Lec 1 introduction to molecular biology

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Lec 1 introduction to molecular biology

  1. 1. Introduction to molecular biology
  2. 2. Subjects overview • The aim of these lectures is to investigate how cells organize their DNA within the cell nucleus, and replicate it during cell division to produce two new copies of the genome. Cellular processes to repair damaged DNA will also be covered. • The mechanism of DNA replication will be discussed, covering the structure of the replication fork, how cells select sites of replication initiation, and how they control whether and when to replicate DNA • The reaction mechanism catalyzed by DNA polymerases causes difficulty in replicating the ends of linear DNA molecules. Various methods have evolved to solve this ‘end-replication problem’. The most common involves the use of an unusual reverse transcriptase, called telomerase • We will discuss genome organization: introns, exons, satellites, repetitive DNAetc • How is the huge amount of genomic DNA packaged to fit within the cell nucleus, and still keeping specific sequences accessible for transcription? We will discuss the structure of the nucleosome and higher levels of chromatin organization and packaging • DNA is often damaged under normal environmental conditions. How can cells repair their genome and what are the consequences if they cannot?
  3. 3. Molecular biology  Is the branch of biology that deals with the molecular basis of biological activity.  This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry.
  4. 4.  Molecular biology chiefly concerns itself with understanding and the interactions between the various systems of a cell, including the interactions between the different types of DNA, RNA and protein biosynthesis as well as learning how these interactions are regulated.
  5. 5.  The field of molecular biology studies macromolecules and the macromolecular mechanisms found in living things, such as the molecular nature of the gene and its mechanisms of gene replication, mutation, and expression.
  6. 6. The genome The totality of genetic information and is encoded in the DNA or RNA for some viruses.
  7. 7. All living things are grouped into three domain: Eukaryotes Prokaryotes Archaea
  8. 8. Eukaryotic cell Eukaryotic cell are generally more advanced than prokaryotic cell.  has a nucleus, which is separated from the rest of the cell by a membrane. The nucleus contains chromosomes, which are the carrier of the genetic material.  The genetic material distributed among multiple chromosome.  Eukaryotic DNA is linear and complexed with protiens called histones.
  9. 9. Prokaryotic cell  Prokaryotes are single-celled organisms  Without nucleus, no nuclear membrane.  DNA is naked, without histones,  Archaea are prokaryotes (without nucleus) but some aspects similar to Eukaryotes.
  10. 10.  Deoxyribonucleic acid (DNA)  The genetic instructions used in the development and functioning of all known living organisms and some viruses.  The main role of DNA molecules is the long-term storage of information.
  11. 11.  DNA is often compared to a set of blueprints or a recipe, or a code, since it contains the instructions needed to construct other components of cells, such as proteins and RNA molecules.
  12. 12. The chromosome The storage place of all genetic information.  The number of chromosome varies from one species to another.
  13. 13. The genes The DNA segments that carry this genetic information are called genes.
  14. 14. • General structure of nucleic acids: • DNA is a long polymer made from repeating units called nucleotides. • The DNA chain is 22 to 26 Å wide (2.2 to 2.6 nano.), and one nucleotide unit is 3.3 Å (0.33 nm) long. Although each individual repeating unit is very small, DNA polymers can be very large molecules containing millions of nucleotides. • Human chromosome number 1, is approximately 220 million base pairs long.
  15. 15. Building Blocks - Nucleotides A nucleotide is composed of three parts: sugar ( Ribose in RNA and Deoxy ribose in DNA), base and phosphate group. If all phosphate groups are removed, a nucleotide becomes a nucleoside.
  16. 16.  The four bases found in DNA are:  Adenine (A),  Cytosine (C),  Guanine (G) and  Thymine (T).  A fifth pyrimidine base, called uracil (U), usually takes the place of thymine in RNA and differs from thymine by lacking a methyl group on its ring.
  17. 17.  These bases are classified into two types; adenine and guanine are fused five- and six-membered heterocyclic compounds called purines,  while cytosine and thymine are six-membered rings called pyrimidines.
  18. 18. • In living organisms, DNA does not usually exist as a single molecule, but instead as a pair of molecules that are held tightly together. These two long strands entwine like vines, in the shape of a double helix. • In a double helix the direction of the nucleotides in one strand is opposite to their direction in the other strand: the strands are antiparallel. • The asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends, with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl group.
  19. 19.  Base pairing Each type of base on one strand forms a bond with just one type of base on the other strand. This is called complementary base pairing. Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T, and C bonding only to G. This arrangement of two nucleotides binding together across the double helix is called a base pair. As hydrogen bonds are not covalent, they can be broken and rejoined relatively easily.
  20. 20. • 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. For example, the sequence of the two strands can be written as • 5' -ACT- 3' • 3' -TGA- 5' • Note that they obey the (A:T) and (C:G) pairing rule. If we know the sequence of one strand, we can deduce the sequence of another strand. For this reason, a DNA database needs to store only the sequence of one strand. By convention, the sequence in a DNA database refers to the sequence of the 5' to 3' strand (left to right).
  21. 21.  Grooves  Twin helical strands form the DNA backbone. Another double helix may be found by tracing the spaces, or grooves, between the strands. As the strands are not directly opposite each other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and the other, the minor groove, is 12 Å wide.
