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University of Ottawa
Kathleen Gilmour

UNIT 1 – STRUCTURE DNA vs RNA -DNA has thymine (which has a methyl group), while RNA has Uracil (no methyl group) -DNA has a ribose (H), RNA has a deoxyribose (OH). Function of protein in DNA: to neutralize the negative charge caused by a negative phosphate backbone. Frederick Griffith Experiment He worked with two forms of Streptococcus pneumoniae. The smooth strain—S—has a polysaccharide capsule surrounding each cell, and is virulent. The rough strain—R—does not have a polysaccharide capsule , and is nonvirulent. Therefore the capsule is responsible for the virulence of the S strain. -killed S bacteria and injected into mice, they lived -heat-killed S and live R forms mixed together and injected into mice, the mice died. Therefore the live R forms must have been transformed into S forms by acquiring the ability to make a polysaccharide capsule. Later it was found out by Oswald Avery that DNA was the transforming agent. He used the same bacteria as Griffith, and mixed heat-killed S forms with R form. Then, when he killed off protein or RNA in the cells, the R form was still being transformed into S form. This meant that DNA was the transforming agent. Hershey and Chase They worked with bacteriophages (viruses that infect cells) because a virus has DNA/RNA inside its protein coat. They tagged DNA and the protein separately with radioactive labels. - DNA entered the cells - Protein coat remained on outside Therefore, DNA was the transforming agent. Watson and Crick DNA contains 4 nucleotides (ATGC) DNA is made of 5 carbon sugar (deoxyribose) + phosphate group +1 of the four nitrogenous bases – ATGC -A and G are purines (2 rings) -T and C are pyrimidines (1 ring) -purines bind with pyrimidines (A-T, G-C) via hydrogen bonding -Erwin Chargaff (A=T, G=C) - DNA has a sugar-phosphate backbone because the nucleotides in one strand (polynucleotide) are joined by phosphates. -5’ end = phosphate group, 3’end = hydroxyl group; strands are antiparallel - Rosalind Franklin used x-ray diffraction to determine that the DNA has a helical structure. She could also tell the the DNA is 2nm in diameter and patterns of atom repeats occurred every 0.34 nm (1 base pair) and 3.4 (1 full helix turn) nm in length.However, she did not have crystals to work with which would have made her results more accurate. Meselson and Stahl - DNA is semiconservative Procedure: Used “heavy” nitrogen isotopes to label the parent strands of the DNA. They transferred the N-15 labeled DNA into a “light” nitrogen isotope medium (N-13) so that the newly synthesized DNA strands would incorporate it. UNIT 2 ­1 DNA Replication DNA Polymerase assembles the complementary strands of DNA from individual nucleotides. More than one kind of DNA polymerase is required for DNA replication in both eukaryotes and prokaryotes. Nucleoside triphosphates (nitrogenous base+deoxyribose sugar+3phosphates=dATP, dGTP, dCTP,dTTP) are substrates for DNA polymerase. DNA polymerase can only add nucleotides to the 3’end, therefore the new strand is synthesized in the 5’ 3’ but is read from 3’5’. DNA helicase unwinds the DNA by using energy from ATP hydrolysis. Single-stranded binding proteins stabilize the DNA for replication so that the unwound DNA does not twist back together. Topoisomerase prevents the DNA from overtwisting Primase lays on the primer so that DNA polymerase can begin synthesis. Later, the primer is removed and replaced by DNA. Leading Strand is synthesized continuously in the direction of unwinding. Lagging Strand is synthesized discontinuously in “okazaki fragments” DNA Polymerase I removes the RNA primer DNA Ligase joins the space between okazaki fragments and closes the nicks. When the initial primer is removed, a gap is left which is not replaced by DNA. This means that with each replication of the DNA, the complementary strand becomes shorter and shorter and eventually this can become lethal for the cell. In most cases, there is a buffer of noncoding DNA at the ends of the strand so as to protect it – called Telomeres (TTAGGG in humans). The enzyme Telomerase helps rebuild the telomeres. Telomerase is not active in all cells. It is active in sperm and egg cells, but not in mature cells. This means the cell eventually dies and is replaced by another one. Therefore, the shortening of telomeres = aging, and constantly active telomeres = cancer. Replication Origins are regions where replication begins. The replication origins are only activated once during S phase as to prevent the same parts of a gene from being replicated multiple times. Proofreading mechanism is a mechanism that proofreads the DNA polymerase’s mistakes. When a mispaired nucleotide is added, the DNA polymerase cannot move forward so it reverses and replaces the nucleotide with the correct one. Any base-pair mismatches that remain after the DNA polymerase has proofread itself are repaired by the DNA repair mechanism. The repair enzymes move along newly replicated DNA molecules, “scanning” the DNA for distortions in the newly synthesized nucleotide chain. If the enzymes encounter a distortion, they remove a portion of the new chain, including the mismatched nucleotides. The gap left by the removal (step 2) is then filled by a DNA polymerase, using the template strand as a guide (step 3). The repair is completed by a DNA ligase, which seals the nucleotide chain into a continuous DNA molecule (step 4) UNIT 2-2 Transcription Transcription is the mechanism by which the information encoded in DNAis made into a complementary RNAcopy. Transcription is the first