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RIBOZYMES from Chapter 3 and more Thanksgiving diversion #2 • Did you know they’re sequencing the turkey genome? • They’ve finished the 1x shotgun sequence phase. This means they have sequences for thousands of fragments scattered throughout its genome, but there are many gaps missing fragments. most important concept: Enzymes control chemical reactions. They replace activation energy and may help transfer energy to/from ATP. most important concept: Enzyme action is determined by enzyme shape; enzyme shape is controlled by its primary structure (its amino acid sequence), all part of the central dogma of modern biology: DNA--->RNA--->protein --> us (Francis Crick) Figure 3.13 A B C Potential energy Transition state Ea without enzyme Ea with enzyme A + BC Reactants AB + C Products Time Box 3.3 Figure 1a Specificity between substrate and enzyme Substrate (glucose) Enzyme (hexokinase) ACTIVE SITE • each enzyme has a four-dimensional space which is like a “glove” that the substrate fits into. most important concept: Enzyme action is determined by enzyme shape; enzyme shape is controlled by its primary structure (its amino acid sequence), all part of the central dogma of modern biology: DNA--->RNA--->protein --> us (Francis Crick) BUT sometimes the enzymes are RNA; then ribozymes control chemical reactions. They replace activation energy and may help transfer energy to/from ATP Freeman’s theme Throughout the textbook, the author shows how the facts of biology and chemistry support evolution, including biochemical evolution in the early chapters-- The RNA world Figure 3.15b Secondary structure of RNA Stem Hairpin Loop 3´ G G C C U A A U C G 5´ Many ribozymes have been identified and studied • intron processing • ribosome activities (protein synthesis) • synthetic RNA • example in textbook chapter 3 Molecules of the “Prebiotic Soup” • Part of Freeman’s theme • Nitrogenous bases: – purines (A, G); – pyrimidines [C, T, U]. – Purines have been synthesized in experiments simulating early earth conditions (like the Miller/Urey flasks), but not pyrimidines so far. Molecules of the “Prebiotic Soup” Nucleotide monomers (ATP, GTP, CTP, UTP) = three phosphate groups bonded to ribose, which is bonded to a nitrogenous base. • In lab simulations they will form RNA without enzymes (no polymerases, etc.) Figure 3.14b – O – O P O The sugar-phosphate spine of RNA O 5´CH O N 2 O O O OH O P O – 5´ The sequence of bases found in an RNA strand is written in the 5´ 3´ direction: NH2 O N CH2 O N N N O OH O P O – O N CH2 O N O NH N NH2 3´ O OH O P O – O NH2 N CH2 O N 3´ OH OH O Freeman’s“Prebiotic Soup” = The RNA world ? • Nucleotide monomers (ATP, GTP, CTP, UTP) = three phosphate groups bonded to ribose, which is bonded to a base. • In lab simulations they will form RNA • And in lab simulations RNA can use the nucleotide monomers to replicate itself. Figure 3.15a RNA basepairing and hydrogen bonds 5´ H Cytosine N H C Sugar-phosphate backbone H C H N C N H H C N N C O C C G N C N H O C C 3´ Guanine N H H Adenine Uracil O C H N N C N H C C A N C C O 3´ H N C U C H C N N H 5´ Figure 3.16, left RNA FORMS A TEMPLATE FOR ITS SYNYTHESIS A G U G 3´ U C 3´ A 5´ C 5´ 1. Complementary bases pair. C G A U G C U G 3´ A C C 5´ 2. Copied strand polymerizes. G A G U C U A G C 3´ 5´ Copied strand G 3´ Template strand 3´ 5´ 3´ Copied strand C 5´ Template strand Template strand 5´ 3. Copy and template separate. Figure 3.16, right A C 5´ 4. Copy serves as new template. 3´ G A U G C U A G C 5´ 5. New copy polymerizes. 3´ C G A New copy strand U C 5´ U G C U A G C 3´ 5´ New template strand 3´ C 3´ New template strand 5´ G U New copy strand G 3´ 5´ A 3´ Copied strand = new template C G 5´ 3´ 5´ 6. New copy is identical to original template. What Constitutes Life? • First living entity was likely some type of self-replicating macromolecule that could: – Act as a template. – Catalyze reactions. • Could that be a protein? • Or . . . The RNA world ? • RNA can be both catalyst and template: • Catalytic RNAs occur in some organisms. • An RNA strand can be a template for forming a The RNA world ? The RNA world ? • A self-replicating RNA could have been the first living entity. • Laboratory experiments can generate self-replicating RNAs. • Selection of self-replicating RNAs demonstrates evolution in vitro. Figure 3.19 HOW RNAs ARE MADE FOR SELECTION EXPERIMENTS Hairpin loop 3´ C A G Tag sequence U G 5´ Oligo substrate 5´ G U C A C Randomly generated sequence 220 nucleotides 3´ Pool RNA 1. Complementary base pairing occurs between oligo substrate and pool RNA. 5´ 3´ CG AU GC UA 220 nucleotides Tag sequence GC 5´ 3´ 2. Ligation occurs via formation of a phosphodiester bond. Tag sequence 5´ CG AU GC UA 220 nucleotides GC 3´ Figure 3.20 EXPERIMENTAL SELECTION OF RIBOZYMES If pool RNA sequence