  22. 22. Sense and antisense A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear.
  23. 23. Supercoiling DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs. If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases.
  24. 24. Alternate DNA structures DNA exists in many possible conformations that include A-DNA, B-DNA, and Z-DNA forms, although, only B- DNA and Z-DNA have been directly observed in functional organisms.
  25. 25. From left to right, the structures of A, B and Z DNA
  26. 26. DNA binding proteins Lec 2
  27. 27. The aims of this lecture is to investigate how cells organize their DNA within the cell nucleus, how is the huge amount of genomic DNA packaged to fit within the cell nucleus, and still keeping specific sequences accessible for transcription?. We will discuss genome organization, satellites, repetitive DNAetc • We will discuss the structure of the nucleosome and higher levels of chromatin organization and packaging.
  28. 28.  Interactions with proteins All the functions of DNA depend on interactions with proteins. These protein interactions can be non-specific, or the protein can bind specifically to a single DNA sequence. Enzymes can also bind to DNA for example the polymerases that copy the DNA sequence in transcription and DNA replication.
  29. 29. • DNA-binding proteins Within chromosomes, DNA is held in complexes with structural proteins. These proteins organize the DNA into a compact structure called chromatin. In eukaryotes this structure involves DNA binding to a complex of small basic proteins called histones, while in prokaryotes multiple types of proteins are involved. The histones form a disk-shaped complex called a nucleosome. These non- specific interactions are formed through basic residues in the histones making ionic bonds to the acidic sugar- phosphate backbone of the DNA.
  30. 30.  Chromatin Chromatin is the complex combination of DNA and protein that makes up chromosomes. It is found inside the nuclei of eukaryotic cells. The major components of chromatin are DNA and histone proteins. The functions of chromatin are to package DNA into a smaller volume to fit in the cell.
  31. 31. • Chromatin is the substance which becomes visible chromosomes during cell division. Its basic unit is nucleosome, composed of 146 bp DNA and eight histone proteins. The structure of chromatin is dynamically changing, at least in part, depending on the need of transcription. In the metaphase of cell division, the chromatin is condensed into the visible chromosome. At other times, the chromatin is less condensed, with some regions in a "Beads-On-a-String" conformation.
  32. 32. • Histones are the proteins closely associated with DNA molecules. They are responsible for the structure of chromatin and play important roles in the regulation of gene expression. Five types of histones have been identified: H1 (or H5), H2A, H2B, H3 and H4. H1 and its homologous protein H5 are involved in higher-order structures of chromatin. The other four types of histones associate with DNA to form nucleosomes.
  33. 33.  Histones (H1, H2A, H2B, H3, H4, and H5) organized into two super classes as follows: Core histones – H2A, H2B, H3 and H4 and linker histones – H1 and H5.  Histones contain a high proportion of basic amino acids (arginine and lysine) that facilitate binding to the negatively charged DNA molecule.
  34. 34.  Two of each of the core histones (H2A, H2B, H3 and H4) assemble to form one nucleosome core particle by wrapping 146 base pairs of DNA around the protein spool in 1.65 left-handed super-helical turn. The linker histone H1 binds the nucleosome and the entry and exit sites of the DNA, thus locking the DNA into place and allowing the formation of higher order structure.
  35. 35. • each nucleosome is associated with an H1 (or H5) to form a solenoid structure. H1 and H5 are called linker histones.
  36. 36.  Chromosomes  A chromosome is an organized structure of DNA and protein that is found in cells. It is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.
  37. 37. • Chromosomes in prokaryotes The prokaryotes – bacteria and archaea – typically have a single circular chromosome, but many variations do exist. Most bacteria have a single circular chromosome that can range in size from only 160,000 base pairs in the endosymbiotic bacterium Candidatus Carsonella ruddii, to 12,200,000 base pairs in the soil-dwelling bacterium Sorangium cellulosum. Spirochaetes of the genus Borrelia are a notable exception to this arrangement, with bacteria such as Borrelia burgdorferi, ontaining a single linear chromosome.
  38. 38. Repetitive DNA Sequences  A stretch of DNA sequence often repeats several times in the total DNA of a cell. For example, the following DNA sequence is just a small part of telomere located at the ends of each human chromosome:
  39. 39. An entire telomere, about 15 kb, is constituted by thousands of the repeated sequence "GGGTTA".
  40. 40. DNA sequences are divided into three classes: • Highly repetitive: About 10-15% of mammalian DNA fragments reassociate very rapidly. This class includes tandem repeats. • Moderately repetitive: Roughly 25-40% of mammalian DNA fragments reassociate at an intermediate rate. This class includes interspersed repeats (also known as mobile elements or transposable elements). • Single copy (or very low copy number): This class accounts for 50-60% of mammalian DNA.
  41. 41.  Tandem repeats are an array of consecutive repeats. They include three subclasses: satellites, minisatellites and microsatellites. The name "satellites" comes from their optical spectra.
  42. 42. Satellites • The size of a satellite DNA ranges from 100 kb to over 1 Mb. In humans, a well known example is the alphoid DNA located at the centromere of all chromosomes. Its repeat unit is 171 bp and the repetitive region accounts for 3-5% of the DNA in each chromosome. Other satellites have a shorter repeat unit. Most satellites in humans or in other organisms are located at the centromere.
