Lesson 7: Nucleic Acids PDF

Biochemistry Lesson 7 Nature of Biomolecules: Nucleic Acids CHAPTER OUTLINE 7.1. STRUCTURAL ELEMENTS: • • • • GENERALITIES NUCLEOTIDES STRUCTURE: PHOSPHATE ESTER BOND NUCLEOSIDES STRUCTURE: N-GLYCOSIDIC BOD NUCLEOTIDES OF BIOLOGICAL INTEREST:  ENERGY  SECOND MESSENGER  COENZYMES 7.2. DNA: •...

Biochemistry Lesson 7 Nature of Biomolecules: Nucleic Acids CHAPTER OUTLINE 7.1. STRUCTURAL ELEMENTS: • • • • GENERALITIES NUCLEOTIDES STRUCTURE: PHOSPHATE ESTER BOND NUCLEOSIDES STRUCTURE: N-GLYCOSIDIC BOD NUCLEOTIDES OF BIOLOGICAL INTEREST:  ENERGY  SECOND MESSENGER  COENZYMES 7.2. DNA: • • • • • PHOSPHODIESTER BOND PRIMARY STRUCTURE SECONDARY STRUCTURE DNA REPLICATION Tm and DENATURATION 7.3. RNA: • TYPES OF RNA: • • • MESSENGER RNA RIBOSOMAL RNA TRANSFERENCE RNA 7.4. GENE CONCEPT: • • PROCARYOTIC AND EUCARYOTIC GENES SPLICING 7.1. STRUCTURAL ELEMENTS GENERALITIES  Biopolymers formed by NUCLEOTIDE and NUCLEOSIDES subunits.  A nucleotide consist of: a nitrogenous base + a sugar (this conjugate is called nucleoside) one or more phosphate groups.  Composed by C, H, N and P atoms  2 types: DNA → deoxyribonucleic acid RNA → Ribonucleic acid  They contain genetic information: Encoded information that allows organisms to develop their biological cycles.  The bases of DNA molecules carry genetic information whereas their sugar and phosphate groups perform a structural role.  Other functions: energy intermediates, coenzymes and second messengers 7.1. STRUCTURAL ELEMENTS STRUCTURE  NUCLEOTIDES are complex molecules containing: Phosphate group + Nitrogenous base + Pentose • NUCLEOSIDES are composed of: Nitrogenous base + Pentose Pentose (Ribose or deoxyribose) the deoxy prefix indicates that this sugar lacks an oxygen atom that is present in ribose. Deoxiribonucleotides Ribonucleotides DNA RNA β-D-Ribose β-D-Deoxyribose 7.1. STRUCTURAL ELEMENTS STRUCTURE Nitrogenous bases: Purine: Adenine (A) and Guanine (G) Pyrimidine: Thymine (T), Cytosine (C) and Uracil (U) Purines Double ring Purine Adenine Guanine Pyrimidines Single ring Pyrimidine Cytosine Uracil Thymine 7.1. STRUCTURAL ELEMENTS STRUCTURE OF A NUCLEOSIDE NB (ex: adenine) Nucleoside (ex: adenosine) N-Glycosidic Bond + N-glycosidic bond Pentose (ex: ribose) Link between C-1’ pentose → N-1 pyrimidine NB N-9 purine NB 7.1. STRUCTURAL ELEMENTS STRUCTURE OF A NUCLEOSIDE Nitrogenous base + sugar = nucleoside Cytosine N-glycosidic bond Deoxycytidine (1-β-deoxyribofuranosil-cytosine) Deoxyribose Nucleoside are named by adding –osine (purine derivative) or –idine (pyrimidine) 7.1. STRUCTURAL ELEMENTS NOMENCLATURE OF A NUCLEOSIDE BASES RIBONUCLEOSIDES DEOXYRIBONICLOSIDES A Adenosine deoxyadenosine G Guanosine deoxyguanosine C Cytidine deoxycytidine U Uridine ----- T ------ deoxythymidine DNA (A, C, G and T) RNA (A, C, G and U) 7.1. STRUCTURAL ELEMENTS STRUCTURE OF A NUCLEOTIDE NUCLEOTIDE It is formed by the binding of a phosphate group to the nucleoside. The phosphoric acid (H3PO4) forms a phosphate ester bond with C-5 of the sugar Such compound is called a nucleoside 5’-phosphate or a 5’-nucleotide Pentose + NB+ phosphate = nucleotide 7.1. STRUCTURAL ELEMENTS STRUCTURE OF A NUCLEOTIDE nucleoside (ex: adenosine) NUCLEOTIDE NUCLEOTIDE (ex: adenosine 5’-monophosphate) N-glycosidic bond Phosphate ion The most common site of esterification in naturally occurring nucleotides is the hydroxyl group attached to C-5 of the sugar Ester bond 7.1. STRUCTURAL ELEMENTS STRUCTURE Ribonucleotides Adenosine 5’-monophosphate (AMP) Guanosine 5’-monophosphate (GMP) Uridine 5’-monophosphate (UMP) Cytidine 5’-monophosphate (CMP) Deoxyribonucleotides deoxyadenosine 5’-monophosphate (dAMP) Deoxyguanosine 5’-monophosphate (dGMP) Deoxythymidine 5’-monophosphate (dTMP) deoxycytidine 5’-monophosphate (dCMP) 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGIC INTEREST