Purine & Pyrimidine

·         Nucleic Acids: Nucleotide linked by3',5' phosphodiester bonnds; Have distinct 3' and5' ends, thus polarity ;Sequence is always specified as5'~3'

·         The flow of information from DNA to RNA to protein is termed the “central dogma” of molecular biology and is descriptive of all organisms, with the exception of some viruses that have RNA as the repository of their genetic information.

·         Certain anticancer drugs, such as dactinomycin (actinomycin D), exert their cytotoxic effect by intercalating into the narrow groove of the DNA double helix, thus interfering with DNA and RNA synthesis.

·         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.

The B form is a right-handed helix with ten residues per 360° turn of the helix, and with the planes of the bases perpendicular to the helical axis. Chromosomal DNA is thought to consist primarily of B-DNA.

 The A form is produced by moderately dehydrating the B form. It is also a right-handed helix, but there are eleven base pairs per turn, and the planes of the base pairs are tilted 20° away from the perpendicular to the helical axis. The conformation found in DNA–RNA hybrids or RNA–RNA double-stranded regions is probably very close to the A form.

Z-DNA is a left-handed helix that contains about twelve base pairs per turn. [Note: The deoxyribose–phosphate backbone “zigzags,” hence, the name “Z”-DNA.] Stretches of Z-DNA can occur naturally in regions of DNA that have a sequence of alternating purines and pyrimidines, for example, poly GC.

·         DNA replication begins at a single, unique nucleotide sequence.This site includes a short sequence composed almost exclusively of AT base pairs. This is referred to as a consensus sequence, because the order of nucleotides is essentially the same at each site.

·         Anticancer agents, such as etoposide target human topoisomerase II.

Bacterial DNA gyrase is a unique target of a group of antimicrobial agents called quinolones, for example, ciprofloxacin.

·         DNA polymerase III has, in addition to its 5′→3′ polymerase activity,

A “proofreading” activity - 3′→5′ exonuclease.

DNA polymerase I also has a 5′→3′ exonuclease activity that is able to hydrolytically remove the RNA primer.

·         A special DNA polymerase called telomerase is responsible for replication of the telomeric DNA. Telomerase contains an RNA molecule that guides the synthesis of complementary DNA. Telomerase is therefore an RNA-dependent DNA polymerase in a category with reverse transcriptase. Telomerase does not require an RNA primer, initiating synthesis of the leading strands at 3’ ends within the telomeric DNA. Synthesis of the lagging strands uses primase, DNA polymerase III, and DNA polymerase I, as with the replication of other chromosomal regions.

·         A defective unwinding protein slows the overall rate of DNA synthesis, but does not alter the size of replicated DNA fragments. Defects in DNA synthesis or transcription may produce a phenotype of accelerated aging, as in Cockayne’s syndrome.

A child presents with severe growth failure, accelerated aging that causes adult complications such as diabetes and coronary artery disease, and microcephaly (small head) due to increased nerve cell death. In vitro assay of labeled thymidine incorporation reveals decreased levels of DNA synthesis compared to controls, but normal-sized labeled DNA fragments.

·         Telomeres may be viewed as mitotic clocks in that their length in most cells is inversely related to the number of times the cells have divided. The study of telomeres is providing insight into the biology of aging and cancer.

·         Reverse transcriptase activity is also seen with transposons, DNA elements that can move about the genome. In eukaryotes, such elements are transcribed to RNA, the RNA is used as a template for DNA synthesis by a reverse transcriptase encoded by the transposon, and the DNA is randomly inserted into the genome. Transposons that involve an RNA intermediate are called retrotransposons. The nucleotide sequences of retrotransposons are similar to those of retroviruses.

·         There are five classes of positively charged histone proteins. Two each of histones H2A, H2B, H3, and H4 form a structural core around which DNA is wrapped creating a nucleosome.

Histone H1, of which there are several related species, is not found in the nucleosome core, but instead binds to the linker DNA chain between the nucleosome beads. H1 is the most tissue-specific and species-specific of the histones. It facilitates the packing of nucleosomes into the more compact structures.

The interconversion of active and inactive forms of chromatin is called chromatin remodeling. A major mechanism by which chromatin is remodeled is through acetylation of lysine residues at the amino terminus of histone proteins. Acetylation eliminates the positive charge on the lysine and thereby decreases the interaction of the histone with the negatively charged DNA. This process of opening up the chromatin and making DNA more accessible for transcription is mediated by histone acetyltransferases. Removal of the acetyl group by histone deacetylases restores the positive charge, and fosters stronger interactions between histones and DNA.