  43. 43. Minisatellites • The size of a minisatellite ranges from 1 kb to 20 kb. One type of minisatellites is called variable number of tandem repeats (VNTR). Its repeat unit ranges from 9 bp to 80 bp. They are located in non-coding regions. The number of repeats for a given minisatellite may differ between individuals. This feature is the basis of DNA fingerprinting. • Another type of minisatellites is the telomere. In a human germ cell, the size of a telomere is about 15 kb. In an aging somatic cell, the telomere is shorter. The telomere contains tandemly repeated sequence GGGTTA.
  44. 44. Microsatellites • Microsatellites are also known as short tandem repeats (STR), because a repeat unit consists of only 1 to 6 bp and the whole repetitive region spans less than 150 bp. Similar to minisatellites, the number of repeats for a given microsatellite may differ between individuals. Therefore, microsatellites can also be used for DNA fingerprinting. In addition, both microsatellite and minisatellite patterns can provide information about paternity.
  45. 45. Interspersed Repeats• Interspersed repeats are repeated DNA sequences located at dispersed regions in a genome. They are also known as mobile elements or transposable elements. A stretch of DNA sequence may be copied to a different location through DNA recombination. After many generations, such sequence (the repeat unit) could spread over various regions. Mobile elements are found in all kinds of organisms. In mammals, the most common mobile elements are LINEs and SINEs.
  46. 46. Thanks for your attention
  47. 47. DNA Replicatio n
  48. 48. Aimes  To understand the DNA replication mechanism in eukaryotes and prokaryotes.  Identifying the DNA polymerases.
  49. 49. DNA replication: The basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA.
  50. 50. "semiconservative"Each strand of the original double-stranded DNA molecule serves as template for the reproduction of the complementary strand.
  51. 51. Replisome The replisome is a complex molecular machine that carries out replication of DNA. It is made up of a number of subcomponents that each provides a specific function during the process of replication.
  52. 52.  Helicase  Gyrase (Topoisomerases)  Primase  DNA pol. III  DNA pol. I  Ligase  SSB (Single strand binding protein).  Exonuclease Major Components of Replisome
  53. 53. • DNA polymerases are a family of enzymes that carry out all forms of DNA replication. • DNA polymerase can only extend an existing DNA strand paired with a template strand; it cannot begin the synthesis of a new strand. • To begin synthesis of a new strand, a short fragment of DNA or RNA, called a primer, must be created and paired with the template strand before DNA polymerase can synthesize new DNA. DNA polymerases
  54. 54. DNA pol III has two key limitations:  It can only add nucleotides to the 3' end of a strand.  2- It cannot start a new strand; it can only extend an existing strand (because it must only add to 3' ends of strand).
  55. 55. Three types of DNA polymerase classified in prokaryotes, Type I, used to fill the gap between DNA fragments of the lagging strand. Type II, involved in the SOS response to DNA damage. Type III, DNA replication is mainly carried out by the DNA polymerase III.
  56. 56. In Eukaryotes • There are five types of DNA polymerases in mammalian cells: a, b, g, d, and e. The (g) subunit is located in the mitochondria, responsible for the replication of mtDNA. Other subunits are located in the nucleus. Their major roles of each subunits are: • a: synthesis of lagging strand. • b: DNA repair. • d: synthesis of leading strand. • e: DNA repair.
  57. 57. • The prokaryotic DNA polymerase III consists of several subunits, with a total molecular weight exceeding 600kD. Among them, a, e, and q subunits constitute the core polymerase. • The major role of b subunits is to keep the enzyme from falling off the template strand. Two b subunits can form a donut-shaped structure to clamp a DNA molecule in its center, and slide with the core polymerase along the DNA molecule. This allows continuous polymerization of up to 5 x 105 nucleotides. In the absence of b subunits, the core polymerase would fall off the template strand after synthesizing 10-50 nucleotides.
  58. 58. DNA replication within the cell Origins of replication • For a cell to divide, it must first replicate its DNA. This process is initiated at particular points within the DNA, known as "origins", which are targeted by proteins that separate the two strands and initiate DNA synthesis. Origins contain DNA sequences recognized by replication initiator proteins (eg. dnaA in E.coli and the Origin Recognition Complex in yeast). These initiator proteins separate the two strands and initiate replication forks.
  59. 59. Origins tend to be "AT-rich" (rich in adenine and thymine bases) to assist this process, because A-T base pairs have two hydrogen bonds (rather than the three formed in a C-G pair)—strands rich in these nucleotides are generally easier to separate because a greater number of hydrogen bonds requires more energy to break them.
  60. 60. The replication fork  When replicating, the original DNA splits in two, forming two "prongs" which resemble a fork (hence the name "replication fork"). Because DNA polymerase can only synthesize a new DNA strand in a 5' to 3' manner, the process of replication goes differently for the two strands comprising the DNA double helix.
  61. 61. Mechanism of DNA Replication Once strands are separated, RNA primers are created on the template strands. DNA Polymerase extends the leading strand in one continuous motion and the lagging strand in a discontinuous motion. Rnase removes the RNA fragments used to initiate replication by DNA Polymerase, and DNA Polymerase I enters to fill the gaps. When this is complete, a single nick on the leading strand and several nicks on the lagging strand can be found. Ligase works to fill these nicks in, thus completing the newly replicated DNA molecule.