The phosphate group in nucleoside 5’-monophosphate can be link to other groups to form the following compounds: - Energy molecules: ATP, ADP - Second messengers: cAMP, cGMP - Coenzyme: Coenzyme A, NAD 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGIC INTEREST ATP: It forms highly energetic bonds between other phosphate groups to form diphosphate and triphosphate nucleotides. The ATP constitutes the link between catabolism and anabolism. It is the energy exchange molecule in the cell. The process of converting ATP to ADP and P is coupled to many metabolic reactions. ATP 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGIC INTEREST ATP: The energy needed in endergonic reactions is obtained by ATP hydrolysis. It forms highly energetic bonds between other phosphate groups to form di- or triphosphate nucleotides. Dephosphorilation Phosphorilation 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGIC INTEREST Second messengers: cAMP: ester bonds with –OH in position 3’, a intramolecular bridge is formed. It is a signaling molecule. INTRAMOLECULAR BRIDGE cAMP 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGIC INTEREST Second messengers: cAMP Ligand (1st messenger) Cyclic adenylate (inactive) Protein receptor Binding site Cyclic adenylate (active) cAMP (2nd messenger) Inactive enzyme G protein ATP Synthesis Active enzyme Ligand + Protein receptor activation G protein G protein activation cyclic adenylate 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGICAL INTEREST Coenzymes: They can be: Coenzymes that participate in Redox reactions: transfer H+ and electrons from one substrate to the other a) Pyrimidine nucleotides: Nicotinamide derivatives: NAD, NADP Nicotinamide nucleotide + adenine nucleotide NAD + phosphate Nicotin-adenindeoxynucleotide NADP (nicotin-adenin-deoxynucleotide phosphate) a) Flavin nucleotides: they have riboflavin (vitamin B2): FMN, FAD 7.1. STRUCTURAL ELEMENTS NUCLEOTIDES OF BIOLOGICAL INTEREST Coenzymes: They can be: Coenzymes that participate in the transfer of chemical groups: Coenzyme A (CoA-SH) which transfer acetyl groups to other substrates. Contains vitamin B5 (pantothenic acid) Vitamins Organic compounds of diverse composition, essential in small quantities. They are synthesized by plants and microrganisms but not by animals. The have to intake in the diet. They act as coenzymes or form part of them. 7.2. DNA DNA structure: Deoxyribonucleic acid Structure Primary Linear macromolecules formed by polymerization of deoxyribonucleo tides 5’monophosphate of: A, G, C, T Secondary Double helix DNA compactation Chromatin Histone association Chromosomes Video 7.2. DNA NH2 Nucleotides are linked by phosphodiester bonds. The 3’ hydroxyl of the sugar moiety of one deoxyribonucleotide is joined to the 5’-hydroxyl of the adjacent sugar by a phosphodiester bridge. H H N O 5’ O P OCH2 O O HH HH 3’ O Phosphodiester bond Video N = Phosphate group: acid, polar N N H OH H 5’ NH2 N O= P OCH2 O O HH HH HO OH N O 7.2. DNA Primary structure The polynucleotide chain presents 2 free-ends: 5’ end Adenine • 5’ linked to phosphate group • 3’ linked to hydroxyl Cytosine Each chain is different in: • size • composition • base sequence Guanine The sequence is named with initial letter of each base from 5’ to 3’ ends: Thymine ACGT 3’ end 7.2. DNA Primary structure There are two different parts: 5’ end Adenine • Backbone of polydeoxyribose-phosphate • Nitrogenous bases Cytosine Guanine Importance The order determines a sequence of DNA. Gene: specific DNA sequence → encodes information for a functional molecule (protein or RNA) The Polynucleotide chain has directionality, individuality and polarity. Thymine 3’ end 7.2. DNA Primary structure Schematic view of DNA strand Primary Lineal polymers of nucleotides Secondary Doble Hélice James Watson and Francis Crick (1953) 3 types DNA: A, B o