Lecture Four Nucleotides & Nucleic Acids Chromatin: In the nuclei of eukaryotes, DNA is closely associated with proteins and RNA. These nucleoprotein complexes, with a DNA proportion of approximately one-third, are known as chromatin. Chromosomes: It is only during cell division that chromatin condenses into that are visible under light microscopy. Histones (B): The proteins contained in chromatin are called Histones (B): are small, strongly basic proteins (high content of lysine and arginine) that are directly associated with DNA. They contribute to the structural organization of chromatin, and their basic amino acids also neutralize the negatively charged phosphate groups, allowing the dense packing of DNA in the nucleus. This makes it possible for the 46 DNAmolecules of the diploid human genome, with their 5*109 base pairs (bp) and a total length of about 2 m, to be accommodated in a nucleus with a diameter of only 10μm. Histones also play a central role in regulating transcription. In the chromosome, the DNA is so densely packed that the smallest human chromosome already contains more than 50 million bp. Bases and nucleotides *The nucleic acids play a central role in the storage and expression of genetic information. They are divided into two major classes: deoxyribonucleic acid (DNA) & ribonucleic acids (RNAs). *All nucleic acids are made up from nucleotide components, which in turn consist of a (nitrogen base, a sugar, and a phosphate residue). Q/What are the differences between RNA & DNA A/ DNA RNA 1-Contain Thiamin base (No Uracil) 1-Contain Uracil base (No Thiamin) 2-Contain 2-Deoxy-ribose sugar 2- Contain ribose sugar 3-Predominantly Double stranded 3-Single chain 4- functions solely in information storage 4- Involved in most steps of gene expression and protein biosynthesis. 1-Sugars: DNA contains 2-Deoxy-ribose sugar while RNA contains ribose sugar. Their structures in addition to phosphate group are shown below: 1 2- Nitrogen bases: The bases that occur in nucleic acids are aromatic heterocyclic compounds derived from either pyrimidine or purine. Five of these bases are the main components of nucleic acids in all living creatures. The purine bases (adenine (abbreviation Ade, not “A”!) and guanine (Gua) and the pyrimidine base cytosine (Cyt), uracil (Ura) is only found in RNA. In DNA, uracil is replaced by thymine (Thy). # the 5-methyl derivative of uracil. 5-methylcytosine also occurs in small amounts in the DNA of the higher animals. A large number of other modified bases occur in tRNA and in other types of RNA. NH2 N O N N NH Purines: N H N H N N Adenine Guanine NH2 O N Pyrimidines: N H NH2 O NH O N H Thymine Cytosine NH O N H O Uracil Note: Small quantities of additional purines and pyrimidines occur in DNA and RNAs. Examples are shown below: Figure: four uncommon nitrogen bases found in human, bacteria & viruses nucleic acids Note: Some naturally occurring purines including caffeine (in coffee & tea), theobromine (in cacao), & theophylline (in tea) 2 Nucleosides: (Nitrogen base+Sugar) When a Nitrogen base is N-glycosidically linked to ribose or 2-deoxyribose , it yields a nucleoside. The nucleoside adenosine (abbreviation: A) is formed in this way from adenine and ribose, for example. The corresponding derivatives of the other bases are called guanosine (G), uridine (U), thymidine (T) and cytidine (C). *When the sugar component is 2-deoxyribose, the product is a deoxyribonucleoside. e. g., 2deoxyadenosine (dA). O NH2 N N NH N N N N HO N NH2 HO O O H H OH H H H H H H OH H H 2-Deoxyguanosine 2-Deoxyadenosine NH2 O O N NH NH N HO O N HO H OH O O H H H H 2-Deoxycytidine O HO O H N O H H OH H H 2-Deoxythymidine H H OH H OH H 2-Deoxyuridine Nucleotides: (Nitrogen base+Sugar+Phosphoric acid) In the cell, the 5-OH group of the sugar component of the nucleoside is usually esterified with phosphoric acid to produce nucleotides.e.g.2-Deoxythymidine (dT) therefore gives rise to 2-deoxythymidine-5-monophosphate (dTMP), one of the components of DNA. *If the 5_phosphate residue is linked via an acid–anhydride bond to additional phosphate residues, it yields nucleoside diphosphates and triphosphates—e. g., ADP and ATP, which are important coenzymes in energy metabolism. In nucleosides and nucleotides, the sugars and bases are linked