  62. 62. The leading strand receives one RNA primer per active origin of replication while the lagging strand receives several; these several fragments of RNA primers found on the lagging strand of DNA are called Okazaki fragments, named after their discoverer.
  63. 63. Leading strand The leading strand is the template strand of the DNA double helix so that the replication fork moves along it in the 3' to 5' direction. This allows the newly synthesized strand complementary to the original strand to be synthesized 5' to 3' in the same direction as the movement of the replication fork.  On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it continuously. This polymerase is DNA polymerase III (DNA Pol III) in prokaryotes and presumably Pol ε in eukaryotes.
  64. 64. Lagging strand The lagging strand is the strand of the template DNA double helix that is oriented so that the replication fork moves along it in a 5' to 3' manner. Because of its orientation, opposite to the working orientation of DNA polymerase III, which moves on a template in a 3' to 5' manner, replication of the lagging strand is more complicated than that of the leading strand.
  65. 65.  On the lagging strand, primase "reads" the DNA and adds RNA to it in short, separated segments. DNA polymerase III or Pol δ lengthens the primed segments, forming Okazaki fragments. Primer removal in eukaryotes is also performed by Pol δ. In prokaryotes, DNA polymerase I "reads" the fragments, removes the RNA by 5'-3' exonuclease activity of polymerase I, and replaces the RNA nucleotides with DNA nucleotides (this is necessary because RNA and DNA use slightly different kinds of nucleotides). DNA ligase joins the fragments together.
  66. 66. In bacteria, which have a single origin of replication on their circular chromosome, this process eventually creates a "theta structure" (resembling the Greek letter theta: θ). In contrast, eukaryotes have longer linear chromosomes and initiate replication at multiple origins within these.
  67. 67. Telomerase and Aging• Synthesis of the lagging strand requires a short primer which will be removed. At the extreme end of a chromosome, there is no way to synthesize this region when the last primer is removed. Therefore, the lagging strand is always shorter than its template by at least the length of the primer. This is the so-called "end-replication problem". • Bacteria do not have the end-replication problem, because its DNA is circular. • In eukaryotes, the chromosome ends are called telomeres which have at least two functions: • to protect chromosomes from fusing with each other. • to solve the end-replication problem.
  68. 68. • In a human chromosome, the telomere is about 10 to 15 kb in length, composed of the tandem repeat sequence: TTAGGG. The telomerase contains an essential RNA component which is complementary to the telomere repeat sequence. Hence, the internal RNA can serve as the template for synthesizing DNA. Through telomerase translocation, a telomere may be extended by many repeats. Aging • In the absence of telomerase, the telomere will become shorter after each cell division. When it reaches a certain length, the cell may cease to divide and die. Therefore, telomerase plays a critical role in the aging process.
  69. 69. Rolling circle replication  Another method of copying DNA, sometimes used in vivo by bacteria and viruses, is the process of rolling circle replication. In this form of replication, a single replication fork progresses around a circular molecule to form multiple linear copies of the DNA sequence. In cells, this process can be used to rapidly synthesize multiple copies of plasmids or viral genomes.
  70. 70. Topoisomerases  During replication, the unwinding of DNA may cause the formation of tangling structures, such as supercoils or catenanes. The major role of topoisomerases is to prevent DNA tangling.
  71. 71. There are two types of topoisomerases: • type I produces transient single-strand breaks in DNA . • types II produces transient double-strand breaks. As a result, the type I enzyme removes supercoils from DNA one at a time, whereas the type II enzyme removes supercoils two at a time.
  72. 72. • In eukaryotes, the topo I and topo II can remove both positive and negative supercoils. • In bacteria, the topo I can remove only negative supercoils. The bacterial topo II is also called the gyrase, which has two functions: (a) to remove the positive supercoils during DNA replication, (b) to introduce negative supercoils (one supercoil for 15-20 turns of the DNA helix) so that the DNA molecule can be packed into the cell. During replication, these negative supercoils are removed by topo I.
  73. 73. Without topoisomerases, the DNA cannot replicate normally. Therefore, the inhibitors of topoisomerases have been used as anti-cancer drugs to stop the proliferation of malignant cells. However, these inhibitors may also stop the division of normal cells. Some cells (e.g., hair cells) which need to continuously divide will be most affected. This explains a noticeable side effect: the hair loss.
  74. 74. DNA Mutation Lecture 4
  75. 75. Lecture overview  One look around a room tells you that each person has slight differences in their physical make up — and therefore in their DNA. These subtle variations in DNA are called polymorphisms (literally "many forms"). Many of these gene polymorphisms account for slight differences between people such as hair and eye color. But some gene variations may result in disease or an increased risk for disease. Although all polymorphisms are the result of a mutation in the gene, geneticists only refer to a change as a mutation when it is not part of the normal variations between people.
  76. 76. Aims  To understand mutation, mutagen, mutants.  Classification of mutations.  Types of mutagens.  Mutagen & carcinogen.
  77. 77. In biology, a mutation is a randomly derived change to the nucleotide sequence of the genetic material of an organism.
  78. 78. • Mutations can be caused by copying errors in the genetic material during cell division( DNA replication), or by exposure to mutagens (chemical, physical or viruses). • In multi-cellular organisms with dedicated reproductive cells, mutations can be subdivided into germ line mutations, which can be passed on to descendants, and somatic mutations, which are not usually transmitted to descendants.
