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Deoxyribonucleic Acid (DNA)
This Sqadia video is the demonstration of Basic Molecular Genetic Mechanisms - I. Macromolecules contain the information for determining the amino acid sequence and hence the structure and function of all the proteins of a cell. The information stored in DNA is arranged in hereditary units, now known as genes, that control identifiable traits of an organism. DNA and RNA are chemically very similar. The primary structures of both are linear polymers composed of monomers called nucleotides. DNA and RNA each consist of only four different nucleotides. All nucleotides consist of an organic base linked to a five-carbon sugar that has a phosphate group attached to carbon 5. The bases adenine (A) and guanine (G) are purines, which contain a pair of fused rings; the bases cytosine (C), thymine (T), and uracil (U) are pyrimidines, which contain a single ring. The unwinding and separation of DNA strands, referred to as denaturation, or “melting,” can be induced experimentally by increasing the temperature of a solution of DNA. The melting temperature ‘’Tm’’ at which DNA strands will separate depends on several factors i.e. proportion of G·C pairs, ion concentration, agents that destabilize hydrogen bonds, such as formamide or urea, and extremes of pH. Single-stranded DNA molecules that result from denaturation form random coils without an organized structure. Denaturation and renaturation of DNA are the basis of nucleic acid hybridization. Many prokaryotic genomic DNAs and many viral DNAs are circular molecules. Circular DNA molecules also occur in mitochondria and chloroplasts. Topoisomerase - I binds to DNA at random sites and breaks a phosphodiester bond in one strand. Topoisomerase - II makes breaks in both strands of a double-stranded DNA and then religates them.
Ribonucleic Acid (RNA)
RNA ribose has hydroxyl group at the 2´ positions. Hydroxyl group on C2 of ribose makes RNA more chemically labile than DNA. RNA is a long polynucleotide that can be double stranded or single-stranded, linear or circular. Hairpins are formed by pairing of bases within ≈5–10 nucleotides of each other, and “stem-loops” by pairing of bases that are separated by >10 to several hundred nucleotides. These simple folds can cooperate to form more complicated tertiary structures, one of which is termed a pseudoknot. mRNA, tRNA, and rRNA are types of RNA. Another type of RNA is Ribozymes. The folded domains of RNA molecules not only are structurally analogous to the α helices and ß strands found in proteins, but in some cases also have catalytic capacities. Such catalytic RNAs are called ribozymes.
Transcription of Protein Coding Genes
Gene is a unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA. During synthesis of RNA, the four-bases of DNA containing A, G, C, and T is simply transcribed, into the four-bases of RNA, which is identical except that U replaces T. In transcription, one DNA strand acts as a template determining the order in which ribonucleoside triphosphate (rNTP) monomers are polymerized to form a complementary RNA chain. Bases in the template DNA strand base-pair with complementary incoming rNTPs, which then are joined in a polymerization reaction catalyzed by RNA polymerase. The equilibrium for the reaction is driven further toward chain elongation by pyrophosphatase. Nucleotide positions in the DNA sequence downstream from a start site are indicated by a positive (+) sign; those upstream, by a negative (-) sign. Transcription has 3 stages i.e. initiation, elongation and termination. The RNA polymerases of bacteria, archaea, and eukaryotic cells are fundamentally similar in structure and function, composed of two related large subunits (ß´ and ß), two copies of a smaller subunit (α), and one copy of a fifth subunit (ω).
Organization of Genes
Transcription of an operon produces a continuous strand of mRNA that carries the message for a related series of proteins. In prokaryotic DNA the genes are closely packed with very few noncoding gaps, and the DNA is transcribed directly into colinear mRNA, which then is translated into protein. Whereas in eukaryotes, each gene is transcribed from its own promoter, producing one mRNA, which generally is translated to yield a single polypeptide. Eukaryotic gene existed in pieces of coding sequence, the exons, separated by non-protein-coding segments, the introns. Introns are present in the DNA of viruses that infect eukaryotic cells and very rare in bacteria. Transcription and translation can occur concurrently in prokaryotes but not in eukaryotes. All eukaryotic pre-mRNAs initially are modified at the two ends, and these modifications are retained in mRNAs. mRNA is modified with the addition of a 5' cap that is 7-methylguanylate and 3' poly-A tail. Cap protects against enzymatic degradation and assist in its transport to cytoplasm. The internal cleavage of a transcript to excise the introns, followed by ligation of the coding exons. Alternative RNA splicing increases the number of proteins expressed from a single eukaryotic gene. In contrast to bacterial and archaeal genes, the vast majority of genes in higher, multicellular eukaryotes contain multiple introns. More than 20 different isoforms of fibronectin have been identified, each encoded by a different, alternatively spliced mRNA composed of a unique combination of fibronectin gene exons.
Control of Gene Expression in Prokaryotes
By controlling transcription initiation, a cell can regulate which proteins it produces and how rapidly. At any given time, a bacterial cell normally synthesizes only those proteins of its entire proteome required for survival under the particular conditions. In multicellular organisms, control of gene expression is largely directed toward assuring that the right gene is expressed in the right cell at the right time during embryological development and tissue differentiation. The lac operon encodes three enzymes required for the metabolism of lactose. The most common one in bacterial cells is ơ70. When E. coli is in an environment that lacks lactose, synthesis of lac mRNA is repressed. In an environment containing both lactose and glucose, E. coli cells preferentially metabolize glucose. Lactose is metabolized at a high rate only when lactose is present, and glucose is largely depleted from the medium. Transcription of the lac operon under different conditions is controlled by lac repressor and catabolite activator protein (CAP). The promoter sequence determines the intrinsic rate at which an RNA polymerase–complex initiates transcription of a gene in the absence of a repressor or activator protein. Promoters that support a high rate of transcription initiation are called strong promoters. Transcription of most E. coli genes is regulated by processes similar to those described for the lac operon. Transcription initiation by RNA polymerases containing these 70-like factors is regulated by repressors and activators that bind to DNA near the region where the polymerase binds, similar to initiation by ơ 70-RNA polymerase itself. In two-component regulatory systems, one protein acts as a sensor monitoring the level of nutrients and other components in the environment. The phosphorylated response regulator then binds to DNA regulatory sequences, thereby stimulating or repressing transcription of specific genes.