·         Some rRNA function as catalysts in protein synthesis. RNA with catalytic activity is termed a ribozyme.

·         If the mRNA carries information from more than one gene, it is said to be polycistronic. Polycistronic mRNA is characteristic of prokaryotes. If the mRNA carries information from just one gene, it is said to be monocistronic and is characteristic of eukaryotes.

·         rRNA-80%

tRNA-15% , Smallest RNA

mRNA-5% , Most Hetrerogenous RNA

·         In contrast to DNA polymerase, RNA polymerase does not require a primer and has no known proofreading activity.

·         Rifampin inhibits the initiation of transcription by binding to the β subunit of prokaryotic RNA polymerase, thus interfering with the formation of the first phosphodiester bond (Figure 30.10). Rifampin is useful in the treatment of tuberculosis.

 Dactinomycin (known to biochemists as actinomycin D) was the first antibiotic to find therapeutic application in tumor chemotherapy.It binds to the DNA template and interferes with the movement of RNA polymerase along the DNA.

·         Pribnow box in prokaryotes & TATA=Hogness Box or GC box in Eukaryotes as promoter consensus sequence.

·         RNA polymerase II is inhibited by α-amanitin—a potent toxin produced by the poisonous mushroom Amanita phalloides (sometimes called “death cap” or “destroying angel”). α-Amanitin forms a tight complex with the polymerase, thereby inhibiting mRNA synthesis and, ultimately, protein synthesis.

·         Transcription factors recognize DNA through a variety of DNA-binding motifs, such as zinc fingers and leucine zippers.

·         The collection of all the precursor molecules for mRNA is known as heterogeneous nuclear RNA (hnRNA).

·         The process of removing introns and joining exons is called splicing. The molecular machine that accomplishes these tasks is known as the spliceosome.

·         Systemic lupus erythematosus, an often fatal inflammatory disease, results from an autoimmune response in which the patient produces antibodies against host proteins, including snRNP.

·         Loss of three nucleotides maintains the reading frame, but can result in serious pathology. For example, cystic fibrosis (CF), a hereditary disease that primarily affects the pulmonary and digestive systems, is most commonly caused by deletion of three nucleotides from the coding region of a gene, resulting in the loss of phenylalanine at the 508th position (ΔF508) in the protein encoded by that gene.

·         FYI : Because the S values are determined both by shape as well as molecular mass, their numeric values are not strictly additive. For example, the prokaryotic 50S and 30S ribosomal subunits together form a 70S ribosome. The eukaryotic 60S and 40S subunits form an 80S ribosome.

·         Binding of the tRNA anticodon to the mRNA codon follows the rules of complementary and antiparallel binding, that is, the mRNA codon is “read” 5′→3′ by an anticodon pairing in the “flipped” (3′→5′) orientation. When writing the sequences of both codons and anticodons, the nucleotide sequence must ALWAYS be listed in the 5′→3′ order.

·         The mechanism by which tRNAs can recognize more than one codon for a specific amino acid is described by the “wobble” hypothesis in which the base at the 5′-end of the anticodon (the “first” base of the anticodon) is not as spatially defined as the other two bases. Movement of that first base allows nontraditional base-pairing with the 3′-base of the codon.

·         The ribosome has three binding sites for tRNA molecules—the A, P, and E sites—each of which extends over both subunits. Together, they cover three neighboring codons.

·         In prokaryotes, three initiation factors are known (IF-1, IF-2, and IF-3), whereas in eukaryotes, there are over ten (designated eIF to indicate eukaryotic origin. There are two mechanisms by which the ribosome recognizes the nucleotide sequence that initiates translation:

1.Shine-Dalgarno sequence: In Escherichia coli, a purine-rich sequence

2.Initiation codon: The initiating AUG is recognized by a special initiator tRNA. Recognition is facilitated by IF-2 (bound to GTP) in prokaryotes and eIF2-GTP (plus additional eIF) in eukaryotes.

·         Proteins that are defective or destined for rapid turnover are often marked for destruction by ubiquitination—the attachment of a small, highly conserved protein, called ubiquitin. Proteins marked in this way are rapidly degraded by a cellular component known as the “proteasome,” which is a complex, ATP-dependent, proteolytic system located in the cytosol.

