Week 9 Biochem Lec NUCLEIC ACIDS PDF

College of Medical Laboratory Science WEEK 9 BIOCHEM LEC: NUCLEIC ACIDS Trixie Anne R. Molina RMT, MLS(ASCPi)cm [email protected] LEARNING OBJECTIVES h At the end of the learning session, the students must be able to: u.p.ed D...

College of Medical Laboratory Science WEEK 9 BIOCHEM LEC: NUCLEIC ACIDS Trixie Anne R. Molina RMT, MLS(ASCPi)cm [email protected] LEARNING OBJECTIVES h At the end of the learning session, the students must be able to: u.p.ed Discuss briefly the importance of nucleic acids. a Explain accordingly the Central Dogma, m ti recombinant DNA and genetic engineering in a f using the genetic code @ lina mo tr CHARACTERISTICS OF NUCLEIC ACIDS Nucleic acids serve as genetic material of living organisms including humans. Biopolymers made up of monomeric units of NUCLEOTIDES Involved in the storage, transfer and expression of genetic information..ph u Contain all the necessary information required for the formation of individual or organism..e d m a Determines physical fitness of an individual to life. a t i f Found in nucleus Two Types of Nucleic Acids DNA: Deoxyribonucleica @ o lin Acid t rm RNA: Ribonucleic Acid CHARACTERISTICS OF NUCLEIC ACIDS Chemical nature of nucleic acids Acidic in nature Both DNA and RNA are polynucleotides They are polymers of nucleotides 3 components.ph Pentose sugar du.e Phosphate group a im Nitrogen-Containing Heterocyclic Bases t fa Phosphodiester linkage a@ olin trm NUCLEOTIDES Pentose Sugar Ribose Structure differences: a —OH group present on carbon 2’ in ribose a —H atom in 2-deoxyribose.p h d u a.e RNA and DNA differ in the identity of the sugar unit in their m nucleotides a t i @ f lina m o tr NUCLEOTIDES Phosphates Derived from phosphoric acid Under cellular pH conditions, the phosphoric acid is fully dissociated to give a hydrogen phosphate ion.ph Nitrogen-Containing Heterocyclic Bases du There are a total five bases a.e Three pyrimidine derivatives tim Two purine derivatives fa a@ olin trm NUCLEOTIDE FORMATION The formation of a nucleotide from sugar, base, and phosphate is visualized below. Phosphate attached to C-5’ and base is attached to C-1’ position of pentose.ph du a.e tim fa a@ olin trm NUCLEOTIDE FORMATION.ph du a.e tim fa a@ olin trm NUCLEOTIDE NOMENCLATURE.ph du a.e tim fa a@ olin trm NUCLEIC ACID STRUCTURE Primary structure of nucleic acids Nucleotide sequence of a polynucleotide It confers individuality to polynucleotide chain Primary structure is due to changes in the bases.ph Polynucleotide chain has direction. They are represented in 5' → 3' direction only. u Phosphodiester linkage runs in 3' → 5' direction. d.e Each poly nucleotide chain has two ends. The 5' end carrying phosphate is shown on a the left hand side and 3' end carrying unreacted hydroxyl is shown on the right hand tim a side. @f Primary structures of DNA and RNA exist in single stranded DNA and RNA organisms. lina mo tr NUCLEIC ACID STRUCTURE Primary structure of nucleic acids.ph du a.e tim fa a@ olin trm NUCLEIC ACID STRUCTURE Primary structure of nucleic acids DNA.ph RNA du a.e a nucleotide polymer in which each of the a nucleotide polymer in which each of the monomers im monomers contains deoxyribose, a phosphate contains ribose, a phosphate group, and one of the at group, and one of the heterocyclic bases adenine, f cytosine, guanine, or thymine heterocyclic bases adenine, cytosine, guanine, or uracil a@ olin trm NUCLEIC ACID STRUCTURE Comparison of the General Primary Structures of Nucleic Acids and Proteins Backbone: -Phosphate-Sugar- Nucleic acids.ph Backbone: -Peptide bonds - Proteins du a.e tim fa a@ olin trm DNA: DOUBLE HELIX Brief History of the Discovery of DNA DNA was initially discovered in cell nuclei by F. Miescher in 1869. Between 1949 and 1953, E. Chargaff and his team used quantitative.p h chromatographic techniques to separate the four DNA bases d u e concluding that A pairs with T, and G pairs with C. R. Franklin and M.H.F. Wilkins identifieda. two forms of DNA, known as the A tim and B conformations. In 1953, J.D. Watson @ fa n a and F.H.C. Crick proposed a three-dimensiona model of DNA’s