by an N-glycosidic bond between the C-1 of the sugar and either the N-9 of the purine ring or N-1 of the pyrimidine ring. 3 O NH2 N N N O -O P N O N -O O P O H O- H OH H H H N NH2 O O H O- NH N H H OH H H 2-Deoxyguanosine-5-monophosphate 2-Deoxyadenosine-5-monophosphate O O NH2 NH NH N N O -O P -O O N O O P O OH OH H H H OH H OH H H N O -O O O H O P O O OH H 2-Deoxycytidine-5-monophosphate 2-Deoxythymidine-5-monophosphate O H H OH H OH 2-Deoxyuridine-5-monophosphate Importance of Nucleotides: nucleotides play a variety of important roles in all cells: 1-They are the precursors of DNA and RNA. 2-They are essential carriers of chemical energy (ATP and to some extent GTP). 3-They are components of the cofactors NAD, FAD, Sadenosylmethionine, and coenzyme A, 4- They are components of activated biosynthetic intermediates such as UDP-glucose and CDP-diacylglycerol. 5- cAMP and cGMP, are cellular second messengers.6- controlling numerous enzymatic reactions through allosteric effects on enzyme activity. 3́, 5́́́́ cyclic AMP (cAMP) methionine Adenosine triphosphate (ATP). S-adenosyl SYNTHETIC NUCLEOTIDE ANALOGS ARE USED IN CHEMOTHERAPY Synthetic analogs of purines& pyrimidines have numerous applications such as 1- The purine analog allopurinol, used in treatment of hyperuricemia and gout, inhibits purine biosynthesis and xanthine oxidase activity, 2- Treatment of cancer Q/Explain how some synthetic analogs of purines & pyrimidines used in the treatment of cancer?Give examples A/ Their toxic effects reflect 1-inhibition of enzymes essential for nucleic acid synthesis or 2their incorporation into nucleic acids with resulting disruption of base-pairing. Oncologists 4 employ 5-fluorouracil and 6-mercaptopurine, which are incorporated into DNA prior to cell division. #polynucleotides _ If the phosphate residue of a nucleotide reacts with the 3́-OH group of a second nucleotide, the result is a dinucleotide with a phosphoric acid diester structure. Dinucleotides of this type have a free phosphate residue at the 5́ -end and a free OH group at the 3́- end. They can therefore be extended with additional mononucleotides by adding further phosphoric acid diester bonds. This is the way in which polynucleotides {ribonucleic acid(RNA)}, while those consisting of deoxyribonucleotide monomers are called {deoxyribonucleicacid (DNA)}. RNA Ribonucleic acids (RNAs) are polymers consisting of nucleoside phosphate components that are linked by phosphoric acid diester bonds. The bases the contain are mainly uracil, cytosine, adenine, and guanine, but many unusual and modified bases are also found in RNAs. Functions: RNAs are involved in all the individual steps of gene expression and protein biosynthesis. Three main types of RNA: A. Ribosomal RNA (rRNA): The great majority of RNA which is a structural and functional component of ribosomes. Ribosomal RNA is produced from DNA by transcription in the nucleolus, and it is processed there and assembled with proteins to form ribosome subunits. The bacterial 16S-rRNA shown in Fig. A, with 1542 nucleotides (nt), is a component of the small ribosomae subunit, while the much smaller 5S-rRNA (118 nt) is located in the large subunit. B. Messenger RNAs (mRNAs): transfer genetic information from the cell nucleus to the cytoplasm. Due to the varying amounts of information that they carry, the lengths of mRNAs also vary widely. Their lifespan is usually short, as they are quickly broken down after translation. C. Transfer RNAs (tRNAs): function during translation. They are small RNA molecules consisting of 70–90 nucleotides, which “recognize” specific mRNA codons with their anticodons through base pairing. At the same time, at their 3_ end they carry the amino acid that is assigned to the relevant mRNA codon according to the genetic code. The molecule contains a high proportion of unusual and modified components. These include pseudouridine (Ψ), dihydrouridine (D), and many methylated nucleotides such as 7methylguanidine (m7G). Note: Small nuclear RNAs (snRNAs): are involved in the splicing of mRNA precursors. They associate with numerous proteins to form “spliceosomes.” 