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Biomolecules Nucleic acids Nucleic acids { { { August2010 Nucleic acids are polynucleotides joined together to make large macromolecules. The important nucleic acids are deoxyribonucleic acid (DNA) and various types of ribonucleic acids (RNA). DNA is the genetic material found in cells and contains instructions that help determine the structure and function of cells HBC108/2010 UON EGK 2 Nucleic acids They are polynucleotides { The primary structure of DNA and RNA is a linear arrangement of nucleotides { Formed by the condensation of two or more nucleotides. { August2010 HBC108/2010 UON EGK 3 Nucleic acids { { { August2010 The condensation most commonly occurs between the 3'-hydroxyl of one nucleotide and the OH of a 5'-phosphate of a second nucleotide with the elimination of H2O, forming a phosphodiester bond Success nucleotides of both DNA and RNA are covalently linked to each other through a phosphate gp HBC108/2010 UON EGK 4 Formation of nucleic acids { The formation of phosphodiester bonds in DNA and RNA exhibits directionality { It proceeds in the 5' ----> 3' direction The primary structure of DNA or RNA molecules is represented with the nucleotide sequences written from left to right { { with the 5' -----> 3' direction as shown: 5'- GATC-3' August2010 HBC108/2010 UON EGK 5 Structure of DNA molecule { { { { August2010 DNA encodes the genetic information It is a helix of two strands wound around each other The chains run in opposite direction (antiparallel). The two strands form a "double helix" structure, which was first discovered by James D. Watson and Francis Crick in 1953. HBC108/2010 UON EGK 6 Mechanism of DNA formation { { { { { August2010 DNA polymerase catalyzes DNA synthesis The enzyme requires a DNA template 3'-OH of one sugar attacks the 5' phosphate of a deoxyribonucleoside triphosphate Each successive nucleotide residue is added to the 3' end of the nucleic acid. Chain growth is always in the 5' -> 3' direction!!! HBC108/2010 UON EGK 7 DNA base pairing { { { August2010 A DNA molecule has two strands, held together by the hydrogen bonding between their bases. Purine bases of one chain form hydrogen bonds with pyrimidines of the other chain in the crucial phenomenon of base pairing Adenine can form two hydrogen bonds with thymine HBC108/2010 UON EGK 8 DNA base pairing Cytosine can form three hydrogen bonds with guanine. { (C:G) and (A:T) base pairs found in natural DNA molecules are very strong { Other base pairs e.g (G:T) and (C:T) may also form hydrogen bonds but are not as strong as those in natural DNA { August2010 HBC108/2010 UON EGK 9 Base pairing G-C Base Pair A-T Base Pair Base pairings In any given molecule of DNA, the concentration of adenine (A) is equal to thymine (T). { and the concentration of cytosine (C) is equal to guanine (G). { This means that A will only base-pair with T, and C with G. { August2010 HBC108/2010 UON EGK 11 Base pairings According to this pattern, known as Watson-Crick base-pairing, the basepairs composed of G and C contain three H-bonds, whereas those of A and T contain two H-bonds. { This makes G-C base-pairs more stable than A-T base-pairs. { August2010 HBC108/2010 UON EGK 12 Complementarity of DNA strands { { { { { August2010 They obey the (A:T) and (C:G) pairing rule. Due to the specific base pairing, DNA's two strands are complementary to each other. Hence, the nucleotide sequence of one strand determines the sequence of another strand. The sequence of the two strands can be written as 5' -ACT- 3‘ OR 3' -TGA- 5‘ Thus the chains are anti-parallel HBC108/2010 UON EGK 13 Base pairing between DNA's two strands Phosphodiester bonds Orientation of the chains The anti-parallel nature of the helix stems from the orientation of the individual strands. { From any fixed position in the helix, one strand is oriented in the 5' ---> 3' direction and the other in the 3' ---> 5' direction. { August2010 HBC108/2010 UON EGK 15 Orientation of the chains The backbone consists of the phosphate gp linking the deoxyribose sugars { The purine and pyrimidine bases face inside the double helix where A-T & G-C base pairs are formed { The double helix of DNA contains two deep grooves between the deoxyribosephosphate chains { August2010 HBC108/2010 UON EGK 16 Double helix structure The normal righthanded "double helix" structure of DNA, also known as the B form. August2010 HBC108/2010 UON EGK 17 The Double Helix-summary 1. The backbone of each strand consists of alternating deoxyribose and phosphate groups. 