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This 7 page Class Notes was uploaded by Udbluehen03 on Sunday September 18, 2016. The Class Notes belongs to BISC401 at University of Delaware taught by Lachke,Salil in Fall 2016. Since its upload, it has received 14 views. For similar materials see Molecular Biology of the Cell in Biology at University of Delaware.
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Date Created: 09/18/16
Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish ES (enzyme-substrate) complex - Enzyme kinetics suggest that enzymes (E) bind substrate molecules (S) at a fixed and limited number of sites – the enzymes’ active site o The bound species is known as an enzyme-substrate (ES) complex - At high concentrations of substrate, all the binding sites of the enzymes have substrate bound, the substrate-binding sites are said to be saturated with substrate - The ES complex is in equilibrium with the unbound enzyme and substrate and is an intermediate step in the conversion of substrate to products (P) - Intermediate structures, such as ES and EX* form at the active site of the enzyme and require the participation of key amino acid residues - Enzymes catalyze the conversion of substrates to products by dividing the process into multiple, discrete chemical reactions that involve multiple, discrete enzyme substrate complexes Optimizing Enzyme Activity - Enzymes taking part in a common metabolic process are generally located in the same cellular compartment, be it in the cytosol, at a membrane, or within a particular organelle. - Within this compartment, products from one reaction can move by diffusion to the next enzyme in the metabolic pathway - Diffusion of products o Random movement o Can be slow, relatively inefficient process for moving molecules between enzymes - Cells evolved mechanisms for bringing enzymes in a common pathway into close proximity - Close association of polypeptides with different catalytic activities cluster closely as subunits of a multimeric enzyme or on a protein scaffold, which makes coupled reactions much more efficient. - Enzymes that have several catalytic domains within the same polypeptide and therefore can catalyze several enzymatic reactions are the pinnacle of efficiency Nucleotides - The monomers from which DNA and RNA polymers are built - All have a common structure Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish o A phosphate group link by a phosphoester bond to a pentose (5-carbon) sugar, which is turned is linked to a nitrogen and carbon-containing ring structure commonly referred to as a base - Function of Nucleic acid: DNA and RNA o Information storage “devices” Genomes (DNA or RNA) contain all the information required to make a new member of a species. DNA is used as a template to direct RNA synthesis. mRNAs are used as templates to direct the synthesis of proteins. o Enzymatic and biosynthetic roles RNA can function as a catalytic enzyme (Self-splicing) RNA plays a major role in the synthesis of proteins (rRNA and tRNA) o Regulate expression of genes Small RNA mediated control of gene expression - Bases found in both DNA and RNA o Adenine o Guanine o Cytosine - Base found in DNA alone o Thymine - Base found in RNA alone o Uracil - Adenine and Guanine o Are purines o Both contain a pair of fused rings - Thymine and Uracil o Pyrimidines o Both contain a single ring - The bases are often abbreviated as o A, G, C, T and U - In nucleotides, the 1’ carbon atom of the sugar (ribose or deoxyribose) is attached to the nitrogen at position 9 of a purine (9 ) or at position 1 of a pyridine 1N ) - The acidic character of nucleotides is due to the phosphate group o Under normal intracellular conditions releases hydrogen ions (H+), leaving the phosphate group negative Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish o Most nucleic acids in cells are associated with proteins, which form ionic interactions with the negatively charged phosphates - Nucleosides o Combinations of a base and a sugar without a phosphate DNA - Deoxyribonucleic acid - Informational molecule that contains in the sequence of its nucleotides the information required to build all the proteins and RNAs of an organism - Extraordinary stable under most terrestrial conditions - 10 nucleotides long 9 - 3 x 10 base pairs in the human genome - The information stored in DNA is arranged in hereditary units, called genes o Control identifiable traits of an organism - In the process of transcription, the information stored in DNA is copied into RNA, which had three distinct roles in protein synthesis - Portions of the DNA nucleotide sequence are copied into messenger RNA (mRNA) o Molecules that direct the synthesis of a specific protein - The nucleotide sequence often mRNA molecule contains into that specifies the correct order of amino acids during the synthesis of a protein - The nucleotide sequence of an mRNA molecule is “read” by a second type of RNA called transfer RNA (tRNA) with the aid of a third type of RNA, ribosomal RNA (rRNA), and associated proteins - The RNA synthesis is called transcription because o The four-base sequence “language” of DNA is precisely copied or transcribed into the nucleotide sequence of an RNA molecule - Protein synthesis is called translation because o The four-base sequence “language” of DNA and RNA is translated into the twenty-amino acid sequence “language” of protein - Proteins are largely responsible for regulating gene expression - The process whereby the information encoded in DNA is decoded into proteins in the correct cells at the correct times in development - Nucleic acids function as the “brains and central nervous system” of the cell. Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish - Central dogma DNA RNA Protein DNA and RNA - All nucleotide consists of an organic base linked to a five carbon sugar that has a phosphate group attached to the 5’ carbon o In RNA, the sugar is ribose o In DNA, the sugar is deoxyribose - A single nucleic acid strand has a backbone composed of repeating pentose-phosphate units from which the purine and pyrimidine bases extend as side groups - Like a polypeptide, a nucleic acid strand has an end to end chemical orientation: the 5’ end has a hydroxyl or phosphate group on the 5’ carbon of its terminal sugar; the 3’ end usually has a hydroxyl group on the 3’ carbon of its terminal sugar o Polynucleotide sequences are written and read in the 5’-> 3’ direction (from left to right) - The chemical linkage between adjacent nucleotides called a phosphodiester bond, usually consists of two phosphoester bonds, one on the 5’ side of the phosphate and another on the 3’ side. - Polynucleotide can twist and fold into