5 Figure: A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine (G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2′-deoxyribosyl moieties attached to the nucleobases by an N-glycosidic bond. Molecular genetics: overview Nucleic acids (DNA and various RNAs) are of central importance in the 1-storage, 2transmission, and 3-expression of genetic information. The decisive factor involved is their ability to enter into specific base pairings with each other. A.Expression and transmission of genetic information: *Storage: The genetic information of all cells is stored in the base sequence of their DNA (RNA only occurs as a genetic material in viruses). *Genes: are transcribable segments of DNA that code for inheritable structures or functions . It is estimated that the mammalian genome contains 30 000–40 000 genes, which together account for less than 5% of the DNA which consists of approximately 5x10 9 base pairs (bp). Most genes code for proteins—i. e., they contain the information for the sequence of amino acid residues of a protein (its sequence). *Codon: consisting of a sequence of three base pairs (a triplet) of DNA. Every amino acid residue is represented in DNA by a codon. e.g. A DNA codon for the amino acid phenylalanine, for example, is thus TTC. 6 I-Replication. During cell division, all of the genetic information has to be passed on to the daughter cells. To achieve this, the whole of the DNA is copied. In this process, each strand serves as a matrix for the synthesis of a complementary second strand. II-Transcription. For the genetic information stored in DNA to become effective, it has to be rewritten (transcribed) into mRNA. DNA only serves as a template and is not altered in any way by the transcription process. As DNA itself is not involved in protein synthesis, the information is transferred from the nucleus to the site of synthesis in the cytoplasm. To achieve this, the template strand in the relevant part of the gene is transcribed and leave the nucleus as messenger RNA (mRNA). *Transcription is catalyzed by DNA–dependent RNA polymerases enzyme. III-Translation. Mature mRNA enters the cytoplasm, where it binds to ribosomes, which convert the mRNA information into a peptide sequence. the DNA codes for the primary structure of proteins. The “language” used in this process has four letters (A, G, C, and T). All of the words (“codons”) contain three letters (“triplets”), and each triplet stands for one of the 20 proteinogenic amino acids. * The ribosomes consist of more than 100 proteins and several RNA molecules (rRNA). rRNA plays a role as 1-a ribosomal structural element and 2-involved in the binding of mRNA to the ribosome and 3-formation of the peptide bond. Amino acid activation. Before binding to the ribosomes, tRNAs are loaded with the correct amino acids by specific ligases. B. Amino acid activation: Some 20 different amino acid tRNA ligases in the cytoplasm each bind one type of tRNA with the corresponding amino acid. 7 Figure: (Amino acid activation; Formation of aminoacyl-tRNA DNA A. DNA: structure: deoxyribonucleic acids (DNAs) are polymeric molecules consisting of nucleotide building blocks. Instead of ribose, however, DNA contains 2́-deoxyribose, and the uracil base in RNA is replaced by thymine. It is observed that the amounts of adenine and thymine are almost equal and the same applies to guanine and cytosine. intact DNA consists of two polydeoxynucleotide molecules (“strands”). Each base in one strand is linked to a complementary base in the other strand by H-bonds. In addition, the two strands have to be intertwined to forma double helix. In all living cells, DNA serves to store genetic information. ■ Genetic information is encoded in the linear sequence of four deoxyribonucleotides in DNA. ■ The double-helical DNA molecule contains an internal template for its own replication and repair. . A two-step reaction, involving the enzymeaminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. A B Figure: A-Base pair rule: Cytosine & Guanosine bind by three H-bonds while Thymidine, or Uracil, & Adenosine bind by two H-bonds. B- Complementarity of strands in the DNA double helix.The complementary antiparallel strands of DNA follow the pairing Rules. 8 Figure: Watson-Crick model for the structure of DNA. Replication of DNA. The preexisting FIGURE: or “parent” strands become separated, and each is the Template for biosynthesis of a complementary “daughter” strand. 9