istructure. l m o t r DNA: DOUBLE HELIX Deoxyribonucleic Acids Nucleic acids have secondary and tertiary structure The secondary structure involves two polynucleotide chains coiled around each other in a helical fashion. ph The relative molecular weight of DNA ranges from 1.6 × 10^6 Daltons. du for bacteriophage DNA to 1× 10^11 Daltons for a human chromosome. a.e Prokaryotes have circular DNAs while eukaryotes have linear DNAs. tim DNA molecules in eukaryotic cells are tightly packed into chromatin fa structures – euchromatin and heterochromatin. a Euchromatin @ olin Less densely packed DNA and consists of active genes. rm Heterochromatin t Consists of more tightly packed DNA Expression of genes in this region is limited DNA: DOUBLE HELIX Deoxyribonucleic Acids The poly nucleotides run anti-parallel (opposite directions) to each other, i.e., 5’ - 3’ and 3’ - 5’ h Adenine is always paired to thymine, and guanine is always u.p paired to cytosine via two hydrogen bonds and three.e d hydrogen bonds, respectively. This is called a Watson-Crick m a i type of Interaction fa Base composition: %A = %T and %Ct = %G) Example: Human DNA a @ contains 30% adenine, 30% thymine, o l 20% guanine andi n20% cytocine t r m DNA: DOUBLE HELIX DNA Sequence The sequence of bases on one polynucleotide is complementary to the other polynucleotide Complementary bases.ph Are pairs of bases in a nucleic acid structure that can hydrogen-bond to each other. Complementary DNA strands du a.e are strands of DNA in a double helix with base pairing such that each base is located tim opposite its complementary base. Example : fa a@ List of bases in sequential order in the direction from the 5’ end to 3’ end of the segment: olin m 5’-A-A-G-C-T-A-G-C-T-T-A-C-T-3’ tr Complementary strand of this sequence will be: 3’-T-T-C-G-A-T-C-G-A-A-T-G-A-5’ DNA: DOUBLE HELIX Replication of DNA Molecules Replication Process by which DNA molecules produce exact duplicates of themselves.p h d u Old strands act as templates for the synthesis DNA polymerase checks the correct.e of new strands m a base pairing and catalyzes ti the formation of phosphodiester linkages a has one new DNA strand and old DNA The newly synthesizedfDNA a @ strand o lin t rm DNA: DOUBLE HELIX Replication of DNA Molecules DNA polymerase enzyme can only function in the 5’-to-3’ direction Therefore one strand (top; leading strand ) grows continuously in the direction of unwinding.p h The lagging strand grows in segments (Okazaki d u fragments) in the opposite direction a.e The segments are latter connected t im by DNA ligase f a at multiple sites within a molecule @ DNA replication usually occurs a li n (origin of replication) o t rm DNA replication Multiple-site is bidirectional from these sites (replication forks) replication enables rapid DNA synthesis DNA: DOUBLE HELIX Chromosomes Upon DNA replication the large DNA molecules interacts with histone proteins to fold long DNA molecules. Histone–DNA complexes.p h d u.e About 15% by mass DNA and 85% by mass Chromosomes occur in matcheda protein tim (homologous) pairs fa a @ o lin tr m DENATURATION OF DNA Melting of DNA Melting point of DNA is known as Tm If the heat denatured DNA is cooled base pairing occurs between.ph strands and reformation of double, stranded molecule takes place. du a.e tim fa a@ olin trm OTHER FORMS OF DNA Most of the DNA in the genome is in B-form Other forms of DNA are: A-DNA When DNA fibre is dehydrated it acquires another form. It is known as A-DNA..ph It is shorter than B-DNA. The base pairs are not perpendicular to the axis they are du e tilted by 19°. a. In A and B forms, glycosidic bonds are in ‘anti’ conformation. Z-DNA tim fa Left handed double helix. a@ A small stretch of Z-DNA can occur in B-DNA. olin Z-DNA is due to the presence of dinucleotides like CG CG CG containing alternate rm purine and pyrimidine bases. t In Z-DNA, glycosidic bonds are in syn conformation OTHER FORMS OF DNA.ph du a.e tim fa a@ olin trm FUNCTIONS OF DNA Genetic material of living systems. The genetic information in DNA is converted to characteristic features of living organisms like colour of the skin and