Z 7.2. DNA Secondary structure In 1953 Watson and Crick published in Nature the model for the molecular structure of DNA. In 1962 they received the Nobel price for their discovery 7.2. DNA Secondary structure Franklin and Wilkins studied the DNA structure using x-ray technology to study DNA fibers. Franklin presented in 1951 data on diffraction patterns of DNA The pattern is in the form of a “maltese cross” Tipical pattern of helical structure. X-ray diffraction pattern produced by hydrated DNA fibers The α helix structure of proteins had already been proposed by Pauling. nucleotide 7.2. DNA DNA is a Double Helix that stores Genetic information Chargaff discovered the DNA base ratios. He found that DNA followed certain rules: • The number of adenine residues is the same as thymine residues; and the number of C residues equals the G residues. A attracts T and G attracts C. • The number of hydrogen bonds depends on the base complementarity A+G =T+ C Adenine Guanine Cytosine 3 hydrogen bonds Thymine 2 hydrogen bonds 7.2. DNA Secondary structure Hydrogen bonds between bases in a DNA helix. The double helix is held together by H bonds between complementary pairs and base-stacking interactions, which are nonspecific and make the major contribution to the stability of the double helix. 7.2. DNA Secondary structure Double helix. X-ray diffraction pattern produced by hydrated DNA fibers • The 2 polynucleotide chains are coiled around a common axis. The chains run in opposite directions. • It has 2 nm (20 Å) diameter • NB are located in the interior linked by hydrogen bonds. • The pair AT has the same size as the pair CG • The backbone of phosphate is accommodated in the exterior. • Dextrorotatory Phosphate Backbone • Each pair of nucleotides is 0.34 nm away from the following. • Each turn has 10 pairs of nucleotides at intervals of 34 Å • Both strands are antiparallel and complementary 7.2. DNA Secondary structure Watson and Crick structure also refered as B form Minor DNA groove Major DNA groove http://highered.mcgrawhill.com/sites/9834092339/student_view0/c hapter14/dna_structure.html B form is the most stable form under physiological conditions. It is the standard form in living organisms 7.2. DNA Secondary structure DNA can occur in different structural forms. DNA is flexible and rotation can occur around a number of bonds in the sugar-phosphate backbone, and thermal fluctuation can produce stretching, bending and melting (unpairing) in the structure. DNA A form is favored in many solutions that are devoid of water. It is shorter and with greater diameter than B form. Found in hybrid RNA-DNA structures. DNA Z form: left-handed helical rotation. The DNA backbone takes on a zig-zag appearance. In sequences were DNA alterns C and G or 5-mC and G → role in gene regulation or genetic recombination 7.2. DNA Replication of DNA The double helix model proposed by Watson and Crick also suggests the mechanism for the transmission of genetic information. The essential feature is the complementarity of the two DNA strands. Making a copy: 1. Separation of the 2 strands 2. Synthesis of a complementary strand for each joining nucleotide in a sequence specified by the base pairing rules. The preexisting strands acts as a template. 7.2. DNA DENATURATION AND Tm DNA strands get denatured by: • High temperature (80-90ºC) → melting temperature, H bonds are broken as well as hydrophobic interactions between bases. The double helical structure melts or unwinds to form two single strands completely separated from each other. • Changes in pH (extrem pH) Denaturing is a reversible process, DNA can be renaturated. When the temperature and pH are returned at physiological range, the unwound segments of the two strands spontaneously rewind or anneal to yield the intact duplex. Melting temperature (Tm) depends on pH and ionic strengh