  79. 79. *By effect on structure 1- Small-scale mutations:- such as those affecting a small gene in one or a few nucleotides, including: A- Point mutations: Exchange a single nucleotide for another, there are different types of point mutation:- *Transitions: Exchanges a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine, (C ↔ T).(Most common) *Transversions: Exchanges a purine for a pyrimidine or a pyrimidine for a purine (C/T ↔ A/G). (Less common) Classification of mutation
  80. 80. *Insertions add one or more extra nucleotides into the DNA. They are usually caused by transposable elements, or errors during replication of repeating elements (e.g. AT repeats). *Deletions remove one or more nucleotides from the DNA.  Ex: original The fat cat ate the wee rat. Point Mutation The fat hat ate the wee rat.
  81. 81. B-Frame-shift mutation In a frame shift mutation, one or more bases are inserted or deleted. This type of mutation disrupt the reading frame thus make the DNA meaningless and often results in a shortened protein. Frame shift mutation can classified to:-
  82. 82.  Deletion Original The fat cat ate the wee rat. Deletion The fat ate the wee rat.  Insertion Original The fat cat ate the wee rat. Insertion The fat cat xlw ate the wee rat.  Inversion In an inversion mutation, an entire section of DNA is reversed. Original The fat cat ate the wee rat. Inversion The fat tar eew eht eta tac.
  83. 83. 2- Large-scale mutations in chromosomal structure, including: A- Deletion of large chromosomal regions, leading to loss of the genes within those regions. B- Translocation: interchange of genetic parts from non-homologous chromosomes. C-Inversion: reversing the orientation of a chromosomal segment. D- Amplifications (or gene duplications) leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them.
  84. 84. Deletion Duplication Inversion
  85. 85.  Loss-of-function mutations are the result of gene product having less or no function.  Gain-of-function mutations change the gene product such that it gains a new and abnormal function.  Lethal mutations are mutations that lead to the death of the organisms which carry the mutations. *By effect on function
  86. 86. In applied genetics it is usual to speak of mutations as either harmful or beneficial.  A harmful mutation is a mutation that decreases the fitness of the organism.  A beneficial mutation is a mutation that increases fitness of the organism, or which promotes traits that are desirable. *By effect on fitness
  87. 87. Conditional mutation is a mutation that has wild-type (or less severe) phenotype under certain "permissive" environmental conditions and a mutant phenotype under certain "restrictive" conditions. For example, a temperature-sensitive mutation can cause cell death at high temperature (restrictive condition), but might have no deleterious consequences at a lower temperature (permissive condition). *Special classes
  88. 88. Causes of mutation Mutations may occur spontaneously (spontaneous mutations) or induced (induced mutations) caused by Mutagens.
  89. 89. *Spontaneous mutations can arise as a result of: 1- DNA replication errors and polymerase accuracy. A- Base alterations Taotomeresim – A base is changed by the repositioning of a hydrogen atom, altering the hydrogen bonding pattern of that base resulting in incorrect base pairing during replication. Deamination - Hydrolysis changes a normal base to an atypical base containing a keto group in place of the original amine group. Examples include C → U and A → HX (hypoxanthine), and 5MeC (5-methylcytosine) → T.
  90. 90. B- Base damage Depurination – Loss of a purine base (A or G) to form an apurinic site (AP site). Alkylation can occur through reaction of compounds such as S-adenosyl methionine with DNA. Alkylated bases may be subject to spontaneous breakdown or mispairing. ** Alkylation, the addition of alkyl (methyl, ethyl, occasionally propyl) groups to the bases or backbone of DNA. 2- Spontaneous genetic rearrengment mutations Deletion, duplication, ……..etc
  91. 91.  Induced mutations on the molecular level can be caused by either Chemical or Physical mutagens. 1- Chemical mutagens The first report of mutagenic action of a chemical was in 1942 by Charlotte Auerbach, who showed that nitrogen mustard (component of poisonous mustard gas used in World Wars I and II) could cause mutations in cells.
  92. 92. A- Base analogs These chemicals structurally resemble purines and pyrimidines and may be incorporated into DNA in place of the normal bases during DNA replication: examples are *bromouracil (BU), resembles thymine (has Br atom instead of methyl group) and will be incorporated into DNA and pair with A like thymine. *aminopurine --adenine analog which can pair with T or with C; causes A:T to G:C or G:C to A:T transitions.
  93. 93. B- Chemicals which alter the structure and pairing properties of bases (base modifiers). Example … *nitrous acid-- formed by digestion of nitrites (preservatives) in foods. It causes C to U, meC to T, and A to hypoxanthine deaminations. *nitrosoguanidine, *methyl methanesulfonate, *ethyl methanesulfonate--chemical mutagens that react with bases and add methyl or ethyl groups. Depending on the affected atom, the alkylated base may then degrade to yield a baseless site, or mispair to result in mutations upon DNA replication.
  94. 94. C- Intercalating agents acridine orange, proflavin, ethidium bromide (used in labs as dyes and mutagens), All are flat, multiple ring molecules which interact with bases of DNA and insert between them. This insertion causes a "stretching" of the DNA duplex and the DNA polymerase is "fooled" into inserting an extra base opposite an intercalated molecule. The result is that intercalating agents cause frameshifts.