three-dimensional conformations stabilized by noncovalent bond - The three-dimensional conformation of DNA and RNA are different Structure and Properties of DNA - DNA consists of two associated polynucleotide strands that wind together to form a double helix - The two sugar-phosphate backbones are on the outside of the double helix and the bases project into the interior Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish - The adjoining bases in each strand stack on top of one another in parallel planes - The orientation of the two strands is antiparallel; that is their 5’-> 3’ directions are opposite o A is paired with T through 2 hydrogen bonds o G is paired with C through 3 hydrogen bonds o Bases are flat, perpendicular to axis of helix - The presence of thousands of hydrogen bonds in a DNA molecule contributes greatly to the stability of the double helix o Hydrophobic and van der Waals interaction between the stacked adjacent base pairs further stabilize the double-helical structure - Watson-Crick base pairs o Association between a larger purine and a smaller pyrimidine - Most DNA in cells take the form of a right-handed helix - The stacked bases are regularly spaced 0.34nm apart along the helix axis - The helix makes a complete turn every 3.4-3.6nm o About 10-10.5 base pairs per turn - B-form of DNA o Normal form present in most DNA stretches in cells - On the outside of the helix, the space between the intertwined strands form two helical grooves of different widths, described as the major groove (22 Å) and the minor groove (12 Å) - The important modifications in the structure of standard B-form DNA come about as a result of protein binding to specific DNA sequences - The double helix is flexible about its long axis - Unlike the α helix in proteins, it has no hydrogen bonds parallel to the axis of the helix - This property allows DNA to bend when complexed with a DNA-binding protein, such as the transcription factor TBP o The conserved C-terminal domain of the TATA box-binding protein (TBP) binds to the minor grove of specific DNA sequence rich in A and T, untwisting and sharply bending the double helix o Transcription of most eukaryotic genes require participation of TBP - Why did DNA, rather than RNA, evolve to be the carrier of genetic information in cell? o The hydrogen at the 2’ position in the deoxyribose of DNA makes it a far more stable molecule than RNA, which instead has a hydroxyl group at the 2’ position of ribose o The 2’ hydroxyl groups in RNA participate in the slow, OH- catalyzed hydrolysis of phosphodiester bonds at neutral pH o The presence of deoxyribose in DNA makes it a more stable molecule Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish o A characteristic that is critical to its function in the long term storage of genetic information DNA Can Undergo Reversible Strand Separation - The unwinding and separation of DNA strands, referred to as denaturation or melting, can be induced experimentally by increasing the temperature of a solution of DNA - As the thermal energy increases, the resulting increase in molecular motion eventually breaks the hydrogen bonds and other forces that stabilize the double helix - Hyperchromicity o A phenomenon useful for monitoring DNA denaturation - The stacked base pairs in duplex DNA absorb less ultraviolet (UV) light than the unstacked bases in single-stranded DNA - The temperature at which DNA denatures increases with the proportion of G•C pairs - Melting of double-stranded DNA can be monitored by its absorption of UV light at 260nm - As regions of double-stranded DNA unpair, the absorption of light by those regions increases almost two fold. - Light absorption by single-stranded DNA changes much less as the temperature is increased GC content of DNA affects its melting temperature - The melting temperature (T ) am which DNA strands separate depends on several factors - Tm= Temperature required to denature (separate) half the number of nucleotides in DNA molecule - Molecules that contain a greater proportion of G•C pairs require higher temperatures to denature because the three hydrogen bonds in G•C pairs make these base pairs more stable than A•T pairs, which have only two hydrogen bonds - The percentage of G•C base pairs in a DNA sample can be estimated from its T m - Agents that destabilize hydrogen bonds, such as formamide or urea also lower the T m - At low (acid) pH, the bases become protonated and thus positively charged, repelling each other - At high (alkaline) pH, the vases lose protons and become negatively charged, again repelling each other because of their similar charges - In cells, pH and temperature are for the most part, maintained at a constant level - The single-stranded DNA molecules that result from denaturation form random coils without an organized structure Class 5 – 9/12/2016, Nucleic Acids and Genomics Dr. Salil Lachke Molecular Cell Biology, 8 edition – Lodish - Lowering the temperature, increasing the ionic concentration, or neutralizing the pH causes two complementary strands to re-associate into a perfect double helix - Two DNA strands that are not related in sequence will remain as random coils and will not renature - Denaturation and renaturation of DNA are the basis of nucleic acid hybridization o A powerful technique used to study the relatedness of two DNA samples and to detect and isolate specific DNA molecules in a mixture containing numerous different DNA sequence Common Forms of DNA in the Lab - Small circular DNA without protein can have supercoils - Localized unwinding of DNA can cause supercoils in other parts of the molecule - Topoisomerase enzyme can relieve this torsional stress by breaking a phosphodiester linkage to enable loss of supercoils, and then rejoins the broken ends RNA Forms Higher Ordered Structures based on H-bonding - Like DNA, RNA is a long polynucleotide that can be double-stranded or single-stranded, linear or circular - RNA molecules are like proteins in that they have structured domains connected by less structured, flexible stretches - Hairpins, stem-loop, and other secondary structures can form by base pairing between distant complementary segments of an RNA molecule - In stem-loops, the single-stranded loop between the base-paired helical stem may be hundreds or even thousands of nucleotides long, whereas in hairpins, the short may contain as few as four nucleotides - Pseudoknots are forms by interactions of loop through base pairing between complementary bases. Use of DNA hybridization 8-37 - Fluorescent in situ hybridization (or FISH) is a technique that allows scientists to visualize the chromosomes that hybridize with fluorescent DNA probes specific to sequences within that chromosome.