and the size and base composition of DNA. As a rule, the higher the rate of CG the higher the Tm 7.2. DNA More complex DNA structures How can 1 m of DNA be packed into a nucleus of 10 µm of diameter? Chromatin Condensation The DNA associates to with special proteins: HISTONES Chromatin is compact DNA = DNA + histones 7.3. RNA STRUCTURE Polyribonucleotide formed by: Pentose: Ribose Nitrogenous bases: A, U, C and G RNA is a single strand molecule (except in some viruses), but rotation of the strand results in regions where nitrogenous bases face each other and are stabilized by H bonds. Double helix region (hairpin) This is responsible for the secondary structure important for the function of the RNA molecule. Complementary bases Loop SEVERAL TYPES OF RNA CONTAIN DIFFERENT STRUCTURES Secondary structures Primary structure Double helix region (hairpin) Loop Secondary structure Complementary bases Loop The association of loops and helix can give a TERTIARY STRUCTURE very complex (example in tRNA) Tertiary structure in a tRNA Complementary region RNA versus DNA Primary structure 7.3. RNA TYPES of RNA • Messenger RNA (mRNA) • Ribosomal RNA (rRNA) • Transfer RNA (tRNA) • Other RNA molecules: - microRNA (miRNA) - small Interference RNA (siRNA or iRNA) - small nuclear RNA (snRNA) - small nucleolar RNA (snoRNA) - large intervening non coding RNA (LincRNA) - iRNA 7.3. RNA MESSENGER RNA • It has only primary structure • Contains the genetic information to be translated into proteins. In Prokaryotes: • It does not have introns • 5’-end has a triphosphate In Eukaryotes: • 5’-end: methyl-guanosine + a triphosphate group: CAP (avoids degradation of the RNA molecule by cellular RNAases and it helps to be recognized by the ribosome to initiate the translation process). • 3’-end: polynucleotide tail: Poly A tail (avoids degradation and drives the RNA molecule migration from nucleus to cytoplasm) • It has exons (sequences with genetic information) and introns (sequences without genetic information). • Undergo a process of splicing (maturation of RNA molecule that eliminates introns) 7.3. RNA MESSENGER RNA Differences in mRNA molecules between prokaryotes and eukaryotes CAP exons eukaryotes introns polyA tail prokaryotes 7.3. RNA MESSENGER RNA in eukaryotic cells Mature mRNA molecule 7.3. RNA MESSENGER RNA in eukaryotes in prokaryotes Sometimes the mRNA molecule has information for more than one gene and a protein precursor is made and then in the cytoplasm is processed into the different protein. 7.3. RNA TRANSFER RNA • It transports the amino acids to the ribosomes • It has secondary structure and tertiary structure • All tRNA share some characteristics: - 5’-end: triplet with guanine and free-phosphoric acid - 3’-end: 3 unpaired bases (CCA). In the end the amino acid is attached - In the Anticodon loop (arm A) there is a triplet of bases called Anticodon, different for each tRNA depending on the amino acid they carry. Anticodon 3` UAC 5’ Codon 5 ‘ AUG 3` aminoacid methionin TRANSFER RNA 7.3. RNA STRUCTURE: The tRNA molecule has a distinctive folded structure with three hairpin loops that form the shape of a threeleafed clover. 1) One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon. 2) 3´end- OH. Each tRNA has its corresponding amino acid attached to its end. FUNCTION: tRNA recognizes and Mature tRNA molecule binds to its corresponding codon in the ribosome, the tRNA transfers the appropriate amino acid to the end of the growing amino acid chain. RIBOSOMAL RNA 7.3. RNA • A group of different RNAs that constitute an 80% of the total cellular RNA • Molecules of different sizes, with secondary structure and tertiary structure in some regions • It forms part of ribosomes. It contributes to have specific sites to fit a mRNA molecule and tRNAs at the same time. • It is responsible for the synthesis of proteins, specifically