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The cell nucleus is one of the Best known but Least Understood of cellular organelles.

The cell nucleus is one of the Best known but Least Understood of cellular organelles.

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Nucleus

  1. 1. Index The Cell Cell Nucleus • Introduction • History Note • Function • Structural Component • Chromatin • Nucleolus • DNA • RNA • Mutation • Genetic Engineering
  2. 2. The cell (from Latin cella, meaning "small room") is the basic structural, functional and biological unit of all known living organisms. Each living cell carries out the tasks of taking food, transforming food into energy, getting rid of wastes, and reproducing. Every living thing - from the tiniest bacterium to the largest whale - is made of one or more cells. The Cell
  3. 3. Principles Of Cell Theory 1. All living things are composed of one or more cells. 2. Cells are organisms’ basic units of structure and function. 3. Cells come only from existing cells. Discovery Of Cells Robert Hooke (1665): Observed a thin slice of cork (dead plant cells) with a microscope. He described what he observed as “little boxes” (cells). The idea that all living things are made of cells was put forward in about 1840 and in 1855 came ‘Cell Theory’ – i.e. ‘cells only come from other cells’ – contradicting the earlier theory of ‘Spontaneous Generation’
  4. 4. Every cell is essentially an assortment of functional parts suspended in a liquid medium and enclosed in a slightly leaky bag. The functional parts are called Organelles. The fluid inside the membrane is called Cytoplasm and is composed of water, salts, and organic molecules. The enclosing material is called the Cell Membrane, which is semi- permeable to allow small molecules and dissolved gases to pass through. An Organelle is a cell component that performs a specific function in that cell.
  5. 5. The Nucleus is a large, often rounded organelle. Most animal and plant cells have a nucleus, which contains a copy of the DNA of the organism (a notable exception would be mammalian red blood cells, which lack a nucleus). Chemically coded on the DNA are the instructions to produce every protein an organism needs to make new cells, digest foods, produce necessary chemicals, move, and all other cell-level life functions. The exact sequences are copied inside the nucleus by molecules of messenger RNA (mRNA), which pass out of the nucleus to ribosomes for production. Ribosomes are able to read the sequence of ingredients on the mRNA and attach amino acids together to form the new protein.
  6. 6. The cell nucleus is one of the Best known but Least Understood of cellular organelles. The structure and functional organization of the nucleus remains a subject of Energetic Debate. At one extreme, the nucleus has been proposed to have its own nucleoskeleton and distinct organelles. At the other, it is viewed as a largely disordered, membrane bound bag of DNA and other molecules, in which all “structures” are no more than transient complexes that form and disperse as a result of Transcription, Replication, & RNA Processing activities in various regions of the genome. Introduction
  7. 7. The nucleus was described by Franz Bauer in 1804. The more detail in 1831 by Scottish Botanist Robert Brown in a talk at the Linnean Society of London. By Robert Brown in 1831 as…. “this areola, or nucleus of the cell as perhaps it might be termed…is more or less distinctly granular…There is no regularity as to its place in the cell; it is not unfrequently, however, central or nearly so.” History Note
  8. 8.  R. Brown, 1831– (Lat. nucleus, kernel; Gr. karyon, nut)  In All Eukaryotic cells – with exception of RBCs  Number– Uninuclear , Binuclear, Multinuclear.  Localization – Centrally or Peripherally  External morphology: Shape – Species-diversified Size – 10% of the cell volume; 5 µm (Spermatozoon), 40 µm (Oogonium) Nucleus : @ a Glance
  9. 9. The Nucleus (House of Genetic Material) contains a Blueprint for all cell structures and activities encoded in the DNA of the chromosomes. It contains the molecular machinery to replicate its DNA and to synthesize RNA. • Pre-rRNA Processing • Ribosomal RNA (rRNA) Transcription • Production of Ribosome Subunit & Exports them into Cytoplasm for Assembly. Interactions and translocation of a large number of proteins and RNAs are conducted and coordinated. Because functional ribosomes do not occur in the nucleus, no proteins are produced here. Macromolecular transfer between the nuclear and cytoplasmic compartments is regulated. Function
  10. 10. The Outer membrane (Cytoplasmic Surface) bound to it and is continuous with the Rough Endoplasmic Reticulum (RER). The Inner membrane (Nuclear Surface) is associated with a Nuclear Lamina (NL) for the attachment of Chromatin. The two membranes fuse at many places to form Nuclear Pores (NP). It controls movement of molecules between Nucleus & Cytoplasm First description : M. Watson , 1955 Nuclear Envelope Nuclear Envelope (NE) that surrounds the nucleus is made of two membranes separated by the Perinuclear Space (PS).
