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Transcript of Nucleotides & Nucleic Acids Student Edition 9/26/13 Version Pharm. 304 Biochemistry Fall 2014 Dr....
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Nucleotides & Nucleic Acids
Student Edition 9/26/13 Version
Pharm. 304 Biochemistry
Fall 2014
Dr. Brad Chazotte 213 Maddox Hall
Web Site:
http://www.campbell.edu/faculty/chazotte
Original material only ©2004-14 B. Chazotte
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Goals• Be familiar with the different biological roles of nucleotides
• Learn the basic structure of nucleotides and nucleosides
• Understand the data that lead to determining the structure of DNA by Watson and Crick
• Understand the role of hydrogen bonding in the base paring of DNA and RNA.
• Understand that DNA can have some variation in structure and that proteins, e.g. histones, do bind to DNA and affect DNA structure such as chromosomes.
• Understand the meaning of DNA, denaturation/renaturation, melting curves and Tm versus base content
• Learn the general structure of tRNAs
• Be aware of the existence of interference RNA (RNAi) molecules.
• Have an awareness of the basics of the genetic code.
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Nucleotide Structure & Function
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The Roles of Nucleotides
• Are the essential “energy currency” in metabolic reactions.
• Are the critical chemical links in cellular response to hormones & extracellular stimuli.
• Are the structural components of an array of enzymes’ cofactors and metabolic intermediates.
• Are the constituents of nucleic acids. (They store and encode the fundamental genetic information, i.e. “blueprints” for life)
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Lehninger 2000 Figure 10.1a
Nucleotide Structure: General
nucleoside
Ester linkage
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Voet, Voet & Pratt 2013 p. 41
Nucleotide Structure: Bases
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Lehninger 2000 Figure 10..2
Purine & Pyrimidine Base Structures
1 1
1
6
6
7
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Nucleotide & Nucleoside Nomenclature
Lodish et al., 2000 Table 4.1
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Nucleotide Structure
Lodish et al., 2000 Figure 4.1
Voet, Voet & Pratt., 2013 Figure p41
Voet, Voet & Pratt., 2013 Figure 3.1
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ATP Nucleotide Structure
Voet, Voet, & Pratt 2013 Fig 14.5
Adenosine
5′
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Nucleotides & Bioenergetics
“All the basic processes by which energy is recovered –whether from the sun, inorganic mineral components, or organic compounds – rely on a series of nucleotide derivatives that transfer energy in discrete quantities.”
Voet, Voet & Pratt 2002 p.45
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Flavin Adenine Dinucleotide (FAD) Structure
Voet, Voet & Pratt 2002 Fig 3.3
Reduction site
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NAD+ and NADP+ Structures
Voet, Voet & Pratt 2002 Fig 3.4
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Coenzyme A Structure
Voet, Voet & Pratt 2002 Fig 3.5
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Stryer et al., 2002 Figure 5.1
Polymeric Structure of Nucleic Acids
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Nucleic Acid Structure
Voet, Voet & Pratt 2013 Fig 3.3a,b
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Chargaff’s Rules1. The base composition of DNA varies from one species to
another.
2. DNA specimens isolated from different tissues of the same species have the same base composition
3. The base composition of DNA in a given species does not change with the age of an organism , its nutritional state or a changing environment.
4. In all cellular DNAs, irrespective of species, the # of adenine residues equals the # of thymidine residues and the # of guanine residues equals the # of cytosine residues. i.e.
A = T & G = C therefore A + G = T + C
(the sum of the purines equals the sum of the pyrimidines)
Lehninger 2000 p. 281
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Stryer et al., 2002 Figure 5.10
X-Ray Diffraction Image of DNA Fiber
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Bases: Tautomeric Forms
Voet, Voet & Pratt 2013 Fig 3.4
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DNA: Building Blocks & Structure
Alberts et al. 2004 Figure 5.2
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Watson-Crick Model1. Two polynucleotide chains wind around a common axis to
form a double helix.
2. The two DNA strands are antiparallel with each forming a right-handed helix.
3. The bases occupy the helix core and the sugar-phosphate chains are on the periphery. The latter minimizes repulsions between phosphate groups. The structure give rise to a major and minor groove.