2. The two strands are "antiparallel” 3. The DNA strands are assembled in the 5′ to 3′ direction 4. Each base forms hydrogen bonds with the one directly opposite it, forming watson-crick pairs Forms of DNA In solution, the double helix of DNA has been shown to exist in several different forms. { This forms depends upon sequence content and ionic conditions { Exists as B or Z-form. { August2010 HBC108/2010 UON EGK 19 B-form of DNA { { { August2010 In B-form structure, DNA prevails under physiological conditions of low ionic strength and a high degree of hydration. Hence, there are about 10 pairs of nucleotides per turn This form make two grooves of different widths, referred to as the major groove and the minor groove, which may facilitate binding with specific proteins. HBC108/2010 UON EGK 20 Z-form In a Z form structure the DNA bases seem to zigzag. { The DNA molecule with alternating G-C sequences in alcohol or high salt solution tends to have such structure. { One turn spans 4.6 nm, comprising 12 base pairs. { August2010 HBC108/2010 UON EGK 21 Forms of DNA Comparison between B form and Z form August2010 HBC108/2010 UON EGK 22 Ribonucleic acid (RNA) { { RNA is a nucleic acid polymer consisting of ribonucleotide monomers RNA plays several important roles in the processes that translate genetic information from deoxyribonucleic acid (DNA) into protein products; z z z August2010 RNA acts as a messenger between DNA and the protein synthesis complexes known as ribosomes, It forms vital portions of ribosomes It acts as an essential carrier molecule for amino acids to be used in protein synthesis HBC108/2010 UON EGK 23 Comparison with DNA RNA and DNA differ in three main ways { Unlike DNA which is double-stranded, RNA is a single-stranded molecule in most of its biological roles and has a much shorter chain of nucleotides { Secondly, while DNA contains deoxyribose, RNA contains ribose sugar { August2010 HBC108/2010 UON EGK 24 Structures of RNA and DNA Comparison with DNA { { { August2010 These hydroxyl groups make RNA less stable than DNA because it is more prone to hydrolysis. Several types of RNA (tRNA, rRNA) contain a great deal of secondary structure, which help promote stability. Thirdly, the base-pair of adenine is not thymine, as it is in DNA, but rather uracil, which is a unmethylated form of thymine. HBC108/2010 UON EGK 26 Messenger RNA (mRNA) { { { August2010 Messenger RNA is RNA that carries information from DNA to the ribosome sites of protein synthesis in the cell. In eukaryotic cells, once mRNA has been transcribed from DNA, it is exported from the nucleus into the cytoplasm, where it is bound to ribosomes and translated into its corresponding protein In prokaryotic cells, mRNA can bind to ribosomes while it is being transcribed from DNA. HBC108/2010 UON EGK 27 Genetic Code Transfer RNA (tRNA) { { { August2010 Transfer RNA is a small RNA chain of about 74-95 nucleotides that transfers a specific amino acid to a growing polypeptide chain at the ribosomal site of protein synthesis during translation. It has sites for amino-acid attachment and an anticodon region for codon recognition that binds to a specific sequence on the messenger RNA chain through hydrogen bonding. It is a type of non-coding RNA HBC108/2010 UON EGK 29 Structure of tRNA: Modified bases such as dihydrouridine (D), pseudouridine (ψ), inosine (I) and methylguanine are present. Ribosomal RNA (rRNA) { { { { { August2010 Ribosomal RNA is the catalytic component of the ribosomes, the protein synthetic factories in the cell. Eukaryotic ribosomes contain different rRNA molecules from prokaryotic ribosomes rRNA molecules are extremely abundant and make up at least 80% of the RNA molecules found in a typical eukaryotic cell. In the cytoplasm, ribosomal RNA and protein combine to form a nucleoprotein called a ribosome. The ribosome binds mRNA and carries out protein synthesis. HBC108/2010 UON EGK 31 Catalytic RNA Certain RNAs are able to catalyse chemical reactions. { These