  11. 11. The Nuclear lamina is formed of filaments proteins, the Lamins, which assemble as a lattice adjacent to the inner nuclear membrane. When the nuclear envelope disperses during early prophase of cell division, at least some Lamin proteins remain attached to the membrane fragments and reassembly of the nuclear lamina immediately after cell division facilitates re-formation of the nuclear envelopes of the two new nuclei. The nuclear lamina also contains Binding sites for Chromatin, helping to organize this material in the nucleus. Nuclear Lamina
  12. 12. A nuclear pore complex (NPC) is membrane-bound channels and a made of transmembrane proteins (Nucleoporins) which form an octagonal annulus or ring, with filaments extending into both the cytoplasm and the nucleus. The Nucleus of a typical mammalian cell contains 3000–4000 pore complexes. Vary in number according to the cell’s function and stage of development. Regulates most Bidirectional Transport between the nucleus and the cytoplasm. The nuclear envelope is impermeable to all sizes of ions and the exchange of substances between the nucleus and the cytoplasm occur only through the nuclear pores. Ions and small molecules pass through the nuclear pore by passive diffusion. Nuclear Pore Complex
  13. 13. The molecules that pass from the cytoplasm into the nucleus are mainly: Ions Nucleotides for DNA synthesis; Proteins for binding to DNA The molecules that pass from the nucleus into the cytoplasm are m-RNA and r-RNA bound to protein as RNA-protein complexes. Protein molecules require a special mechanism to pass through the nuclear pores. Passage of Substances Through Nuclear Pore
  14. 14. Nucleosome (U-body) : Basic Structural unit of Chromatin Core of Histone Octamers. Two Loops of DNA – 146 Base Pairs Composition Coiled Strands of DNA & Proteins: Histone Proteins (80%) –Basic Proteins only in the Nucleus H2A, H2B, H3, H4 – Nucleosome Core H1–Keeping in place the wrapped DNA, Binds to the “Linker DNA" Non-Histone Proteins (20%) – Acid Proteins Nuclear protein matrix Chromatin
  15. 15. Chromatin, in non-dividing nuclei, is in fact the Chromosomes in a different degree of uncoiling. According to the degree of chromosome condensation, two types of chromatin, (Heterochromatin & Euchromatin) can be distinguished with both the light and electron microscopes. Heterochromatin (Condensed) - 90% Gr. heteros, other, + chroma, color Heterochromatin is electron dense, appears as coarse granules in the electron microscope and as basophilic clumps in the light microscope. • Marginal chromatin – Nuclearmembrane • Chromocenters – nucleoplasm • Nucleolar-associated chromatin • Constitutive heterochromatin – Inactive, around the chromosome centromere. • Facultative heterochromatin – Sex chromatin Chromatin
  16. 16. Euchromatin ( Extended) - 10% Gr. eu, good, true + chroma, color A lightly packed form of chromatin (DNA, RNA and Protein) Comprises the most active portion of the Genome – Replication and Transcription Euchromatin is the less coiled portion of the chromosomes, visible as a finely dispersed granular material in the electron microscope and as lightly stained basophilic areas in the light microscope. The proportion of heterochromatin to euchromatin accounts for the light-to-dark appearance of nuclei in tissue sections as seen in light and electron microscopes. Chromatin is composed mainly of coiled strands of DNA bound to basic proteins called histones and to various non-histone proteins. Chromatin
  17. 17. The basic structural unit of chromatin is the Nucleosome which consists of • A core of four types of Histones: two copies each of histones H2A, H2B, H3, and H4, around which are wrapped 146 DNA Base Pairs. • An additional 48-base pair segment forms a link between adjacent nucleosomes, and another type of histone (H1 or H5) is bound to this DNA. Chromatin
  18. 18. The presence of a prominent nucleolus indicates that the cell is actively synthesizing protein. A prominent nucleolus is present in protein-secreting cells such as those of the pancreas, plasma cells, developing blood cell precursors. Medical Application The intensity of nuclear staining of the chromatin is frequently used to distinguish and identify different tissues and cell types in the light microscope. This organization of chromatin has been referred to as "Beads-on-a-String." Non-Histone Proteins are also associated with chromatin, but their arrangement is less well understood. Chromatin
  19. 19. Denomination: W. von Waldeyer, 1888 Definition : a very long DNA molecule (that contains many genes) and associated proteins, carrying portions of the hereditary information of an organism. Total number in humans – 46 (2n) (23 homologous pairs) 22 pairs of Autosomes 1 pair of Gonosomes (sex chromosomes) – X and Y Size: length– 0.1 to 30 µm (average 3-8µm) (51 million to 245 million base pairs) Thickness – 0.5-2 µm Single DNA molecule– 1.7-8.5 cm Total length– 1.7 m Chromosome
  20. 20. A chromosomal aberration is a major change in DNA that can be observed at the level of the chromosome. There are four types of aberrations: Inversions, Translocations, Duplications, and Deletions. An Inversion occurs when a chromo- some is broken and a piece becomes reattached to its original chromosome but in reverse order—it has been cut out and flipped around. A Translocation occurs when one broken segment of DNA becomes integrated into a different chromosome. Duplications occur when a portion of a chromosome is replicated and attached to the original section in sequence. Deletion aberrations result when a broken piece becomes lost or is destroyed before it can be reattached. Chromosomal Aberrations
  21. 21. Nucleolus The nucleolus is a generally spherical, highly basophilic structure present in the nuclei of cells active in protein synthesis. Fibrillar Centre – Pale-stained region: • RNA polymerase I Fibrillar part, pars fibrosa: • Nucleolonema • Newly synthesizes rRNA Granular part, pars granulosa: • Ribonucleoprotein particles It is the site of rRNA Transcription and Processing, & of Ribosome Assembly.