4. Each base-pair is H-bonded to a base in the opposite strand to form planar base pairs. The structure can only accommodate A – T binding and G- C binding, i.e. complementary base pairing
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Base-Paring holds the DNA Helix together
Alberts et al. 2004 Figure 5.7Alberts et al. 2004 Figure 5.6
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DNA: Complementary Strand Structure
Voet, Voet & Pratt 2008 Fig 3.8Voet, Voet & Pratt 2013 Fig 3.8
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DNA: 3-D Structure
Voet, Voet & Pratt 2013 Fig 3.6, 3.7
3.4 Å
20 Å
10 nucleotides per helix turn ~34 Å
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Biological Implication of the Watson-Crick Structure
Each DNA strand can act as a template for the synthesis of it complementary strand and hence that hereditary information is encoded in the sequence of bases in either strand.
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Models of Known DNA Structures
Lodish et al., 2000 Figure 4.6
Voet, Voet, & Pratt 2013 Table 24.1
Voet, Voet, & Pratt 2013 Fig 24.5
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Bending of DNA Due to Protein Binding
Lodish et al., 2000 Figure 4.7
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Chromosomes: Folded DNA Structures
Chromatin – the complex of histones or NHC-proteins with DNA.
Chromatin is the material that comprises a chromosome.
Euchromatin – loosely packed transcribable DNA
Heterochromatin – tightly packed DNA – “transcriptionally silent”
Nucleosomes – Histones (H2A, H2B, H3 & H4) associate with DNA to form nucleosomes, i.e. unit particles of chromatin. Nucleosomes look like “beads on a string” In some models of gene activation and repression nucleosomes are repositioned by transcription factors. Nucleosomes can obscure a “promoter region” or by preventing binding of the transcription factor in the first place.
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Histones and Non-Histone Chromosomal Proteins
H1, H2A, H2B, H3 and H4 are typical histones in eukaryotic cells. Evolutionarily well conserved.
H1 involved in chromosome folding.
Lodish et al., 2004 Figure 5.22Voet, Voet, & Pratt 2013 Fig. 24.41
Voet, Voet, & Pratt 2013 Fig. 24.45
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DNA Denaturation
Matthews et al. 1999 Figure 4.31
Electrostatic repulsion of backbone phosphate groups favors denaturation. (Conditions of high ionic strength favors double helix)
Entropy is higher in the random coil (denatured) DNA – favors denaturation
G=H-TS
Helix Random Coil
For RC S >0
& Helec < 0
But:
H > 0 due to base H-bonds & base van der Waals.
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GC content vs Tm
Matthews et al. 1999 Figure 4.32
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Denaturation and Renaturation of DNA
Lodish et al., 2000 Figure 4.8
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RNAs
Ribosomal RNA (rRNA) are the structural components of ribosomes where proteins are synthesized.
Messenger RNA (mRNA) are intermediates that carry the genetic information from one to a few genes to a ribosome. The template for protein synthesis
Transfer RNA (tRNA) are “adapter” molecules that faithfully translate the information in mRNA into a specific sequence of amino acids.
Interference RNAs (RNAi) a naturally occurring class of short (21-33 nt) single (miRNA) or double stranded RNAs (siRNA) involved in post-transcriptional regulation of gene expression.
Long Noncoding RNAs (lncRNA) extensively transcribed RNAs that do NOT code for proteins that form extensive networks of ribonucleoprotein complexes (RNPs) with numerous chromatin regulators that then target these enzymatic activities to appropriate locations in the genome (Rinn & Chang
Annu. Rev. Biochem. 81: 145-166, 2012).
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Brief Summary of DNA & RNA Differences1. DNA deoxyribose sugar. RNA ribose sugar with 2’ OH group
2’OH makes RNA more polar & more chemically active; causes slight structural change (twist/pucker) in ribose sugar. (Structure affects function and interactions)
2. DNA uses thymine base. RNA uses uracil baseThymine - important protector from spontaneous damage (see DNA Replication
lecture; DNA base excision repair).
3. DNA (B-form) and RNA (A form) double helix secondary structure differ. 2’-OH prevents RNA from forming the less compact DNA B-form. The pucker of the ribose sugar affects the interphosphate distance. In the A-form helix the major groove is deep and narrow making the bases fairly inaccessible for protein interactions.
4. RNA can form tertiary structures. DNA does not. RNA forms a number of shorter helices interspersed by single stranded regions
5. DNA replication forms two complementary strands. Transcription makes single RNA strands from one template strand. Thus RNA does not have a long complementary RNA strand with which to form a double helix like DNA (Also DNA strands are replicated to form identical ds-DNA molecules and during transcription DNA stands are only transiently separated).
