two polypeptide chains 3. Hemoglobin is formed by four polypeptide chains! FOUR! V. Translation 4. The biological activity is related to its primary, secondary, tertiary and quaternary structure 5. certain treatments can alter the structure permanently and change how the protein functions denaturing the protein i. pH changes ii. Temperature extremes