  22. 22. Transcription & Processing of rRNA The nucleolus, which is not surrounded by a membrane, is organized around the chromosomal regions that contain the genes for the 5.8S, 18S, & 28S rRNAs. Eukaryotic ribosomes contain Four types of RNA, designated the 5S, 5.8S, 18S, and 28S rRNAs. The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit within the nucleolus by RNA Polymerase I, yielding a 45S ribosomal precursor. Transcription of the 5S rRNA, which is also found in the 60S ribosomal subunit, takes place outside of the nucleolus and is catalysed by RNA Polymerase III. The 45S pre-rRNA is processed to the 18S rRNA of the 40S (Small) ribosomal subunit and to the 5.8S and 28S rRNAs of the 60S (Large) ribosomal subunit.
  23. 23. Transcription & Processing of rRNA
  24. 24. The formation of ribosomes involves the assembly of the ribosomal precursor RNA with both Ribosomal Proteins and 5S rRNA. The genes that encode Ribosomal Proteins are transcribed outside of the nucleolus by RNA polymerases II, yielding mRNAs that are translated on cytoplasmic ribosomes. Ribosomal Proteins are imported to the nucleolus from the cytoplasm and begin to assemble on pre-rRNA to form preribosomal particles prior to its cleavage. The genes for 5S rRNA are also transcribed outside of the nucleolus, in this case by RNA polymerase III, 5S rRNAs similarly are assembled into preribosomal particles within the nucleolus. Ribosomal proteins and the 5S rRNA are incorporated into preribosomal particles as cleavage of the pre-rRNA proceeds. Ribosome Assembly
  25. 25. The final steps of maturation follow the export of preribosomal particles to the Cytoplasm, yielding the 40S and 60S ribosomal subunits. The Smaller Ribosomal Subunit (40S), which contains only the 18S rRNA, matures more rapidly than the Larger Subunit (60S), which contains 28S, 5.8S, and 5S rRNAs.
  26. 26. A highly Dynamic structure Composition – Amorphous • Nucleoskeleton -- Proteins+ RNA • Nuclear Lamina • Numerous enzymes • Metabolites • Ions • Crystalline inclusions • Viruses and • Other inclusions Nuclear Matrix (Nucleoplasm)
  27. 27. Barr Body In 1949, Murray Barr and Ewart Bertram found that staining cat cells with Feulgen, a stain that binds to DNA, often resulted in the appearance of a "drumstick". They identified a highly condensed structure in the interphase nuclei of somatic cells in female cats that was not found in male cats. This structure became known as the Barr body. In 1960, Susumu Ohno correctly proposed that the Barr body is a highly condensed X chromosome.
  28. 28. Medical Application  Diagnostics in Endocrinology  Forensic Medicine Practice  Study of Inherited Chromosome Anomalies – Klinefelter's & Turner Syndromes.  Disclosure of the Genetic sex– In Hermaphroditism & Pseudohermaphroditism Barr Body
  29. 29. DNA: A Nucleic Acid that contains the Genetic Information (Genes) Chemical composition: Long Polymers of Nucleotides Phosphate Group Deoxyribose - Five-Carbon Sugar Nitrogenous Bases Purine –Adenine and Guanine Pyrimidine – Thymine and Cytosine Structure Arranged in the form of a Double Helix, Two anti-parallel strands It looks like a Twisted Ladder: The sides of the ladder are composed of nucleotides The strands of the ladder are bonds between the bases where Adenine only forms a bond with Thymine, and Guanine with Cytosine. DNA
  30. 30. BASE+ SUGAR = NUCLEOSIDE BASE + SUGAR + PHOSPHAT= NUCLEOTIDE Nitrogenous Bases Purine –Adenine and Guanine Pyrimidine – Thymine and Cytosine Deoxyribose - Five-Carbon Sugar Phosphate Group
  31. 31. Nucleotides are joined together by a phosphodiester linkage between 5 ' and 3' carbon atoms to form nucleic acids. The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated by a one-letter code, A-G-C-T-T-A-C-A, with the 5‘ end of the chain at the left. The relationship between Genetic information carried in DNA and proteins
  32. 32. The basic chemical composition of nucleic acids was elucidated in the 1920s through the efforts of P. A.Levene. Despite his major contributions to nucleic acid chemistry, Levene mistakenly believed that DNA was a very small molecule, probably only four nucleotides long, composed of equal amounts of the four different nucleotides arranged in a fixed sequence. Further advances in our understanding of DNA structure awaited the development of significant new analytical techniques in chemistry. One development was the invention of paper chromatography by Archer Martin and Richard Synge between 1941 and 1944. By 1948 the chemist Erwin Chargaff had begun using paper chromatography to analyse the base composition of DNA from a number of species. He soon found that the base composition of DNA from genetic material did indeed vary among species just as he expected. Furthermore, the total amount of purines always equalled the total amount of pyrimidines; and the adenine/thymine and guanine/ cytosine ratios were always 1. These findings, known as Chargaff’s rules, were a key to the understanding of DNA structure. The Elucidation of DNA Structure
  33. 33. Purines = Pyrimidines A/T = 1 & G/C = 1 Chargaff’s rules
  34. 34. Another turning point in research on DNA structure was reached in 1951 when Rosalind Franklin arrived at King’s College, London, and joined Maurice Wilkins in his efforts to prepare highly oriented DNA fibers and study them by X-ray crystallography. By the winter of 1952–1953, Franklin had obtained an excellent X-ray diffraction photograph of DNA.
