BMB 462 Lecture 27 Outline of Last Lecture I. Review of Nucleotide Excision RepairII. Direct RepairIII. Recombinational RepairIV. Error-Prone Repair Mechanismsa. Nonhomologous End Joining (NHEJ)b. SOS repairV. Damage that must be Repaired in the cellVI. DNA Recombinationa. Homologous Recombinationb. Site Specific RecombinationOutline of Current Lecture I. Overview of the Central Dogma of Molecular BiologyII. Differences between DNA and RNAIII. Structure of RNAa. Primaryb. Secondaryc. Tertiaryd. Non-Watson-Crick base pairingIV. RibozymesV. RNA genomesCurrent LectureConcepts to remembers from previous courses/lectures:-I. Overview of the Central Dogma of Molecular Biologya. Information flows from DNA, the storehouse of information. When making proteins, the DNA is then transcribed to mRNA. That gets translated on ribosomes into a polypeptide form that gets folded into its final conformation.II. Comparing DNA and RNAThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.a. Both are composed of a sugar-phosphate backbone with bases attached. b. DNA has thymine while RNA has uracil. (The difference between these 2 bases is the methyl group found on the thymine)i. Adding that methyl group takes a little more energy, so it's more expensive for the cell to make thymine. But because deamination of cytosine results in uracil, if the DNA used uracil as well, the cell would have no way of distinguishing replication errors. On the other hand, the RNA doesn't have to be repaired because they are transient molecules in the cell. The DNA is the storehouse and the source of information and must be preserved.1. There are even certain RNAs that are intentionally deaminated to change its function (This is called RNA editing)c. The deoxyribose in DNA has a hydrogen atom on the 2' Carbon. RNA's ribose has a hydroxyl group at that position. That hydroxyl group is important in chemical reactions; in vitro, it makes RNA less stable in alkaline conditions. In the presenceof hydroxyl groups, the 2' hydroxyl becomes more reactive, causing the phosphodiester bond to break. This quality can be used in research to break down the RNA strands so they are easier to separate from DNA.d. In vivo, the RNA is less stable because it is susceptible to attack from ribonucleases (RNases). RNA is able to form secondary and tertiary structures, though, which protect the strands from attack and make it more stable.III. Structure of RNAa. Primaryi. RNA's primary structure forms a right-handed helix due to base stacking. You can determine it's a right-handed helix due to the fact that the molecule is spiraling around the axis in a counterclockwise direction.ii. Once the primary structure is established, secondary structure can form. If there are complementary sequences running in an antiparallel fashion, they can pair much like DNA. These complementary sequences form A-helices, not the B-helices found in DNA.iii. A-form helices are a little shorter, more dehydrated, than the b-form. The A-form double helices are quite stable, increasing the stability of RNA double helices.b. Secondaryi. Structural elements of Complementary RNA sequences:1. Bulge - a single base is mismatched, causing a kink in the helix.2. Internal loop - several base pairs are mismatched.3. Hairpin or Stem loop - formed by inverted complementary strands base pairing.a. Inverted repeats in DNA, when transcribed, are what allow the hairpins to form in RNA.ii. Tertiary RNA structure can be visualized using tRNA1. The secondary structure looks like a cloverleaf, with a stem and 3 "leaves" formed by hairpins.2. In RNA, it is very common for G to pair with U, instead of the usualA-U pairing. The G-U pairing forms 2 H-bonds, just like the A-U pairing does.iii. The 3' end of the stem is where the amino acid attaches. The anticodon is found on the leaf directly opposite that. The anticodon is what pairs to the codon in the mRNA.iv. This structure is RNase P, a ribozyme - it is an RNA sequence associated with a protein component. It is the RNA part that does the catalysis in a reaction (the fact that the RNA does the catalysis is what makes it a ribozyme)1. Pairing does not have to occur between consecutive sequences. Pairing between distal parts is what allows the formation of tertiary sequences.c. Tertiaryi. The tertiary structure of tRNA in the cell is L-shaped, not the secondary clover shape. ii. The functional ends of the molecule are at the ends of the L-shape.iii. Tertiary folding creates a very compact, highly-complementary, stable structure1. Part of the tertiary structure is formed bya. Adenine can bond with another purine, dimethylguanine.b. Another example of an unusual hydrogen bond occurs between a hydroxyl group in a ribose and a Nitrogen atom in a base.iv. Unusual hydrogen bonding and base modification give variety and stability to the structure of tRNAs as well as other RNA molecules.d. Non-Watson-Crick base pairingi. There are 40-50 tRNA molecules in the cell1. Molecules called amino acyl tRNA synthases have to recognize theright tRNA and attach the correct amino acid to it.a. All tRNA are in the L-shape; the modified bases give the molecules variety so the enzymes can recognize them in addition to recognizing the anticodons.b. The modified bases also add stability to the RNA molecules; methylating bases makes them morehydrophobic, which allows them to stack better on top of each other, increasing stability.IV. Ribozymesa. A ribozyme is an RNA (such as RNase P) that folds into a distinct tertiary structure, bringing functional groups (like Hydroxyl groups) into position to bind asubstrate and catalyze a reaction, much as an enzyme does. The difference is thatthe RNA carries out the catalytic function.b. RNA World hypothesis - because of the structural and functional diversity found in RNA, with structures like ribozymes, it is thought that RNAs might have been the first catalysts and first genes. Before life as we know it, RNAs might have been evolving and gaining catalytic function. This could offer a solution to the Chicken-egg problem - you need the enzymes to replicate DNA but the DNA to code for the enzymes. Perhaps neither of these came first; the RNA first acted as catalysts.c. Steps of Creation in the RNA World hypothesisi. Creation of a prebiotic soup, including nucleotides from components of Earth's primitive atmosphere.ii. Production of
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