can catalyze ligation reaction... GC UA CG AU CG Tag 5´ 5´ Tag Oligo substrate Oligo substrate GC UA CG AU CG 3´ 220 nucleotides CG AU GC UA GC Tag 5´ …any ligated pool RNA sequences will adhere to the affinity column and be saved for further experimentation. Affinity column 3´ 220 nucleotides Pool RNA If pool RNA sequence cannot catalyze ligation reaction... C A G U G 5´ Tag …unligated pool RNA sequences will pass through column and be thrown away. 220 nucleotides 3´ Pool RNA 5´ G U C A C 3´ Mutation step in the The RNA world ? When new ribozymes are made, some of them have mutations Figure 3.20 EXPERIMENTAL SELECTION OF RIBOZYMES If pool RNA sequence can catalyze ligation reaction... GC UA CG AU CG Tag 5´ 5´ Tag Oligo substrate Oligo substrate GC UA CG AU CG 3´ 220 nucleotides CG AU GC UA GC Tag 5´ …any ligated pool RNA sequences will adhere to the affinity column and be saved for further experimentation. Affinity column 3´ 220 nucleotides Pool RNA If pool RNA sequence cannot catalyze ligation reaction... C A G U G 5´ Tag …unligated pool RNA sequences will pass through column and be thrown away. 220 nucleotides 3´ Pool RNA 5´ G U C A C 3´ Figure 3.21 101 100 Ligation rate (per hour) 10-1 10-2 10-3 10-4 10-5 10-6 0 1 2 3 4 5 6 Round of experiments 7 8 9 10 Figure 3.22 HOW RNA REPLICASE COULD EVOLVE First living entity (RNA replicase) Ribonucleotides Exact copies Mutant copies Mutant copies Different (mutant) bases These copies work at the same rate as the original. Most mutants work less well than the original. A few mutants work better than the original. Few or no copies. Few copies. Copies of original RNA replicase become outnumbered by the more efficient form. Many copies–some of these have mutations that allow replicase to work better yet. Many copies of most efficient RNA replicase are made. The RNA world ? • Chemical Evolution would be Overtaken by Biological Evolution • Natural selection becomes the engine that drives further evolution. • The RNA world eventually becomes the DNA world ? most important concept: Enzyme action is determined by enzyme shape; enzyme shape is controlled by its primary structure (its amino acid sequence), all part of the central dogma of modern biology: DNA--->RNA--->protein --> us (Francis Crick) BUT sometimes the enzymes are RNA; then ribozymes control chemical reactions. They replace activation energy and may help transfer energy to/from ATP Help Session • This week featuring Cell structure • Thursday @ 3pm Next: MEMBRANES http://www.queens.edu/faculty/jannr/bio103/helpPages/c04membrane.htm especially membrane proteins! And especially the enzymes and transport proteins both with active sites MEMBRANE PROCESS TABLE Download from Friday’s help page QUIZ on everything except the “How” column PROCESS ENERGY SOURCE HOW IT HAPPENS DIFFUSION Random molecular motion (passive) Dissolved substances become evenly dispersed = net movement from areas of high concentration to areas of lower concentration for each substance independently. Molecules move through membrane faster when they're smaller and when the temperature rises. Like regular diffusion except ________(fill it in)______ Facilitated Diffusion and Channel-mediated Diffusion ________(fill it in)______ Osmosis ________(fill it in)______ Active Transport Cell energy sources (usually ATP) Endocytosis (active) (phagocytosis) Exocytosis (active) (secretion) Cell energy sources ________(fill it in)______ Like regular diffusion except ________(fill it in)______ Dissolved substances become_______ dispersed = net movement is often from areas of _____ concentration to areas of _________ concentration Membrane surrounds and engulfs substances, usually (maybe always) attached to receptors on external face of membrane________(fill it in)______ Reverse of endocytosis: ________(fill it in)______ WHAT MOVES THIS WAY Small uncharged molecules, larger nonpolar molecules: Oxygen, ethanol, steroids materials which fit carriers or channels: Sugar, ions, ________(fill it in)______ ________(fill it in)______ ________(fill it in)______ ________(fill it in)______ _______(fill it in)______ MEMBRANE PROCESS TABLE PROCESS ENERGY SOURCE HOW IT HAPPENS Endocytosis (active) (phagocytosis) Cell energy sources Membrane surrounds and engulfs substances, usually (maybe always) attached to receptors on external face of membrane________(fill it in)______ Reverse of endocytosis: ________(fill it in)______ Exocytosis (active) (secretion) ________(fill it in)______ Do the last two rows on your own WHAT MOVES THIS WAY ________(fill it in)______ _______(fill it in)______ Next: MEMBRANES http://www.queens.edu/faculty/jannr/bio103/helpPages/c04membrane.htm especially membrane proteins! And especially the enzymes and transport proteins both with active sites ACTIVE SITE: The glove and the “hand” are near-perfect matches –geometrically –electrochemically –for hydrophobicity etc. –for motions and vibrations –for van der Walls forces. ACTIVE SITE: The glove and the “hand” are near-perfect matches • THE near-perfect match depends on exact amino acid sequence in the active site and in any part of the enzyme controlling substrate/active site access. ACTIVE SITE: the near-perfect match depends on exact amino acid sequence • Mutations can change the amino acid sequence • * • ** • *** • **** ACTIVE SITE: its shape can also be changed reversibly by other factors • Inhibitors can block the active site • Inhibitors can pull on another part of the enzyme and stretch the active site out of shape • phosphates and other factors can pull on another part of the enzyme to pull the active site into the correct shape Reversible changes in protein shape = The cell’s method of adjusting to its needs • time for hydrolysis? – or synthesis? • Time to take in sugar? – Or time to dump it out? • Time to send an electrical signal? – Or time to restore gradients? ACTIVE SITE: its shape can also be changed reversibly by other factors • Inhibitors and phosphates and hormones and other factors pull on another part of the enzyme to pull the active site into or out of the correct shape • usually reversible because the factors interact with the enzyme’s hydrogen and ionic bonds, not the covalent bonds ACTIVE SITE: its shape can also be changed by other factors • pH changes can change the amino acid interactions so that the shape is wrong or right. • Again, affecting ionic hydrogen bonds, not covalent bonds • So is a pH effect permanent? Figure 2.17 [H3O+] pH 10-14 14 10-13 Oven cleaner 13 10-12 12 Household bleach Household ammonia 10-11 11 Basic Neutral Acidic Milk of magnesia 1010 10 10-9 9 10-8 8 10-7 7 10-6 6 Human blood Pure water Milk Urine 10-5 5 Black coffee 10-4 4 10-3 3 10-2 2 Tomatoes Wine Vinegar, soft drinks, beer Lemon juice 10-1 1 10 0 Baking soda Seawater Stomach acid ACTIVE SITE: its shape can also be changed reversibly by other factors • pH changes can change the amino acid interactions so that the shape is wrong or right. • Why does stomach fluid have a low pH? • Why does the small intestine have a high pH? Figure 3.3b, row 4, left Each amino acid has a different side chain. H H3N+ O C H3N+ C H H C CH2 NH +NH H3N+ C C – – O O O CH2 O – CH2 CH2 CH2 CH2 CH2 CH2 NH +NH 3 C C O +NH 2 NH2 Histidine (H) His Lysine (K) Lys Basic side chains Arginine (R) Arg Figure 3.3b, row 4, right Each amino acid has a different side chain. H H3N+ H O C H3N+ C O C C – – CH2 O CH2 C – O CH2 O C – O Aspartate (D) Asp O Glutamate (E) Glu Acidic side chains O Table 3.1 TABLE 3.1 HOW AMINO ACIDS INTERACT WITH WATER In this table, the 20 amino acids are ranked according to how likely they are to interact with water. The ranking is from least likely to most likely. Highly hydrophobic Least likely Isoleucine Valine Leucine Phenylalanine Methionine Less hydrophobic Alanine Glycine Cysteine Tryptophan Tyrosine Proline Threonine Serine Interaction with water Highly hydrophilic Histidine Glutamate Asparagine Glutamine Aspartate Lysine Arginine Most likely Mutations are not reversible • mutations change the amino acid sequence. A different amino acid has a different side chain. • Changing a side chain may change the enzyme’s shape and therefore its action. • Changing a side chain may change the transport protein’s shape and therefore its action MEMBRANE PROCESS TABLE Download from Friday’s help page QUIZ on everything except the “How” column PROCESS ENERGY SOURCE HOW IT HAPPENS DIFFUSION Random molecular motion (passive) Dissolved substances become evenly dispersed = net movement from areas of high concentration to areas of lower concentration for each substance independently. Molecules move through membrane faster when they're smaller and when the temperature rises. Like regular diffusion except ________(fill it in)______ Facilitated Diffusion and Channel-mediated Diffusion ________(fill it in)______ Osmosis ________(fill it in)______ Active Transport Cell energy sources (usually ATP) Endocytosis (active) (phagocytosis) Exocytosis (active) (secretion) Cell energy sources ________(fill it in)______ Like regular diffusion except ________(fill it in)______ Dissolved substances become_______ dispersed = net movement is often from areas of _____ concentration to areas of _________ concentration Membrane surrounds and engulfs substances, usually (maybe always) attached to receptors on external face of membrane________(fill it in)______ Reverse of endocytosis: ________(fill it in)______ WHAT MOVES THIS WAY Small uncharged molecules, larger nonpolar molecules: Oxygen, ethanol, steroids materials which fit carriers or channels: Sugar, ions, ________(fill it in)______ ________(fill it in)______ ________(fill it in)______ ________(fill it in)______ _______(fill it in)______ Chapter 2