  35. 35. The same year that Franklin began work at King’s College, the American biologist James Watson went to Cambridge University and met Francis Crick. Although Crick was a physicist, he was very interested in the structure and function of DNA, and the two soon began to work on its structure. Their attempts were unsuccessful until Franklin’s data provided them with the necessary clues. Her photograph of fibrous DNA contained a crossing pattern of dark spots, which showed that the molecule was Helical. The dark regions at the top and bottom of the photograph showed that the purine and pyrimidine bases were stacked on top of each other and separated by 0.34 nm. Franklin had already concluded that the phosphate groups lay to the outside of the cylinder.
  36. 36. Finally, the X-ray data and her determination of the density of DNA indicated that the helix contained two strands, not three or more as some had proposed. Without actually doing any experiments themselves, Watson and Crick constructed their model by combining Chargaff’s rules on base composition with Franklin’s X-ray data and their predictions about how genetic material should behave. By building models, they found that a smooth, two-stranded helix of constant diameter could be constructed only when an adenine hydrogen bonded with thymine and when a guanine bonded with cytosine in the centre of the helix. They immediately realized that the double helical structure provided a mechanism by which genetic material might be Replicated. The two parental strands could unwind and direct the synthesis of complementary strands, thus forming two new identical DNA molecules. Watson, Crick, and Wilkins received the Nobel Prize in 1962 for their discoveries. Franklin could not be considered for the prize because she had died of cancer in 1958 at the age of thirty-seven.
  37. 37. DNA has four properties that enable it to function as genetic material. It can…  Store Information that determines the characteristics of cells and organisms;  Use this information to Direct the Synthesis of structural and regulatory proteins essential to the operation of the cell or organism;  Mutate, or chemically change, and transmit these changes to future generations; and  Replicate by directing the manufacture of copies of itself. Each strand of the DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Each strand can therefore act as a template for the synthesis of a new complementary strand.
  38. 38. Central Dogma The Central Dogma of Molecular Biology is an explanation of the flow of genetic information within a biological system. It was first stated by Francis Crick in 1958 and re-stated in a Nature paper published in 1970. DNA contains the complete genetic information that defines the structure and function of an organism. Proteins are formed using the genetic code of the DNA. Three different processes are responsible for the inheritance of genetic information and for its conversion from one form to another. Replication : A double stranded nucleic acid is duplicated to give identical copies. This process propagates the genetic information. Transcription : A DNA segment that constitutes a gene is read and transcribed into a single stranded sequence of RNA. The RNA moves from the nucleus into the cytoplasm. Translation : The RNA sequence is translated into a sequence of amino acids as the Protein is formed. During translation, the ribosome reads three bases (Codon) at a time from the RNA and translates them into one amino acid.
  39. 39. Central Dogma
  40. 40. Before a cell divides, its DNA must replicate so that each daughter cell receives the same set of genetic instructions. Clues to the self-replication mechanism came from Watson and Crick’s report on DNA’s chemical structure. The paper ends with the tantalizing statement, “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” They envisioned DNA unwinding, exposing unpaired bases that would attract their complements, and neatly knitting two double helices from one. This route to replication is called Semiconservative because each DNA double helix conserves half of the original molecule Bidirectional Replication. Replication
  41. 41. The copying of genetic information by DNA replication. In this process, the two strands of a DNA double helix are pulled apart, and each serves as a template for synthesis of a new complementary strand.
  42. 42. Each strand of the DNA double helix contains a sequence of nucleotides that is exactly complementary to the nucleotide sequence of its partner strand. Each strand can therefore act as a template for the synthesis of a new complementary strand. The process of DNA replication is begun by initiator proteins that bind to the DNA and pry the two strands apart, breaking the hydrogen bonds between the bases. The positions at which the DNA is first opened are called Replication Origin. They usually marked by a particular sequence of nucleotides. Replication DNA Synthesis Begins at Replication Origins
  43. 43. During the DNA replication it is possible to see Y-shaped junctions in the DNA, called REPLICATION FORKS. At these forks, the replication machine is moving along the DNA, opening up the two strands of double helix and using each strand as a template to make new daughter strand. Two replication forks are formed starting from each replication origin, and they move away from the origin in both directions, unzipping DNA as they go. Enzymes called Helicases unwind and hold apart replicating DNA The 5’-to-3’ direction of the DNA polymerization mechanism poses a problem at replication fork One new DNA is being made on a template that runs in one direction (3’to 5’), whereas the other new strand is being made on a template that runs in the opposite direction (5’ to 3’). The Replication Fork Is Asymmetrical New DNA Synthesis Occurs at Replication Forks
  44. 44. DNA Polymerase, however, can catalyse the growth of the DNA chain in only one direction: it can add subunits only to the 3’end of the chain. As a result, a new DNA chain can be synthesized only in 5’ to 3’ direction. The DNA strand whose 5’ end must grow is made DISCONTINUOUSLY, in successive separate small pieces. These small pieces are called Okazaki fragments. The DNA strand that is synthesized discontinuously in this way is called Lagging strand; the strand that is synthesized continuously is called Lagging strand. DNA Polymerase
  45. 45. There is another important restriction for DNA polymerase. It can only add a nucleotide to a polynucleotide that is already correctly paired with the complementary strand. This means that DNA polymerase cannot actually initiate synthesis of a DNA strand by joining the first nucleotides. Nucleotides must be added to the end of an already existing chain, called primer. The primer is not a DNA, but short stretch of RNA. Still another enzyme, Primase, makes the primer. Priming A Primase enzyme builds a short complementary piece of RNA, an RNA Primer, at the start of each DNA segment to be replicated. The RNA primer attracts DNA polymerase, the enzyme that adds new DNA nucleotides complementary to the bases on the exposed strand. The primer is necessary because DNA polymerase can only add nucleotides to an existing strand. As the new DNA strand grows hydrogen bonds form between the complementary bases.