eye, height, intelligence,.p h ability to metabolize particular substance, ability to with stand stress, susceptibility to disease and unable to produce d u or synthesize certain substances etc. a.e tim fa Source of information for the synthesis of all cellular proteins. @ DNA is transmitted from parent to off spring and hence DNA flows from a l in one generation to other o in a given species. t rm OVERVIEW OF PROTEIN SYNTHESIS Protein synthesis is directly under the direction of DNA Proteins are responsible for the formation of skin, hair, enzymes, hormones, and so on Protein synthesis can be divided into two phases..p h d u Transcription – A process by which DNA directs the synthesis of mRNA molecules a. e tim fa Translation – a process in which mRNA is deciphered to synthesize a protein molecule a @ o lin t r m DIFFERENCES BETWEEN DNA AND RNA.ph du a.e tim fa a@ olin trm RIBONUCLEIC ACIDS (RNAS) Present in nucleus and cytoplasm of eukaryotic cells. They are involved in the transfer and expression of genetic information. They act as primers for DNA formation..p h d u.e Some RNA act as enzymes as well as coenzymes. RNA also function as genetic materiala tim for viruses. f a a @ o lin tr m RIBONUCLEIC ACIDS (RNAS) Chemical nature of ribonucleic acids RNAs are also polynucleotides. In RNA polymer, purine and pyrimidine nucleotides are linked together through phosphodiester linkage. The sugar present in a RNA is ribose..ph du e Three Major classes Messenger RNA or m-RNA a. tim Ribosmal RNA or r-RNA fa Transfer RNA or t-RNA a @ Minor Classes o lin m Heterogeneous nuclear RNA (hnRNA) r t nuclear RNA (snRNA) small small cytoplasmic RNA (scRNA) MAJOR TYPES OF RNA MOLECULES Messenger RNA or m-RNA It accounts for 1-5% of cellular RNA Structure: Majority of mRNA has primary structure. ph They are single-stranded linear molecules.. du mRNA molecules have free or phosphorylated 3’ and 5’ end..e mRNA molecules have different life spans. a im Eukaryotic mRNA are more stable than prokaryotic mRNA. fat The mRNA nucleotide sequence is complementary from which it is synthesized or copied. a@ lin Some prokaryotic mRNA has secondary structure. o Intrastrand base paring among complementary bases allows folding of liner trm molecule. As a result hairpin, or loop like secondary structure is formed. MAJOR TYPES OF RNA MOLECULES Messenger RNA or m-RNA Functions: Carries instructions for protein synthesis (genetic information) from DNA. The molecular mass of mRNA varies with the length of the protein ph Usually a molecule of mRNA contains information required for the formation of one. protein molecule. du.e Genetic information is present in mRNA in the form of genetic code. a im Some times single mRNA may contain information for the formation of more than one protein. fat a@ olin trm MAJOR TYPES OF RNA MOLECULES Transfer RNA t-RNA accounts for 10-15% of total cell RNA Structure Smallest of all the RNAs Single strand molecules.ph du Many unusual bases 7-15 per molecule.e Methylated adenine, guanine, cytosine and thymine, dihydrouracil, pseudo a im uridine, isopentenyl adenine etc. fat These unusual bases are important for binding of t-RNA to ribosomes @ About half of the nucleotides in t-RNA are involved in intrachain base pairing a lin Double helical segments o Some bases are not involved in the base pairing trm Loops and arms formation in t-RNA Folding in primary structure which generate secondary structure MAJOR TYPES OF RNA MOLECULES Transfer RNA Secondary structure of t-RNA Form of clover leaf Tertiary structure of t-RNA X-ray diffraction analysis ph Three-dimentional structure of t-RNA looks like inverted or tilted L. u Functions:.ed Carrier of amino acids to the site of protein synthesis. a There is at least one t-RNA molecule to each of 20 amino acids required for protein synthesis. m ti Eukaryotic t-RNAs are less stable where as prokaryotic RNAs are more stable fa a@ olin trm MAJOR TYPES OF RNA MOLECULES Ribosomal RNA Accounts for 80% of total cellular RNA It is present in ribosomes r-RNA is found in combination with protein. ph The length of r-RNA ranges form 100-600 nucleotides. du Both