include cutting and ligating other RNA molecules and also the catalysis of peptide bond formation in the ribosome Double-stranded RNA { Double-stranded RNA (or dsRNA) is RNA with two complementary strands, similar to DNA { dsRNA forms the genetic material of some viruses { August2010 HBC108/2010 UON EGK 32 Chemical Stability of Nucleic Acids Hydrolysis by acids and alkali { DNA is generally quite stable. It will resist attack in acid and alkali solutions. { However, in mild acid solutions - at pH4 - the β-glycosidic bonds to the purine bases are hydrolyzed { Protonation of purine bases (N7 of guanine, N3 of adenine) occurs at this pH August2010 HBC108/2010 UON EGK 33 Chemical Stability of Nucleic Acids { { { August2010 Protonated purines are good leaving groups hence the hydrolysis In contrast to DNA, RNA is very unstable in alkali solutions due to hydrolysis of the phophodiester backbone. The 2'OH group in ribonucleotides renders RNA molecules susceptible to strand cleavage in alkali solutions HBC108/2010 UON EGK 34 Hydrolysis by enzymes Enzymatic hydrolysis of DNA { DNA is hydrolyzed by deoxyribonucleases. { These enzymes hydrolyze the phosphodiester linkages { These enzymes may digest a DNA strand from the end(s) - exonucleases - or internally endonucleases. Enzymatic hydrolysis of RNA { Ribonucleases are enzymes that cleave RNA August2010 HBC108/2010 UON EGK 35 Thermal properties of DNA DNA can be copied (replicated). { Each daughter cell acquires the same amount of genetic material as the mother cell. { The two strands of the helix must first be separated, in a process termed denaturation. { August2010 HBC108/2010 UON EGK 36 Denaturation of DNA This process can also be carried out in vitro by extreme pH and Temperature (80-90ºC). { If a solution of DNA is subjected to high temperature, the H-bonds between bases become unstable and the strands of the helix separate. { The process is referred to as thermal denaturation. { August2010 HBC108/2010 UON EGK 37 Melting temperatures During thermal denaturation, a point is reached at which 50% of the DNA molecule exists as single strands. { This point is the melting temperature (TM), and is characteristic of the base composition of that DNA molecule { August2010 HBC108/2010 UON EGK 38 Annealing When thermally melted DNA is cooled, the complementary strands will again reform the correct base pairs, in a process is termed annealing or hybridization { The rate of annealing is dependent upon the nucleotide sequence of the two strands of DNA. { August2010 HBC108/2010 UON EGK 39 Base compositions The base composition of DNA varies widely from molecule to molecule and even within different regions of the same molecule. { Regions of the duplex that have predominantly A-T base-pairs will be less thermally stable than those rich in G-C base-pairs. { August2010 HBC108/2010 UON EGK 40 Spontaneous causes of Base alterations Tautomerization { The bases of DNA are subject to spontaneous structural alterations called tautomerization { These bases are capable of existing in two forms between which they interconvert. { The various tautomer forms of the bases have different pairing properties { For example, guanine and Thymine can exist in keto or enol forms { The keto form of guanine is favored August2010 HBC108/2010 UON EGK 41 Tautomerization { If G is in the enol form, it base pair with T instead of the normal C . The result is a G:C to G:T transition Spontaneous Base alterations Deamination { Another mutagenic process occurring in cells is spontaneous base degradation. { The deamination of cytosine to uracil happens at a significant rate in cells. { Deamination can be repaired by a specific repair process which detects uracil, not normally present in DNA { otherwise the U will cause A to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated. August2010 HBC108/2010 UON EGK 43 Spontaneous base alterations { { { { August2010 Deamination of methylcytosine to thymine also occurs. Methylcytosine occurs in the human genome If the meC is deaminated to T, there is no repair system which can recognize and remove it (because T is a normal base in DNA). This means that wherever CpG occurs in genes it is a "hot spot" for change. HBC108/2010 UON EGK 44 August2010 HBC108/2010 UON EGK 45