Molecular Biology (BIO 206) PDF

MOLECULAR BIOLOGY [BIO 206] Nucleic Acids Structure By Dr. Maha M Salah Assistant Professor of Medical Biochemistry and Molecular Biology By the end of this lecture, the student should be able to: 1. Describe the primary, secondary and tertiary structure of DNA. 2. Describe the composition and...

MOLECULAR BIOLOGY [BIO 206] Nucleic Acids Structure By Dr. Maha M Salah Assistant Professor of Medical Biochemistry and Molecular Biology By the end of this lecture, the student should be able to: 1. Describe the primary, secondary and tertiary structure of DNA. 2. Describe the composition and structure of DNA, based on the Watson–Crick model, including the concepts of directionality and complementarity in DNA structure. 3. Define DNA denaturation and melting temperature. 4. Identify the differences between the A, B and Z-forms of DNA. 5. Mention the types of DNA supercoiling. 6. Explain briefly the structure and importance of the mitochondrial DNA. BIO 206 Molecular Biology § It deals with biological phenomena at the molecular level. § It is the study of DNA and RNA, proteins and other macromolecules involved in genetic information and cell function. § DNA stores the genetic information that controls all cellular processes. BIO 206 Central Dogma NUCLEIC ACIDS BIO 206 Introduction § Nucleic acids are polymers of nucleotides. § There are 2 main types of nucleic acids: a) Deoxyribonucleic acids (DNA) b) Ribonucleic acids (RNA). § Nucleic acids are responsible mainly for storage and transfer of information needed for production of proteins. BIO 206 Deoxyribonucleic Acids (DNA) - It consists of two strands of polynucleotides, and it has the following primary, secondary and tertiary structures: I- DNA Primary Structure 1. Primary structure describes the sequence of nucleotides in each strand. 2. The nucleotides that form DNA primary structure are mainly four: dAMP, dGMP, dCMP and dTMP. 3. In each strand, the nucleotides are linked together by phosphodiester bonds between the 5`-hydroxyl of one nucleotide and 3`-hydroxyl of the next nucleotide. BIO 206 4. The alternating sugar phosphate units form the backbone of each DNA strand (5`-P-S-P-S-3`). 5. The nitrogenous bases, which are linked to the pentoses, are projecting to the inside of the two strands of DNA at right angle. 6. The sequence of bases determines the coding structure of DNA (genetic information). 7. Each polynucleotide strand has two terminals. One end has a free phosphate group attached to 5`-hydroxyl group of the terminal pentose and the other end has a free 3`- hydroxyl group. BIO 206 8. The order of nucleotides in any DNA strand is always written in the 5` to 3` direction, which is the direction of synthesis e.g., AGCT or 5ˋAGCT-3ˋ for the following chain in the figure below. II- DNA Secondary Structure : Double Helix (dsDNA) q Watson and Crick proposed a structure for DNA in the form of a double helix and it is now referred to as B-form of DNA, which is the most common physiological form. BIO 206 q It has the following characters: 1- Two antiparallel strands form a RightHanded Helix ü The two strands of DNA are paired to each other and coil around a common axis to form a right-handed helix. The sugar phosphate backbone lies on the outside of the molecules, while the nitrogenous bases project inward perpendicular to the long axis. ü The two strands run antiparallel, i.e., one runs in the 5` to 3` direction and the other in the 3` to 5` direction. BIO 206 2- Complementary base pairing ü The two strands are held together by the complementary base pairing through specific hydrogen bonds. ü Adenine pairs with thymine through two hydrogen bonds, and guanine pairs with cytosine through three hydrogen bonds. ü Therefore, the number of adenine bases equals the number of thymine bases, and the number of guanine bases equals the number of cytosine bases in DNA. ü Accordingly, the sequence of the two strands is complementary and the sequence of one strand determines the sequence of the second one. ü This is important during DNA replication as each of the original DNA strands acts as a template for synthesis of a new complementary strand to form two daughter DNA molecules. BIO 206 3- Base stacking ü The base pairs inside the helix are stacked above each other by Van der Waals forces and hydrophobic interactions. ü The hydrogen bonding between complementary base pairs and the Van der Waals forces and hydrophobic interactions of stacked base pairs provide the stability of the double helix. ü The excessive