prokaryotic and eukaryotic ribosomes contain r-RNA molecules.e 4 types of r-RNAs in eukaryotes. a im 5, 5.8, 18 and 28S r-RNA molecules fat 3 types of r-RNA molecules in Prokaryotes @ 5, 16 and 23S r-RNA molecules a Structure: olin Secondary structure trm Intra strand base pairing between complementary base generates double helical segments or loops. MAJOR TYPES OF RNA MOLECULES Ribosomal RNA Functions Combines with specific proteins to form ribosomes r-RNAs are required for the formation of ribosomes.ph 16S RNA is involved in initiation of protein synthesis. du a.e tim fa a@ olin trm MINOR TYPES OF RNAS Heterogeneous nuclear RNA (hnRNA) Formed directly by DNA transcription. Post-transcription processing converts the hnRNA to mRNA small nuclear RNA (snRNA).ph Facilitates the conversion of hnRNA to mRNA. du e Contains from 100 to 200 nucleotides a. small cytoplasmic RNA (scRNA) tim a A class of small, non-coding RNA molecules that are found in the cytoplasm of @f eukaryotic cells. lina Primarily in the regulation of RNA and protein synthesis. mo tr TRANSCRIPTION: RNA SYNTHESIS Transcription: A process by which DNA directs the synthesis of mRNA molecules Gene: A segment of a DNA base sequence responsible for the production of a specific hnRNA/mRNA molecule Genome: All of the genetic material (the totalu h.p contained in d DNA) the chromosomes of an organism a.e tim fa a @ o l in trm STEPS IN TRANSCRIPTION PROCESS Unwinding of DNA double helix to expose some bases (a gene): The unwinding process is governed by RNA polymerase Alignment of free ribonucleotides along the exposed DNA strand (template) forming new base pairs.p h d u RNA polymerase catalyzes the linkage of ribonucleotides one by one to form mRNA molecule a.e tim Transcription ends when the RNA polymerase enzyme encounters a fa @ stop signal on the DNA template: The newly formedaRNA molecule and the RNA polymerase enzyme areo lin tr m released POST-TRANSCRIPTION PROCESSING: FORMATION OF MRNA Involves conversion of hnRNA to mRNA Splicing: Excision of introns and joining of exons Exon - a gene segment that codes for genetic information.p h Intron – a DNA segments that interrupt a genetic message The splicing process is driven by snRNA d u Alternative splicing a.e A process by which severalim f a t different protein variants are @ produced from a single gene a lin The process involves o excision of one or more exons t rm TRANSCRIPTOME Transcriptome: All of the mRNA molecules that can be generated from the genetic material in a genome. Transcriptome is different from a genome.p h d u Responsible for the biochemical complexity created by splice variants obtained by hnRNA. a.e tim fa a @ o lin tr m THE GENETIC CODE The base sequence in a mRNA determines the amino acid sequence for the protein synthesized. The base sequence of an mRNA molecule involves only 4 different bases Codon.ph A three-nucleotide sequence in an mRNA molecule that codes for a specifi c amino acid du Genetic code a.e tim The assignment of the 64 mRNA codons to specific amino acids (or stop signals) fa a@ 3 of the 64 codons are termination codons (“stop” signals) olin trm CHARACTERISTICS OF GENETIC CODE The genetic code is highly degenerate: Many amino acids are designated by more than one codon. Arg, Leu, and Ser - represented by six codons. Most other amino acids - represented by two codons.ph Met and Trp - have only a single codon. du e Codons that specify the same amino acid are called synonyms a. There is a pattern to the arrangement of synonyms in the genetic code table. tim All synonyms for an amino acid fall within a single box in unless there are fa @ more than four synonyms ina The significance of the “single box” pattern - the first two bases are the same l o For example, the four synonyms for Proline - CCU, CCC, CCA, and CCG. m tr CHARACTERISTICS OF GENETIC CODE The genetic code is almost universal: With minor exceptions the code is the same in all organisms The same codon specifies the same amino acid whether the cell is a bacterial cell, a corn plant cell, or a human cell. An initiation codon exists:.ph du e The existence of “stop” codons (UAG, UAA, and