Elliot & Ladomery “Molecular Biology of RNA” 2011, p.23
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RNA vs DNA Structure
Elliot & Ladomery “Molecular Biology of RNA” 2011
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RNA Secondary Structures
Elliot & Ladomery “Molecular Biology of RNA” 2011
Stable RNA secondary structures can be promoted in solution by positively charged molecules such as metal ions, Mg2+ in particular. They balance the strong negative charges of the phosphodiester backbone to allow the molecules to come together
Individual RNAs often will contain a number of these secondary structural motifs. Folding patterns of RNAs can be quite complex.
Stemloop
Stryer et al., 2002 Figure 5.19
Elliot & Ladomery “Molecular Biology of RNA” 2011 Figs. 2.13 & 2:16
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RNA Structure
Lodish et al., 2000 Figure 4.12
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RNA Tertiary StructureRNA molecules employ three primary strategies to pack together negatively charged helices. Coaxial Stacking: shorter RNA helices are stacked on top of each other to yield more stable helical structures.Hydrogen Bonding: Base Triplets – In addition to Watson-Crick base “normal” base-pairing. Hoogstein bonds – additional hydrogen bonds can attach a 3rd base – builds up groups of interacting nucleotides Ribose zippers – Involve H-bonding of Ribose 2’-OH groups (2’-OH groups project outside helix) important in holding together RNA tertiary structures.Metal Ions Magnesium Ions have the most important role in RNA tertiary structure; sodium ions next. Play a key role in gluing RNA secondary structures into compact globular tertiary structures. Ions are also able to bind specific sites in RNA tertiary structures to hold helices together.
Base Triplets
Important Note: RNAs can fold into complex tertiary structures whose intricacy can rival those of proteins. RNA tertiary structure is held together via the interactions of distant regions of RNA. Metal ions at specific binding sites provide a positively charged core around which RNA molecules can fold.
Elliot & Ladomery “Molecular Biology of RNA” 2011
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Voet, Voet & Pratt., 2013 Fig 24.24
RNA Molecule Structure
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Catalytic RNAs
Elliot & Ladomery “Molecular Biology of RNA” 2011
Ribozymes: enzymatically active RNA molecules. They can speed up chemical reactions as much as protein-based enzymes. However, unlike enzymes not all ribozymes remain unchanged after their reaction.
2’-OH group oxygen can function as a nucleophile. Many reactions of ribozymes involve an attack on the phosphodiester bonds. The bases in RNA have nitrogens that can act as hydrogen donors or acceptors in acid-base catalysis.
We shall see in “Translation” (protein synthesis) that peptide bond formation is actually catalyzed by rRNA.
Some introns have been found to function as self-splicing ribozymes
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Transfer RNA (tRNA) Structure
Lehninger 2000 Figure 10-28a
Lodish et al., 2004 Figure 4.26
Voet, Voet, & Pratt 2013 Figure 24.25
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Stryer et al., 2002 Figure 5.31
Aminoacyl t-RNA
Symbolic Diagram
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RNAi Definitions• RNAi Ribonucleic Acid interference – small noncoding RNAs (see
Fire et al 1998)
• siRNA Short interfering RNA. These are dsRNA 21-25bp in length with a 3’ overhang that are processed from longer RNAs by the enzyme “Dicer”. Synthetic siRNA can be introduced in mammalian cells to
produce interference.• shRNA Short (interfering) hairpin RNA. Used to supply siRNAs with
vector-based approaches to produce stable gene silencing
• miRNA microRNA. ssRNA 19-23nt long that originate from ss precursor transcripts characterized by imperfectly base-paired hairpins. Function as a silencing complex.
• RISC RNA-induced silencing complex. A nuclease complex of proteins and siRNA that targets and cleaves mRNAs complementary to the siRNA in the RISC complex.
RNAi & Epigentics Sourcebook, Invitrogen 2010 Ann. Rev. Cell Biol. 25 355-376 2009
• piRNA PIWI-interacting RNAs 24 -32 nt long involved in germ line cells.
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lncRNA• Involved in Epigenetic Regulation – how particular
stretches of DNA are transcribed into RNA. Loosely packed chromatin, euchromatin, can be transcribed. Tightly packed chromatin, heterochromatin, is transcriptionally silent. • Methylation of DNA’s cytosine and methylation and
acetylation of histones control chromatin packing: drive heterochromatin formation.• lncRNA are part of a diverse group of RNAs which
regulate diverse epigenetic aspects, e.g., gene expression during animal development or equalizing sex chromosome gene expression between males and females
Elliot & Ladomery “Molecular Biology of RNA” 2011, p.389
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Stryer et al., 2002 Table 5.4
The Genetic Code
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End of Lectures