  46. 46. DNA polymerase “proofreads” as it goes, discarding mismatched nucleotides and inserting correct ones. At the same time, another enzyme removes each RNA primer and replaces it with the correct DNA nucleotides. Enzymes called Ligases form covalent bonds between the resulting DNA segments. Three enzymes that function in DNA synthesis: DNA polymerase, ligase, and primase. Many other proteins also participate, and here we examine two of them: helicase and single- strand binding proteins. Helicase is an enzyme that works at crotch of the replication fork, untwisting that double helix and separating the two old strands. Single Strand Binding Protein then attach in chains along unpaired DNA strands, holding these templates straight until new strand can be synthesized.
  47. 47. Helicase unwinds double helix. Primase adds short RNA primer to template strand. Binding proteins stabilize each strand. DNA polymerase binds nucleotides to form new strands. Ligase joins Okazaki fragments and seals nicks in sugar phosphate backbone.
  48. 48. DNA replication is incredibly accurate; after proofreading, DNA polymerase incorrectly incorporates only about 1 in a billion nucleotides. Other repair enzymes help ensure the accuracy of DNA replication by cutting out and replacing incorrect nucleotides. Nevertheless, mistakes occasionally remain. The result is a Mutation, which is any change in a cell’s DNA sequence.
  49. 49. Until September 11, 2001, the most challenging application of DNA profiling had been identifying plane-crash victims, a grim task eased by having lists of passengers. The terrorist attacks on the World Trade Centre provided a staggeringly more complex situation, for several reasons: the high number of casualties, the condition of the remains, and the lack of a list of who was actually in the buildings. Overall, the disaster yielded more than a million DNA samples In the days following September 11, researchers at Myriad Genetics, Inc., in Salt Lake City, who usually analyse DNA for breast cancer genes, received frozen DNA from soft tissue recovered from the disaster site. The laboratory also received cheek scrapings from relatives of the missing, and tissue from the victims’ toothbrushes, razors, and hairbrushes. 9/11
  50. 50. The workers used PCR to determine the numbers of copies of four-base sequences of DNA, called short tandem repeats, or STRs, at 13 locations in the genome. The chance that any two individuals have the same 13 markers by chance is one in 250 trillion. If the STR pattern of a sample from the crime scene matched a sample from a victim’s toothbrush, identification was fairly certain. DNA extracted from tooth and bone bits was sent to Celera Genomics Corporation in Rockville, Maryland. Here, DNA sequences were analysed from mitochondria, which can survive incineration. The labs used DNA to identify about 850 of the more than 2700 people reported missing. It was a very distressing experience for the technicians and researchers, whose jobs had suddenly shifted from detecting breast cancer and sequencing genomes to helping in recovery. 9/11
  51. 51. We now turn our attention to RNA, which differs from DNA in three respects. First, the backbone of RNA contains Ribose rather than 2-deoxyribose. That is, ribose has a hydroxyl group at the 2nd position. Second, RNA contains Uracil in place of thymine. Uracil has the same single-ringed structure as thymine, except that it lacks the 5-methyl group. Third, RNA is usually found as a Single Polynucleotide Chain. RNA
  52. 52. Except for the case of certain viruses, RNA is not the genetic material and does not need to be capable of serving as a template for its own replication. As a consequence of being a single strand, RNA can fold back on itself to form short stretches of double helix between regions that are complementary to each other.
  53. 53. RNA RNA allows a greater range of base pairing than does DNA. Thus, as well as A:U and C:G pairing, U can also pair with G. This capacity to form a non-Watson-Crick base pair adds to the propensity of RNA to form double helical segments. Freed of the constraint of forming long range regular helices, RNA can form complex tertiary structures, which are often based on unconventional interactions between bases and between bases and the sugar-phosphate backbone. The main role of RNA is to transfer the genetic code need for the creation of proteins from the nucleus to the ribosome. This process prevents the DNA from having to leave the nucleus, so it stays safe. Without RNA, proteins could never be made.
  54. 54. RNA Polymerase RNA polymerase I catalyses the transcription of all rRNA genes except 5S. These rRNA genes are organized into a single transcriptional unit and are transcribed into a continuous transcript. This precursor is then processed into three rRNAs: 18S, 5.8S, and 28S. The transcription of rRNA genes takes place in a specialized structure of the nucleus called the nucleolus , where the transcribed rRNAs are combined with proteins to form ribosomes. RNA polymerase II is responsible for the transcription of all mRNAs. Many Pol II transcripts exist transiently as single strand precursor RNAs (pre-RNAs) that are further processed to generate mature RNAs. For example, precursor mRNAs (pre-mRNAs) are extensively processed before exiting into the cytoplasm through the nuclear pore for protein translation. RNA polymerase III transcribes small non-coding RNAs, including tRNAs, 5S rRNA, U6 snRNA, SRP RNA, and other stable short RNAs such as ribonuclease P-RNA.