UGA) suggests the existence of “start” codons. a. tim The codon - coding for the amino acid methionine (AUG) functions as fa @ initiation codon. lina mo tr ANTICODONS AND TRNA MOLECULES During protein synthesis amino acids do not directly interact with the codons of an mRNA molecule. tRNA molecules as intermediaries deliver amino acids to mRNA. Two important features of the tRNA structure.ph The 3’ end of tRNA is where an amino acid is covalently bonded to the tRNA. du e The loop opposite to the open end of tRNA is the site for a sequence of three a. bases called an anticodon. Anticodon tim fa @ a three-nucleotide sequence on a tRNA molecule that is complementary to a ina codon on an mRNA molecule. l mo tr TRANSLATION: PROTEIN SYNTHESIS Translation Process in which mRNA codons are interpreted to create a protein. Ribosome an rRNA–protein complex.ph Serves as the site of protein synthesis du e Contains 4 rRNA molecules and ~80 proteins - packed into two rRNA-protein a. subunits (one small and one large) tim ~65% rRNA and 35% protein by mass fa @ A ribosome’s active site – Large subunit ina Ribosome is a RNA catalyst l o The mRNA binds to the small subunit of the ribosome. m tr FIVE STEPS OF TRANSLATION PROCESS Activation of tRNA Addition of specific amino acids to the 3’-OH group of tRNA. Initiation of protein synthesis: Begins with binding of mRNA to small ribosomal subunit such that its first.ph codon (initiating codon AUG) occupies a site called the P site du e Elongation: a. Adjacent to the P site in an mRNA–ribosome complex is A site and the next tim tRNA with the appropriate anticodon binds to it. fa @ Termination: ina The polypeptide continues to grow via translocation until all necessary amino l o acids are in place and bonded to each other. m tr Post-translational processing Gives the protein the final form it needs to be fully functional EFFICIENCY OF MRNA UTILIZATION Polysome (polyribosome) Complex of mRNA and several ribosomes Many ribosomes can move simultaneously along a single mRNA molecule The multiple use of mRNA molecules reduces the amount of resources and.ph energy that the cell expends to synthesize needed protein. du e In the process – several ribosomes bind to a single mRNA - polysomes. a. tim fa a@ olin trm MUTATION An error in base sequence reproduced during DNA replication Errors in genetic information is passed on during transcription. The altered information can cause changes in amino acid sequence during h protein synthesis and thereby alter protein function u.p Such changes have a profound effect on an organism..ed ma ati @f lina mo tr MUTAGENS Mutations are caused by mutagens A mutagen is a substance or agent that causes a change in the structure of a gene: h Radiation and chemical agents are two important types of mutagens u.p Ultraviolet, X-ray, radioactivity and cosmic radiation are mutagenic.e d Chemical agents can also have mutagenic effects m a i Under normal conditions mutations are repaired by repair enzymes f a t a @ o li n trm NUCLEIC ACIDS AND VIRUSES Viruses Tiny disease causing agents with outer protein envelope and inner nucleic acid core.p h They can not reproduce outside their host cells (living organisms) d u Invade their host cells to reproduce and in the process disrupt the.e Virus invade bacteria, plants animals, a normal cell’s operation tim and humans fa a @ o lin t r m NUCLEIC ACIDS AND VIRUSES Viruses attach to the host cell on the outside cell surface and proteins of virus envelope catalyze the breakdown of the cell membrane and forms a hole Viruses then inject their DNA or RNA into the host cell.p h d u The viral genome is replicated, proteins coding for the viral envelope are produced in hundreds of copies. a.e tim Hundreds of new viruses are produced using the host cell replicated f a genome and proteins in short time a @ o lin t rm VACCINES Inactive virus or bacterial envelope Antibodies produced against inactive viral or bacterial envelopes will kill the active bacteria and viruses.ph du a.e tim fa a@ olin trm RECOMBINANT DNA AND GENETIC ENGINEERING DNA molecules that have been synthesized by splicing a sequence of