·         DNA sequences flanking a gene are called cis-acting because they influence expression of genes only on the same chromosome. A trans-acting factor is the regulatory molecule itself, which can diffuse through the cell from its site of synthesis to its DNA-binding site.

The binding of proteins to DNA is through structural motifs such as the zinc finger, leucine zipper, or helix-turn-helix in the protein.

·         OPERON: The genes are thus coordinately controlled, that is, turned on or off as a unit. This entire package is referred to as an operon.

The lactose (lac) operon codes for three proteins involved in the catabolism of the disaccharide, lactose: The lacZ gene codes for β-galactosidase, which hydrolyzes lactose to galactose and glucose; the lacY gene, which codes for a permease that facilitates the movement of lactose into the cell; and the lacA gene that codes for thiogalactoside transacetylase whose exact physiologic function is unknown. All of these proteins are produced when lactose is available to the cell but glucose is not.

The tryptophan (trp) operon codes for five proteins that are required for the synthesis of the amino acid, tryptophan. As with the lac operon, the trp operon is subject to both positive and negative control. Repression by trp is not always complete, however, and so unlike the lac operon, the trp operon is also regulated by a process known as attenuation. With attenuation, transcription is initiated but is terminated well before completion.

Transcriptional attenuation can occur in prokaryotes because translation of an mRNA begins before its synthesis is complete. In eukaryotes this does not occur because, as a result of having a membrane-bound nucleus, transcription and translation are spatially and temporally separate processes.

·         RNA interference: The presence of double-stranded (ds)RNA in a eukaryotic cell can trigger a process known as RNA interference or RNAi (also known as RNA silencing, or RNA inactivation)

RNAi is thought to be a part of the body's natural immune system evolved as a defense against retroviruses, such as the human immunodeficiency virus (HIV), that store their genetic information in dsRNA.

RNAi THERAPY: The first clinical trial - age-related macular degeneration (AMD

In patients with AMD, too much of this protein leads to the sprouting of excess blood vessels behind the retina. The blood vessels leak, clouding and often entirely destroying vision; hence, AMD is also referred to as “wet” macular degeneration. The siRNA drug—a 21-nucleotide dsRNA that specifically targets the mRNA of VEGF—is injected into the eye. This direct delivery helps ensure that siRNA can reach their target intact and with minimal effects on other tissues. The siRNA promotes the cleavage of mRNA for VEGF. One siRNA molecule can destroy hundreds of mRNA, resulting in the suppression of thousands of VEGF proteins, thus suppressing damaging angiogenesis in the retina. The first clinical trial, involving about two dozen patients, showed promising results.

·         Transposons (Tn) are mobile segments of DNA that move in an essentially random manner from one site to another on the same or a different chromosome. Movement is mediated by transposase, an enzyme encoded by the Tn itself.

·         A DNA library is a collection of cloned restriction fragments of the DNA of an organism.

Two kinds of libraries: genomic libraries and complementary DNA (cDNA) libraries.

·         Restriction endonucleases recognize short stretches of DNA (generally four or six base pairs) that contain specific nucleotide sequences. These sequences, which differ for each restrictionendonuclease, are palindromes, that is, they exhibit twofold rotational symmetry.

·         In order for RFLP to be able to detect and follow the inheritance of these genes, the detected mutation must be at or closely linked to an altered restriction site. Mutations within the restriction sites change the size of restriction fragments. The different-sized fragments migrate in different positions during electrophoresis of bands visualized by Southern blot analysis, which utilizes fluorescent or radiolabeled DNA probes.

·         At a physiologic pH of 7.4, mRNAs (like DNA) are polyanionic owing to the negatively charged phosphate hydroxyl groups.

·         ATP is required for the esterification of amino acids to their corresponding tRNAs. This reaction is catalyzed by the class of enzymes known as aminoacyl-tRNA synthetases. Each one of these enzymes is specific for one tRNA and its corresponding amino acid.

·         DNA methylation occurs mainly at CpG dinucleotides that often cluster in at the upstream promoter regions of genes (CpG islands).

·         The major effects of radiation are to damage cellular DNA by opening purine rings and rupturing phosphodiester bonds. Chemical agents such as formaldehyde can cross-link DNA, and inhibitors of DNA methylation, such as methotrexate (an inhibitor of folic acid), were the first anticancer drugs.