  95. 95. D- Agents altering DNA structure Includes a variety of different kinds of agents. These may be:  Large molecules which bind to bases in DNA and cause them to be noncoding "bulky" lesions (eg. NAAAF).  agents causing intra- and inter-strand crosslinks (eg. psoralens--found in some vegetables and used in treatments of some skin conditions).  chemicals causing DNA strand breaks (eg. peroxides)
  96. 96. Physical mutagens (Radiation)
  97. 97. Natural sources of radiation produce so- called background radiation. These include cosmic rays from the sun and outer space, radioactive elements in soil and terrestrial products (wood, stone) and in the atmosphere (radon). One's exposure due to background radiation varies with geographic location. Sources of radiation
  98. 98. Artificial sources: humans have created artificial sources of radiation which contribute to our radiation exposure. Among these are medical testing (diagnostic X-rays and other procedures), nuclear testing and various other products (TV's, smoke detectors, airport X-rays).
  99. 99. Types of radiation Ionizing radiation - Alpha, Beta, Neutron, X-ray and Gamma  Non-ionizing radiation (electromagnetic radiation) - Visible light, Infrared, Microwave, Radio waves, Very low frequency (VLF), Extremely low frequency (ELF), Thermal radiation (heat) and Black body radiation.
  100. 100. Non Ionizing radiation  1. EM spectrum Visible light and other forms of radiation are all types of electromagnetic radiation (consists of electric and magnetic waves). The length of EM waves (wavelength) varies widely and is inversely proportional to the energy they contain: this is the basis of the so-called EM spectrum.
  101. 101. •UV (ultraviolet) UV radiation is less energetic, and therefore non-ionizing, but its wavelengths are preferentially absorbed by bases of DNA and by aromatic amino acids of proteins, so it has important biological and genetic effects.
  102. 102. UV is normally classified in terms of its wavelength:  UV-C (180-290 nm)--"germicidal"--most energetic and lethal, it is not found in sunlight because it is absorbed by the ozone layer.  UV-B (290-320 nm)--major lethal/mutagenic fraction of sunlight.  UV-A (320 nm--visible)--"near UV"--also has deleterious effects (because it creates oxygen radicals) but it produces very few pyrimidine dimers.
  103. 103.  The major lethal lesions are pyrimidine dimers in DNA (produced by UV-B and UV-C)--these are the result of a covalent attachment between adjacent pyrimidines in one strand. These dimers, like bulky lesions from chemicals, block transcription and DNA replication and are lethal if unrepaired. They can stimulate mutation and chromosome rearrangement as well.
  104. 104.  . Ionizing radiation X- and gamma-rays are energetic enough that they produce reactive ions (charged atoms or molecules) when they react with biological molecules; thus they are referred to as ionizing radiation. Intense exposure (high dose rate) causes burns and skin damage versus a long-term weak exposure (low dose rate) which would only increase risk of mutation and cancer.
  105. 105. • Biological effects of radiation Ionizing radiation produces a range of damage to cells and organisms primarily due to the production of free radicals of water (the hydroxyl or OH radical). Free radicals possess unpaired electrons and are chemically very reactive and will interact with DNA, proteins, lipids in cell membranes, etc. Thus X-rays can cause DNA and protein damage which may result in organelle failure, block cell division, or cause cell death. The rapidly dividing cell types (blood cell- forming areas of bone marrow, gastrointestinal tract lining) are the most affected by ionizing radiation and the severity of the effects depends upon the dose received.
  106. 106.  Genetic effects of radiation Ionizing radiation produces a range of effects on DNA both through free radical effects and direct action:  Breaks in one or both strands (can lead to rearrangements, deletions, chromosome loss, death if unrepaired; this is from stimulation of recombination).  Damage to/loss of bases (mutations).  cross linking of DNA to itself or proteins
  107. 107. Thanks
  108. 108. Introduction  DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1 million individual molecular lesions per cell per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions induce potentially harmful mutations in the cell's genome, which affect the survival of its daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. When normal repair processes fail, and when cellular apoptosis does not occur, irreparable DNA damage may occur, including double- strand breaks and DNA crosslinkages
  109. 109. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. A cell that has accumulated a large amount of DNA damage, or one that no longer effectively repairs damage incurred to its DNA, can enter one of three possible states:  an irreversible state of dormancy, known as senescence  cell suicide, also known as apoptosis or programmed cell death  unregulated cell division, which can lead to the formation of a tumor that is cancerous 
  110. 110.  Since many mutations are deleterious,, DNA repair systems are vital to the survival off all organisms  – Living cells contain several DNA repair systems that can fix different type of DNA alterations
  111. 111. DNA Repair  DNA repair mechanisms are placed into different categories on the basis of the way they operate  Direct correction or direct reversal- reversing the damage  Excise the damaged areas and then repair the gap by new DNA synthesis
  112. 112. Basic mechanism of repairing DNA  In most cases,, DNA repair is a multi-step process 1. An irregularity in DNA structure is detected and removed 2. Normal DNA is synthesized DNA 3. Ligation
  113. 113. Direct Reversal of DNA Damage  Mismatch Repair by DNA Polymerase Proofreading.  Repair of UV-Induced Pyrimidine Dimers (reverted by exposure to near-UV light-activates photolyase – not found in humans, It splits the dimers restoring the DNA to its original condition).  Repair of Alkylation Damage (by O6-methylguanine methyltransferase encoded by ada gene, It transfers the methyl or ethyl group from the base to a cysteine  side chain within the alkyltransferase protein)
  114. 114. Base Excision Repair and Repair Involving Excision of Nucleotides There are three major DNA repairing mechanisms: 1- Base excision 2- Nucleotide excision 3- Mismatch repair
  115. 115. • Base excision DNA bases may be modified by deamination or alkylation. The position of the modified (damaged) base is called the "abasic site" or "AP site". In E.coli, the DNA glycosylase can recognize the AP site and remove its base. Then, the AP endonuclease removes the AP site and neighboring nucleotides. The gap is filled by DNA polymerase I and DNA ligase.