catalyses the formation of peptide bond (catalytic activity) Ribosome RNAs in a ribosome 7.3. RNA RIBOSOMAL RNA Processing of pre-rRNA and assembling of ribosomes. rRNA precurssors are synthetized in the nucleolus 7.3. RNA OTHER RNA molecules 1. Nucleolar RNA: It associates with different proteins to form the nucleolus. ribozyme 2. RNA in ribozymes (catalytic function) 3. Ribonucleoproteins: ex. RNA in SRP (signal recognition particle) important in the transport of proteins into the Endoplasmic reticulum (ER) SRP 7.3. RNA OTHER RNA molecules • iRNA: dsRNA (double stranded RNA) that interferes with gene expression. They are essential in: * Gene expression regulation * Defense mechanisms against exogen nucleic acids (pathogens). * Therapeutic potential. • miRNAs: Family of endogenous RNAs (more than 400 in humans). Regulatory role. 7.4 GENE CONCEPT What is a gene? In molecular terms, a gene is the entire nucleic acid sequence that is necessary for the synthesis of one functional molecule and that is inherited. DNA Transcription RNA PROTEINS Translation FLOW OF GENETIC INFORMATION Transcription Translation 7.5 GENE CONCEPT A GENE includes: -sequences that encode for the protein and/or a functional RNA- CODING REGION - sequences required for the synthesis of any RNA-- NON CODING REGION located in the 5´end and in the 3´-end (Enhancer, promoter, poly A sites), splicing sites and could include introns (eukaryotic). Diagram of an eukaryotic gene 7.4 GENE CONCEPT Genes in eukaryotes contain introns are usually monocistronics and Monocistronic: the coding region encodes information for only one protein. Gene 1 Gene 2 Gene X Gene DNA segment Chromosome Introns: sequences of DNA that are removed during RNA processing in the nucleus before the mRNA is exported to the cytosol. Usually introns are bigger in size than exons. Genes in eukaryotes are usually monocistronics and contain introns. In many cases introns in a gene are considerably longer that the exons. The median intron length in human is 3.3 Kb. In comparison most human exons contain only 50-200 base pairs. The typical human gene encoding a protein (50,000 pb long), 95% of that sequence consits of introns and flanking 5´end and 3´end. Genes in prokaryotes are polycistronic and do not contain introns. Polycistronic genes: include the coding region for several proteins that function together in a common biological proces The Chromosome in prokaryotes is circular without introns PROKARYOTIC The cluster of genes that forms a bacterial operon comprises a SINGLE TRANSCRIPTION UNIT that is transcribed from a specific promoter. In this case a single transcription unit contains several genes. EUKARYOTIC In eukaryotics, there are simple and complex eukaryotic transcription units. A simple transcription unit includes a region that encodes one protein, and introns are removed. 90% of human transcription units are complex. The primary mRNA transcript can be processed in more than a way-- ALTERNATIVE SPLICING In eukaryotic cells: Exon splicing is used to produce several mRNA transcripts for the same gen in different tissues- the number of proteins is expanded in higher organisms Splicing only occurs in eukaryotic genes Eukaryotic Prokaryotic 7.5 LOCATION OF NUCLEIC ACIDS IN A EUKARYOTIC CELL BASIC CONCEPTS • MONOMERIC UNITS FOUND IN NUCLEIC ACIDS: 1) Differences between nucleosides and nucleotides 2) Type of nucleotides according to its composition 3) Chemical bonds implicated in its formation 4) Recognition of the different nitrogen bases • DNA: structure, types, composition, cellular location and function. Types of bonds defining the DNA structure. Denaturalization of DNA • RNA: structure, types, composition, cellular location and function • Concept of gene: Differences in prokaryotes in Eukaryotes • Differences in prokaryotes and eukaryotes in the DNA and mRNA