segment DNA (usually a gene) from one organism to the DNA of another organism Genetic Engineering (Biotechnology): p The study of biochemical techniques that allow.the h d u transfer of a “foreign” gene to a host organism and a.e produce the protein associated with the added gene tim Bacterial strains such as E.acoli inserted with circular plasmids, @ f in a and/or yeast cells carrying l vectors containing foreign genes are o used for this purpose m tr Plasmids (double stranded DNA) replicate independently in bacteria or yeast RECOMBINANT DNA AND GENETIC ENGINEERING Transformed cell can reproduce a large number of identical cells – clones: Clones are the cells that have descended from a single cell and have identical DNA Given bacteria grow very fast, within few hours 1000s.p h d u of clones will be.e Each clone can synthesize the protein a produced t im directed by foreign gene it carries f a a @ o lin trm RECOMBINANT DNA PRODUCTION USING A BACTERIAL PLASMID Dissolution of cells: E. coli cells of a specific strain containing the plasmid of interest are treated with chemicals to dissolve their membranes and release the cellular contents.p h Isolation of plasmid fraction: d u a.e The cellular contents are fractionated to obtain plasmids Cleavage of plasmid DNA: t im Restriction enzymes are used fa to cleave the double-stranded DNA a @ o lin t r m RECOMBINANT DNA PRODUCTION USING A BACTERIAL PLASMID Gene removal from another organism: Using the same restriction enzyme the gene of interest is removed from a chromosome of another organism Gene–plasmid splicing: p The gene (from Step 4) and the opened plasmid.(from h d u Step 3) are a mixed in the presence of the enzyme DNA.e ligase to splice them together. Uptake of recombinant DNA: a ti m The recombinant DNA @ f lin a prepared in step 5 are transferred to a live E. m o coli culture where they can be replicated, transcribed and t r translated. POLYMERASE CHAIN REACTION The polymerase chain reaction (PCR) a method for rapidly producing multiple copies of a DNA nucleotide sequence (gene). This method allows to produce billions of copies of a specific gene in a few hours..p h d u e PCR is very easy to carryout and the requirements are: Source of gene to be copied a. t im a(dATP, dGTP, dCTP and dTTP) Thermostabel DNA polymerase @ Deoxynucleotide triphosphatesf lin a A set of two oligonucleotides with complementary sequence to the m gene (primers)o r t plastic container Thermostable Source of heat DNA SEQUENCING DNA sequencing is a method by which the base sequence in a DNA molecule (or a portion of it) is determined. Discovered in 1977 by Fredrick Sanger Concept in DNA sequencing: Selective interruption of polynucleotide.ph synthesis using 2’,3’- du e dideoxyribonucleotide triphosphates (ddNTPs). a. tim fa a @ o lin tr m DNA SEQUENCING This interruption of synthesis leads to the formation of every possible nucleotide site mixture. These nucleotides are labeled using radioactive dNTP during their synthesis..ph The radiolabeled nucleotides are then separated on a gel by du e electrophoresis a. tim fa a@ o lin trm BASIC STEPS INVOLVED IN DNA SEQUENCING Step 1: Cleavage of DNA using restriction enzymes: Restriction enzymes are used to cleave the large DNA molecule into smaller fragments (100–200 base pairs). Step 2: Separation into individual components:.p h The mixture of small DNA fragments generated by the restriction du e enzymes is separated into individual components via gel electrophoresis techniques. a. Step 3: Separation into single strands:im f t a into its two strands by chemical @ A given DNA fragment is separated methods to use it as aa o lin template in step 4. t rm BASIC STEPS INVOLVED IN DNA SEQUENCING.ph du a.e tim fa a@ olin trm REFERENCES Rao, M.N. Medical Biochemistry, 2nd edition, New Age International Publishers, 2006 Stoker, S.H. General, Organic, and Biological Chemistry, 6th edition, 2013, (pp. 734-771), Brooks/COLE Cengage Learning  College of Medical Laboratory Science QUESTIONS? Trixie Anne R. Molina RMT, MLS(ASCPi)cm  College of Medical Laboratory Science THANK YOU SO MU- Trixie Anne R. Molina RMT, MLS(ASCPi)cm