  116. 116.  These enzymes can recognize a single damaged base and cleave the bond between it and the sugar in the DNA.  Removes one base, excises several around it, and replaces with several new bases using Pol adding to 3’ ends then ligase attaching to 5’ end  Depending on the species,, this repair system can eliminate abnormal bases such as  – Uracil; Thymine dimers  – 3-methyladenine; 7-methylguanine
  117. 117. • Nucleotide excision In E. coli, proteins UvrA, UvrB, and UvrC are involved in removing the damaged nucleotides (e.g., the dimer induced by UV light). The gap is then filled by DNA polymerase I and DNA ligase. In yeast, the proteins similar to Uvr's are named RADxx ("RAD“ for "radiation"), such as RAD3, RAD10. etc.
  118. 118. An important general process for DNA repair is nucleotide excision repair (NER)  Nicks DNA around damaged base and removes region  Then fills in with Pol on 3’ends, and attaches 5’ end with ligase This type off system can repair many types off DNA damage,, including – Thymine dimers and chemically modified bases NER is found in all eukaryotes and prokaryotes (However, its molecular mechanism is better understood in prokaryotes).
  119. 119. Nucleotide excision repair (NER) of pyrimidine dimer and other damage induced distortions of DNA.
  120. 120. Several human diseases have been shown to involve inherited defects in genes involved in NER – These include xeroderma pigmentosum (XP) and Cockayne syndrome (CS) " A common characteristic off both syndromes is an increased sensitivity to sunlightt – Xeroderma pigmentosum can be caused by defects in seven different NER genes
  121. 121. Skin lesions of Xeroderma Pigmentosum Caused by homozygosity For a recessive mutation in A repair gene. One example of a DNA-repair genetic disease
  122. 122. Mismatch Repair System If proofreading fails, the methyl-directed mismatch repair system comes to the rescue --This repair system is found in all species --In humans, mutations in the system are associated with particular types of cancer. Methyl-directed mismatch repair recognizes mismatched base pairs, excises the incorrect bases, and then carries out repair synthesis.
  123. 123. • Mismatch repair To repair mismatched bases, the system has to know which base is the correct one. In E. coli, this is achieved by a special methylase called the "Dam methylase", which can methylate all adenines that occur within (5')GATC sequences. Immediately after DNA replication, the template strand has been methylated, but the newly synthesized strand is not methylated yet. Thus, the template strand and the new strand can be distinguished.
  124. 124. --The repairing process begins with the protein MutS which binds to mismatched base pairs. Then, MutL activates MutH which binds to GATC sequences. --Activation of MutH cleaves the unmethylated strand at the GATC site. Subsequently, the segment from the cleavage site to the mismatch is removed by exonuclease (with assistance from helicase II and SSB proteins).
  125. 125. If the cleavage occurs on the 3' side of the mismatch, this step is carried out by exonuclease I (which degrades a single strand only in the 3' to 5' direction). * If the cleavage occurs on the 5' side of the mismatch, exonuclease VII or RecJ is used to degrade the single stranded DNA. The gap is filled by DNA polymerase III and DNA ligase.
  126. 126. Mechanism of mismatch repair. The mismatch correction enzyme recognizes which strand the base mismatch is on by reading the methylation state of a nearby GATC sequence. If the sequence is unmethylated, a segment of that DNA strand containing the mismatch is excised and new DNA is inserted.
  127. 127. Mismatch Repair in Eukaryotes Eukaryotes also have mismatch repair, but it is not clear how old and new DNA strands are identified. – Four genes are involved in humans, hMSH2 and hMLH1, hPMS1, and hPMS2 – All of these are mutator genes
  128. 128. In humans, mutations in any one of the four human mismatch repair genes confers a phenotype of hereditary predisposition to a form of colon cancer called hereditary nonpolyposis colon cancer
  129. 129. Proteins involved in DNA repairing of E. coli
  130. 130. Thanks
  131. 131. Gene Expression
  132. 132.  Overview of Gene Expression  An organism may contain many types of somatic cells, each with distinct shape and function. However, they all have the same genome. The genes in a genome do not have any effect on cellular functions until they are "expressed". Different types of cells express different sets of genes, thereby exhibiting various shapes and functions.
  133. 133. Essential steps involved in the expression of the genes.
  134. 134.  Gene expression Is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA (rRNA), transfer RNA (tRNA) or small nuclear RNA (snRNA) genes, the product is a functional RNA.