step in a process whereby particular genes are expressed in any given cell at a given time. Some of those genes are protein-coding genes that encode mRNAs to be translated; others are non–protein-coding genes that encode RNAs that are never translated, such as ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and small nuclear RNAs (snRNAs). → RNApolymerase creates the complementary RNAsequence following complementary base pairing → The RNAtranscribed from a gene encoding a polypeptide is called messenger RNA(mRNA). Process Prokaryote Eukaryote Transcription and Translation Occur at the same time. No Separate processes pre-mRNA (transcription in nucleos, translation in cytoplasm) Pre-mRNA Initiator codon AUG AUG Promoter In prokaryotes, the promoters The promoters of protein- are immediately upstream of coding genes are immediately where transcription initiates upstream of the transcription start point and are typically more complex than in prokaryotes. Transcription factors bind to the TATA box and recruit RNApolymerase # RNApolymerases Since all the other types of different polymerases for genes in prokaryotes (for transcribing different types of example, tRNAand rRNA genes. RNApolymerase II genes) have similar promoters, transcribes protein-coding the same RNApolymerase genes. RNApolymerases I and complex can transcribe them III transcribe genes for non- all. protein-coding RNAs. Termination are two types of specific DNA no equivalent “transcription sequences called terminators terminator” sequences. The 3′end that signal the end of of the gene is a sequence that is transcription of the gene. Both to be transcribed into the pre- types of terminator sequences mRNA. Proteins bind to act after they are transcribed. Inthis polyadenylation signal and the first case, the terminator cleave the pre-mRNA at that sequence on the mRNAbase- point. This signals the RNA pairs with itself to form a polymerase to stop transcription. “hairpin.” In the second case, a protein binds to the terminator sequence on the mRNA. Both of these mechanisms trigger the termination of transcription and the release of the RNAand RNA polymerase from the template. → Genetic code – nucleotide information that specifies that aa sequence of a polypeptide → Codon – three bases that “code” for a word. By convention, scientists write the codons in the 5′→ 3′ direction as they appear in mRNAs. o Initiator codon – AUG (methionine) is the first codon translated in any mRNA o One of these codons,AUG, specifies the amino acid methionine. It is the first codon translated in any mRNAin both prokaryotes and eukaryotes. o Stop codon (nonsense or termination codons) – (UAA, UAG, UGA) doesn’t specify an aa and that act as indicators of the end of the polypeptide sequence Only two amino acids, methionine and tryptophan, are specified by a single codon. All the rest are represented by at least two, some by as many as six. In other words, there are many synonyms in the nucleic acid code, a feature known as degeneracy (or redundancy). For example, UGU and UGC both specify cysteine, whereas CCU, CCC, CCA, and CCG all specify proline. → There is only one reading frame for each mRNA(have to start at correct initiator codon and read 3 bases at a time) → The code is also universal. With a few exceptions, the same codons specify the same amino acids in all living organisms, and also in viruses. SET RULES OF TRANSCRIPTION: • in a given gene, only one of the two DNAnucleotide strands acts as a template for synthesis of a complementary copy, instead of both, as in replication. • only a relatively small part of a DNAmolecule—the sequence encoding a single gene— serves as a template, rather than all of both strands, as in DNAreplication. • RNApolymerases catalyze the assembly of nucleotides into an RNAstrand, rather than the DNA polymerases that catalyze replication. • the RNAmolecules resulting from transcription are single polynucleotide chains, not double ones, as in DNAreplication. STEPS IN TRANSCRIPTION 1. helicase unwinds the DNA 2. DNApolymerize can start synthesis WITHOUT a primer, by binding to a promoter region and synthesizing in the 5’-3’direction while reading the strand 3’-5’. 3. The part of the gene that is to be transcribed into RNAis called the transcription unit. 4. DNApolymerase reaches a terminating sequence and the completed RNAtranscript and RNApolymerase are released from the DNA Pre-mRNAhas exons and introns, while mRNAonly has exons. Introns = noncoding Exons = coding Aeukaryotic protein-coding gene is typically transcribed into a precursor-mRNA (pre-mRNA) that must be processed in the nucleus to produce translatable mRNA. The mature mRNAexits the nucleus and is translated in the cytoplasm. MODIFICATIONS At the 5′ end of the pre-mRNAis the 5′ cap, consisting of a guanine-containing nucleotide that is reversed so that its 3′-OH group faces the beginning rather than the end of the molecule.Acapping enzyme adds the 5′cap to the pre-mRNA. The cap, which is connected to the rest of the chain by three phosphate groups, remains when pre-mRNAis processed to mRNA. The cap functions as the initial attachment site for mRNAs to ribosomes to allow translation. The termination of transcription of a eukaryotic protein-coding gene is different from that of a prokaryotic gene in that there is no terminator sequence at the end of the gene in the DNA. Instead, at the 3′end of the gene is a sequence that is to be transcribed into the pre-mRNA. Proteins bind to this polyadenylation signal and cleave the pre-mRNA at that point. This signals the RNApolymerase to stop transcription. Then the enzyme poly(A) polymerase