  55. 55. Transcription & Processing of rRNA Eukaryotic transcription occurs within the nucleus where DNA is packaged into nucleosomes and higher order chromatin structures. Transcription is the process of copying genetic information stored in a DNA strand into a transportable complementary strand of RNA. Transcription takes place in the nucleus of the cell and proceeds in basic three sequential stages: Initiation, Elongation, and Termination. The transcriptional machinery that catalyses this complex reaction has at its core three multi-subunit RNA polymerases. Transcription is divided into  Pre-initiation  Initiation  Promoter clearance  Elongation and  Termination
  56. 56. Transcription & Processing of rRNA Initiation The initiation of gene transcription in eukaryotes occurs in specific steps . First, an RNA polymerase along with general transcription factors binds to the promoter region of the gene to form a closed complex called the Preinitiation Complex. The subsequent transition of the complex from the closed state to the open state results in the melting or separation of the two DNA strands and the positioning of the template strand to the active site of the RNA polymerase. Without the need of a primer, RNA polymerase can initiate the synthesis of a new RNA chain using the template DNA strand to guide ribonucleotide selection & polymerization chemistry. Polymerase II-transcribed genes contain a region in the immediate vicinity of the transcription start site (TSS) that binds and positions the preinitiation complex. This region is called the core promoter because of its essential role in transcription initiation. Different classes of sequence elements are found in the promoters. For example, the TATA box is the highly conserved DNA recognition sequence for the TATA box binding protein (TBP) whose binding initiates transcription complex assembly at many genes.
  57. 57. Transcription & Processing of rRNA General transcription factors are a group of proteins involved in transcription initiation and regulation.These factors typically have DNA-binding domains that bind specific sequence elements of the core promoter and help recruit RNA polymerase to the transcriptional start site. General transcription factors for RNA polymerase II include TFIID, TFIIA, TFIIB, TFIIF, TFIIE and TFIIH. Promoters & General Transcription Factors Assembly of Preinitiation Complex To prepare for transcription, a complete set of general transcription factors and RNA polymerase need to be assembled at the core promoter to form the Preinitiation Complex. For example, for promoters that contain a TATA box near the TSS, the recognition of TATA box by the TBP subunit of TFIID initiates the assembly of a transcription complex. The next proteins to enter are TFIIA and TFIIB, which stabilize the DNA-TFIID complex and recruit Polymerase II in association with TFIIF and additional transcription factors. TFIIF serves as the bridge between the TATA-bound TBP and polymerase. One of the last transcription factors to be recruited to the preinitiation complex is TFIIH, which plays an important role in promoter melting and escape.
  58. 58. Transcription & Processing of rRNA Promoter melting and open complex formation Abortive initiation Once the initiation complex is open, the first ribonucleotide is brought into the active site to initiate the polymerization reaction in the absence of a primer. This generates a nascent RNA chain that forms a hetero-duplex with the template DNA strand. However, before entering the elongation phase, polymerase may terminate prematurely and release a short, transcript. This process is called abortive initiation. Promoter escape When a transcript attains the threshold length of ten nucleotides, it enters the RNA exit channel.The polymerase breaks its interactions with the promoter elements and any regulatory proteins associated with the initiation complex that it no longer needs. Promoter melting in eukaryotes requires hydrolysis of ATP and is mediated by TFIIH. TFIIH is a ten-subunit protein, including both ATPase and Protein Kinase activities.
  59. 59. Transcription & Processing of rRNA Elongation After escaping the promoter and shedding most of the transcription factors for initiation, the polymerase acquires new factors for the next phase of transcription: Elongation Double stranded DNA that enters from the front of the enzyme is unzipped to avail the template strand for RNA synthesis. For every DNA base pair separated by the advancing polymerase, one hybrid RNA:DNA base pair is immediately formed. DNA strands and nascent RNA chain exit from separate channels; the two DNA strands reunite at the trailing end of the transcription bubble while the single strand RNA emerges alone.
  60. 60. Transcription & Processing of rRNA Elongating polymerase is associated with a set of protein factors required for various types of RNA processing. mRNA is capped as soon as it emerges from the RNA-exit channel of the polymerase. RNA Processing
  61. 61. Transcription & Processing of rRNA RNA Processing One of the most significant differences between prokaryotic and eukaryotic transcription is that the protein-coding region of the prokaryotic DNA is continuous, while in eukaryotic cells, it is not. A eukaryotic gene begins with a promoter region and an initiation code and ends with a termination code and region. However, the intervening gene sequence contains patches of nucleotides that apparently do not code for protein but do serve important roles in maintaining the cell. If they were used in protein synthesis, the resulting proteins would be worthless. To remedy this problem, eukaryotic cells prune these segments from the mRNA after transcription. When such split genes are transcribed, RNA polymerase synthesizes a strand of pre-mRNA that initially includes copies of both Exons (meaningful mRNA coding sequences) and Introns (intervening DNA sequences).