stacking of DNA is balanced by the repulsive forces of negative charges present on phosphate groups. BIO 206 4- Spiral staircase ü The double helix of DNA appears much like spiral staircase, in which there are 10.4 base pairs or steps for each complete turn of the helix. ü Each complete turn is 3.4 nm long and 2 nm wide. ü The size of DNA is usually expressed as the number of base pairs (bp). BIO 206 5- Dimensions ü The B-form of DNA is 2 nm wide. ü From outside of the helix, two grooves are apparent, a major groove (2.2 nm) and a minor groove (1.2 nm). ü Through these grooves many drugs and proteins can contact the nitrogenous bases without any need to open the helix. BIO 206 q Structural forms of the double helix: - There are three major structural forms of DNA: • The B form, described by Watson and Crick in 1953, the A form, and the Z form. 1- A form of DNA • It is thicker and shorter than the B- form, which affects the size of the major and minor grooves. • Such variations may be important in regulation of gene expression, since they influence the extent of DNA binding with various types of regulatory proteins. • Found in moderately dehydrated chromatin, also found in DNA-RNA BIO 206 hybrids and in RNA-RNA double stranded regions. 2- B-DNA: • Chromosomal DNA is thought to consist primarily of B-DNA. 3- Z Form of DNA • This is a special form of left-handed double helical DNA which is formed mainly of alternating cytosine and guanine bases (CGCGCG). BIO 206 Z Form of DNA • It is longer and thinner than that of B-DNA. • Small stretches of Z-DNA (12 – 24 bp long) are commonly found at the 5` ends of genes, which are the regions that regulate transcriptional activities. BIO 206 III- DNA Tertiary Structures • DNA is mainly linear or sometimes it is circular as in DNA of mitochondria, chloroplasts (in plants) and bacteria. • The packing of large DNA molecules into cells requires a process called “DNA supercoiling”. • Supercoiling may be positive (more tight) or negative (less tight). • Negative supercoils are more commonly present under normal physiological state. BIO 206 • Types of supercoiling in linear DNA: a) Toroidal: The DNA may coil into a series of spirals around a cylinder-shaped object. b) Interwound coil: The DNA may also cross over and under itself repeatedly. • Types of supercoiling in circular DNA: a) Right-handed supercoil. b) Left-handed supercoil. BIO 206 III- DNA Tertiary Structures (Cont.) • Normally, left-handed winding of DNA around histones (toroidal supercoil) makes it negatively supercoiled. • Replication and transcription requires unwinding of DNA. • Unwinding is easier if DNA is negatively supercoiled. • Thus, negatively supercoiled form of DNA is biologically more favored. BIO 206 • Eukaryotes have closed circular DNA molecules in their mitochondria (multiple copies are present per mitochondrion). • A prokaryotic organism contains a single, double-stranded, supercoiled, circular chromosome. • Most species of bacteria also contain small, circular, extrachromosomal DNA molecules (carry genes that confer antibiotic resistance to bacteria) called plasmids. • Some viruses have a circular DNA molecule; others have linear molecules. Only a few viruses have single-stranded DNA molecules. BIO 206 Mitochondrial DNA (mtDNA) § Mitochondrial DNA forms 0.3 to 1% of the total cellular DNA. § Human mtDNA is present in the form of small double stranded circular supercoil (16,569 bp), many copies per mitochondrion. § It is responsible for controlling synthesis of 2 rRNA, 22 tRNA and 13 proteins. Most of these mitochondrial proteins are involved in oxidative phosphorylation (electron transport chain and ATP synthesis). § Mitochondria are provided by the ovum, thus mtDNA molecules are maternally inherited and are used as maternal lineage. § Mutations of mtDNA are associated with certain types of myopathies. BIO 206 q Denaturation of DNA ü Heating of DNA produces rupture of hydrogen bonds and separation of the two strands of DNA or DNA denaturation. ü This process absorption is of accompanied UV light with at 260 increased nm or hyperchromicity in addition to decreased viscosity. ü The temperature that produces loss of 50% of DNA helical form temperature (Tm). BIO 206 is termed the melting q Denaturation of DNA ü Tm decreases if: a) DNA contains a higher proportion of A-T base pairs. b) If the medium is made more alkaline. c) If salt concentration is decreased. ü Cooling of denatured DNA results in reformation of the double helix (Renaturation or Reannealing). BIO 206 THANK YOU