adds a chain of 50 to 250 adenine nucleotides, one nucleotide at a time, to the newly created 3′end of the pre-mRNA. The string of adenine nucleotides, called the poly(A) tail, enables the mRNA produced from the pre-mRNAto be translated efficiently and protects it from attack by RNA-digesting enzymes in the cytoplasm. SPLICING Aprocess called mRNAsplicing, which occurs in the nucleus, removes introns from pre-mRNAs and joins exons together. How does this occur? mRNAsplicing occurs in a spliceosome, a complex formed between the pre-mRNA and a handful of small ribonucleoprotein particles (snRNPs) Alternative Splicing. The removal of introns from a given gene is not absolute. That is, in certain tissues, or under certain environmental conditions, exons may be joined in different combinations to produce different mRNAs from a single DNAgene sequence. The mechanism, called alternative splicing, greatly increases the number and variety of proteins encoded in the cell nucleus without increasing the size of the genome. This process produces different mRNAs fro the same pre-mRNAby removing different combinations of exons along with the introns. Exon Shuffling. different exons either within a gene or between two nonallelic genes are occasionally mixed. Evolution of new proteins by this mechanism would produce changes much more quickly than by changes in individual amino acids at random points. UNIT 2-3 TRANSLATION Translation is the use of the information encoded in the RNAto assemble amino acids into a polypeptide. Prokaryotes Eukaryoes Translation Occurs all over cell Occurs mostly in cytoplasm mRNAavailability Available right away because it’s Must first exit the nucleus not confined to a nucleus Translation initiation the rRNAof the ribosomal rRNAscans the mRNAfrom 5’– subunit finds the region with the 3’searching for the start codon start codon directly by base pairing with a specific ribosome binding site on the mRNAjust upstream of the start codon. The large ribosomal subunit then binds to the small one to complete the ribosome. Elongation 15-20 times/sec 1-3 times/sec → In translation, an mRNAassociates with a ribosome, a particle on which amino acids are linked into polypeptide chains. As the ribosome moves along the mRNA, the amino acids specified by the mRNAare brought by tRNAs and joined one by one to form the polypeptide encoded by the gene. tRNASTRUCTURE → All tRNAs can base-pair with themselves to wind into four double-helical segments, forming a cloverleaf pattern in two dimensions.At the tip of one of the double-helical segments is the anticodon, the three-nucleotide segment that pairs with a codon in mRNAs.At the other end of the cloverleaf is a double-helical segment that links to the amino acid corresponding to the anticodon. → wobble hypothesis proposed that the complete set of 61 sense codons can be read by fewer than 61 distinct tRNAs because of the particular pairing properties of the bases in the anticodons. That is, the pairing of the anticodon with the first two nucleotides of the codon is always precise, but the anticodon has more flexibility in pairing with the third nucleotide of the codon. → The process of adding an amino acid to a tRNAis called aminoacylation(literally, the addition of an amino acid) or charging (because the process adds free energy as the amino acid–tRNA combinations are formed). The final tRNAwith an amino acid is called an aminoacyl–tRNA. Enzymes called aminoacyl–tRNAsynthetases catalyze aminoacylation RIBOSOMES → Ribosomes Are rRNA–Protein Complexes that Work as Automated ProteinAssembly Machines. They translate the mRNAinto a sequence of amino acids. → Chloroplasts and mitochondria each have their own ribosomes in addition to those in the cytoplasm. Structure of ribosomes: → Afinished ribosome is made up of two parts of dissimilar size, called the large and small ribosomal subunits → The Asite (aminoacyl site) is where the incoming aminoacyl–tRNA(carrying the next amino acid to be added to the polypeptide chain) binds to the mRNA. The Psite (peptidyl site) is where the tRNAcarrying the growing polypeptide chain is bound. The E site (exit site) is where an exiting tRNAbinds as it leaves the ribosome. STEPS OF TRANSLATION 1. Initiation - assembly of all the translation components on the start codon of the mRNA. In translation initiation, a large and small ribosomal subunit associates with an mRNA molecule and the first aminoacyl–tRNAof the new protein chain becomes bound to theAUG start codon. This is now called the initiator tRNA with an anticodon to the methionine-specifyingAUG start codon. Each step in translation initiation is aided by proteins called initiation factors. → The complex binds to the mRNAat the 5′cap and then moves along the mRNA—a process called scanning—until it reaches the firstAUG codon (step 2). This is the start codon, and it is recognized by the Met–tRNA's anticodon. The large ribosomal subunit then binds, completing the ribosome (step 3).At the end of initiation, the initiator Met– tRNAis in the P site. 2. Elongation - Elongation involves reading the string of codons in the mRNAone at a time while assembling the specified amino acids into a polypeptide. → The P site, with one exception, can only bind to a peptidyl–tRNA—a tRNAlinked to a growing polypeptide chain containing two or more amino acids. The exception is the initiator tRNA, which is recognized by the P site as a peptidyl–tRNAeven though it carries only a single amino acid, methionine. TheAsite can bind only to an aminoacyl– tRNA. The tRNApreviously in the P site binds to the E
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