  62. 62. Transcription & Processing of rRNA RNA Processing Pre-mRNA is used to describe the nuclear transcript that is processed by modification and splicing to give an mRNA. Soon after its manufacture, Post-transcriptional modifications (i.e. Splicing) remove introns before shipping the final mRNA to the cytoplasm. RNA splicing is the process of excising the sequences in RNA that correspond to introns, so that the sequences corresponding to exons are connected into a continuous mRNA. Spliceosomes are organelles in which the excision and splicing reactions that remove introns from pre-mRNA occur. In humans, it has been found that the exons of a single gene may be spliced together in three different ways, resulting in the production of three different, mature messenger RNAs. The final RNA processing event that is coupled with the termination of transcription.
  63. 63. Transcription & Processing of rRNA Termination The last stage of transcription is termination, which leads to the dissociation of the complete transcript and the release of RNA polymerase from the template DNA. The process differs for each of the three RNA polymerases. The mechanism of termination is the least understood of the three transcription stages.
  64. 64. Transcription & Processing of rRNA This is a summary of the events that occur in the nucleus during the manufacture of mRNA in a eukaryotic cell. Notice that the original nucleotide sequence is first transcribed into pre-RNA molecule that is later “clipped” and then rebonded to form a shorter version of the original. It is during this time that the introns are removed. Transcription of mRNA in Eukaryotic Cells
  65. 65. Reverse Transcription Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA. HIV has an RNA genome that is reverse transcribed into DNA. The resulting DNA can be merged with the DNA genome of the host cell. The main enzyme responsible for synthesis of DNA from an RNA template is called reverse transcriptase. In the case of HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand (cDNA) to the viral RNA genome. The enzyme ribonuclease H then digests the RNA strand, and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure ("cDNA"). The cDNA is integrated into the host cell's genome by the enzyme integrase , which causes the host cell to generate viral proteins that reassemble into new viral particles. In HIV, subsequent to this, the host cell undergoes programmed cell death, or apoptosis of T cells .However, in other retroviruses, the host cell remains intact as the virus buds out of the cell.
  66. 66. Reverse Transcription Reverse Transcription is widely used in the laboratory to convert RNA to DNA for use in molecular cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.
  67. 67. Transfer RNA (tRNA) A Transfer RNA is an adaptor molecule composed of RNA, typically 73 to 94 nucleotides in length, that serves as the physical link between the nucleotide sequence of nucleic acids and the amino acid sequence of proteins. It does this by carrying an amino acid to the protein synthetic machinery of a cell (ribosome) as directed by a three-nucleotide sequence (codon) in a messenger RNA (mRNA).
  68. 68. Transfer RNA (tRNA) As such, tRNAs are a necessary component of protein translation, the biological synthesis of new proteins according to the genetic code. An anticodon is a unit made up of three nucleotides that correspond to the three bases of the codon on the mRNA. Each tRNA contains a specific anticodon triplet sequence that can base-pair to one or more codons for an amino acid. Some anticodons can pair with more than one codon due to a phenomenon known as wobble base pairing. In the genetic code, it is common for a single amino acid to be specified by all four third- position possibilities, or at least by both Pyrimidines and Purines; for example, the amino acid glycine is coded for by the codon sequences GGU, GGC, GGA, and GGG.
  69. 69. Translation Translation is the process of using the information in RNA to direct the ordered assembly of amino acids. The word “translation” refers to the fact that nucleic acid language is being changed to protein language. To translate mRNA language into protein language, a translation dictionary is necessary.
  70. 70. A 3-nucleotide combination is correlated to the single amino acid that is required in the process of translation. Each 3-nucleotide combination is called a Codon. Consider that each codon codes for one, and only one, amino acid. The codon UUU corresponds to only phenylalanine (Phe). However, notice that more than one mRNA codon may code for the same amino acid. Phenylalanine (Phe) can be coded for by the UUU codon and the UUC codon. This is possible because there are only 20 Amino Acids and 64 Different Codons.
  71. 71. The construction site of the protein molecules (i.e., the translation site) is on the Ribosome in the cytoplasm of the cell. The ribosome is a cellular organelle that serves as the meeting place for mRNA and the tRNAs that carry amino acid building blocks. There are many ribosomes in a cell. The mRNA and the tRNAs were synthesized by the process of transcription in the cell’s nucleus and then moved to the cell’s cytoplasm.. The process of translation can be broken down into three basic processes.. • Initiation • Elongation • Termination
  72. 72. Translation The covalent attachment to the tRNA 3’ end is catalysed by enzymes called aminoacyl-tRNA synthetases. During protein synthesis, tRNAs with attached amino acids are delivered to the ribosome by proteins called elongation factors (eEF-1 in eukaryotes), which aid in decoding the mRNA codon sequence. If the tRNA's anticodon matches the mRNA, another tRNA already bound to the ribosome transfers the growing polypeptide chain from its 3’ end to the amino acid attached to the 3’ end of the newly delivered tRNA, a reaction catalysed by the ribosome. Aminoacylation 1. Amino Acid + ATP → Aminoacyl-AMP + PPi 2. Aminoacyl-AMP + tRNA → Aminoacyl-tRNA + AMP Initiation
  73. 73. Translation The ribosome has three binding sites for tRNA molecules that span the space between the two ribosomal subunits:  A Site (Aminoacyl Site)  P Site (Peptidyl Site)  E Site (Exit Site) A Start codon (AUG) complements with the Methionine (Met) tRNA in the ribosome, constituting the translation initiation complex. A new anticodon will land in the A site, and its amino acid will join Met. The tRNA will slide to the P site leaving the A site free for another anticodon.
  74. 74. Translation
  75. 75. Translation Once protein synthesis is initiated, the ribosome, mRNA, and tRNA undergo a repetitive series of events to bring in each subsequent amino acid of the protein. To provide a one-to-one correspondence between tRNA molecules and codons that specify amino acids, 61 types of tRNA molecules would be required per cell. The order for the entry of amino acids into the growing protein is dictated by base-pairing rules between the codon of the mRNA and the anticodon of the tRNA. Elongation
  76. 76. Translation Reading Frames and Their Importance. The place at which DNA sequence reading begins determines the way nucleotides are grouped together in clusters of three (outlined with brackets), and this specifies the mRNA codons and the peptide product. In the example, a change in the reading frame by one nucleotide yields a quite different mRNA and final peptide.
  77. 77. Translation The mRNA coding for a protein is not read to its end to finish protein synthesis. Just as there was initiation codon for starting protein synthesis, there are also termination Codons that allow protein synthesis to end in the middle of the mRNA molecule. A stop codon (UAG, UAA, or UGA) signals the end of the mRNA molecule. A release factor triggers the disassembling of the two ribosomal units and the mRNA molecule. Termination
  78. 78. Translation
  79. 79. Any change in the nucleotide sequence of DNA—the genetic information—is a mutation. Some mutations result in changes in the proteins produced and some do not. As stated earlier, as a genetic material, DNA must be able to change, or mutate. Different types of changes can occur and these changes have a potential impact on protein synthesis. Mutagenic agents are substances or conditions that can cause the sequence of DNA to change. Agents known to cause damage to DNA are certain viruses (e.g., papillomavirus), weak or “fragile” spots in the DNA, radiation (X rays or UV light), and chemicals found in foods and other products such as nicotine in tobacco. Mutation
  80. 80. Forward Mutation 1. Single Nucleotide Base Pair Substitution ( At DNA Level) Transition: purine --> purine or pyrimidine --> pyrimidine A--> G or G--> A T--> C or C--> T Transversion: purine --> pyrimidine or vice versa A--> T, C; G -->T,C; T-->A, G; C-->A,G Causes…… (At Protein Level) • Silent Mutation • Neutral or Splicing Mutation • Missense Mutation • Nonsense Mutation.
  81. 81. What types of DNA mutations can be the most devastating to protein synthesis? Consider that any change in the start codon will prevent the protein from being correctly synthesized. Also consider that inserting or deleting a single nucleotide in the protein coding sequence will cause the ribosome to read the wrong set of 3 nucleotides as the codon. This type of mutation is called a Frameshift Mutation. To identify the impact that a mutation can have on a protein and, ultimately, the whole organism, consider sickle-cell anemia. In some individuals, a single nucleotide of the gene may be changed. This type of mutation is called Point Mutation.
  82. 82. Genetic Engineering & Biotechnology Biotechnology includes the use of a method of splicing genes from one organism into another, resulting in a new form of DNA called recombinant DNA. Organisms with these genetic changes are referred to as genetically modified (GM), or transgenic organisms. These organisms or their offspring have been engineered so that they contain genes from at least one unrelated organism, which could be a virus, a bacterium, a fungus, a plant, or an animal.
  83. 83. Gene cloning is accomplished by using enzymes that are naturally involved in the DNA replication process and other enzymes that are naturally produced by bacteria. When genes are spliced from different organisms into host cells, the host cell replicates these new “foreign” genes and synthesizes proteins encoded by them. Gene splicing begins with the laboratory isolation of DNA from an organism that contains the desired gene—for example, from human cells that contain the gene for the manufacture of insulin. If the gene is short enough and its base sequence is known, it can be synthesized in the laboratory from separate nucleotides. If the gene is too long and complex, it is cut from the chromosome with enzymes called restriction endonucleases. Gene cloning
  84. 84. The polymerase chain reaction (PCR) is a biochemical technology in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include……  DNA cloning for Sequencing, DNA-based Phylogeny, or Functional Analysis of Genes;  The diagnosis of Hereditary Diseases;  The identification of Genetic Fingerprints (used in forensic sciences and paternity testing)  The detection and Diagnosis of Infectious Diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR. Polymerase Chain Reaction (PCR)
  85. 85. Because every person’s DNA is unique …  when samples of an individual’s DNA are subjected to restriction enzymes, the cuts will occur in different places and DNA chunks of different sizes will result.  Restriction enzymes have the ability to cut DNA at places where specific sequences of nucleotides occur. When the chunks are caused to migrate across an electrophoresis gel.  The smaller fragments migrate farther than the larger fragments, producing a pattern known as a “DNA fingerprint”  Because of individual differences in DNA sequences, these sites vary from person to person. As a result, the DNA fingerprint that separates DNA fragments on the basis of size can appear different from one person to another. DNA Fingerprinting
  86. 86. Bibliography: • Hams Histology • Bruce Alberts: Molecular.Biology.Of.The.Cell.5th.Ed • Arthur C. Guyton; John E. Hall. Text book of Medical Physiology. Tenth edition.

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