  135. 135. Steps of gene expression Several steps in the gene expression process may be modulated, including the Transcription RNA splicing Translation Post-translational modification of a protein.
  136. 136. A DNA strand is used as a template to synthesize a complementary RNA strand, which is called the primary transcript. Transcription
  137. 137. Schematic illustration of transcription. (a) DNA before transcription. (b) During transcription, the DNA should unwind so that one of its strand can be used as template to synthesize a complementary RNA.
  138. 138. The function of RNA polymerases Both RNA and DNA polymerases can add nucleotides to an existing strand, extending its length. However, there is a major difference between the two classes of enzymes: RNA polymerases can initiate a new strand but DNA polymerases cannot. RNA Polymerases
  139. 139. In prokaryotes RNA polymerase is composed of five subunits: • Two α subunits • one for each β, β´ • δ subunit. Several different forms of δ subunits have been identified, with molecular weights ranging from 28 kD to 70 kD. The δ subunit is also known as the sigma factor (δ factor).
  140. 140. δ factor plays an important role in recognizing the transcriptional initiation site, and also possesses the helicase activity to unwind the DNA double helix. Tow α subunits, β, β´ carry out nucleotide synthesis.
  141. 141. Core RNA polymerase RNA polymerase without sigma factor (α2 subunits, β, β´), carry out nucleotide synthesis. Holoenzyme refers to a complete and fully functional RNA polymerase. The holoenzyme includes the core polymerase and the δ factor.
  142. 142. In Eukaryotes • There are three classes of eukaryotic RNA polymerases: I, II and III, each comprising two large subunits and 12-15 smaller subunits. • The two large subunits β and β' subunits. • Two smaller subunits α subunit. • The eukaryotic RNA polymerase does not contain any sigma factor. • Therefore, in eukaryotes, transcriptional initiation should be mediated by other proteins.
  143. 143.  RNA polymerase II is involved in the transcription of all protein genes and most snRNA genes.  The other two classes transcribe only RNA genes. RNA polymerase I is located in the nucleolus, transcribing rRNA genes except 5S rRNA.  RNA polymerase III is located outside the nucleolus, transcribing 5S rRNA, tRNA, U6 snRNA and some small RNA genes.
  144. 144.  In prokaryotes, binding of the polymerase's δ factor to promoter can catalyze unwinding of the DNA double helix. The most important δ factor is Sigma 70.  Promoter: a short nucleotide sequence that is recognized by an RNA polymerase enzyme as a point at which to bind to DNA in order to start transcription. Promoters occur upstream of the gene. Transcription Mechanisms in Prokaryotes
  145. 145.  Identifiable steps during transcription: 1- Promoter recognition. 2- Chain initiation. 3- Chain elongation. 4- Chain termination
  146. 146. 1- promoter recognition: δ factor directs RNA polymerase to specific sequences in the DNA called promoters so that transcription initiates at the proper place. Prokaryotic polymerases can recognize the promoter and bind to it directly. Promoters contain two distinct sequence motifs that reside ~10 bases and ~35 bases upstream of the transcriptional start site or first base of the RNA.
  147. 147. • The transcriptional start site is known as the +1 site. All of the bases following the +1 site are transcribed into RNA and are numbered with positive numbers. • The bases prior to the +1 site are numbered with negative numbers. • The promoter sequence consists of tow motifs a ~10 bases upstream of the +1 site is called the -10 box (Pribnow box) and ~35 bases upstream of the +1 is called the -35 box. • δ 70 recognizes promoters with a consensus sequence consisting of TAATAT at the -10 region and -35 region.
  148. 148. The following steps occur before initiation: RNA polymerase recognizes and specifically binds to the promoter region on DNA. At this stage, the DNA is double-stranded ("closed"). This wound-DNA structure is referred to as the closed complex. •The DNA is unwound and becomes single-stranded ("open") in the vicinity of the initiation site (defined as +1). This unwound-DNA structure is called the open complex. • RNA polymerase incorporate the first few nucleotides to the +1 region. • sigma disassociate from the promoter.
  149. 149.  Chain initiation: Unwinding (melting) of the DNA double helix. The enzyme which can unwind the double helix is called helicase. Prokaryotic RNA polymerases have the helicase activity.  Chain elongation: Synthesis of RNA based on the sequence of the DNA template strand. RNA polymerases use nucleoside triphosphates (NTPs) to construct a RNA strand.
  150. 150. Chain termination: Prokaryotes and eukaryotes use different signals to terminate transcription. Transcription in eukaryotes is much more complicated than in prokaryotes, partly because eukaryotic DNA is associated with histones, which could hinder the access of polymerases to the promotor.
  151. 151. Transcriptional Termination in Prokaryotes In prokaryotes, the transcription is terminated by two major mechanisms:  Rho-independent  Rho-dependent.
  152. 152. The Rho-independent termination signal is a stretch of 30-40 bp sequence (terminator sequence), consisting of many GC residues followed by a series of T ( "U" in the transcribed RNA). The resulting RNA transcript will form a stem-loop structure (hairpin) to terminate transcription.
  153. 153. The stem-loop structure of the RNA transcript as a termination signal for the transcription of the trp operon.
  154. 154. • Rho-dependent mechanism • Rho is a ~ 50 kD protein, involved in transcription terminations. Six Rho proteins form a hexamer to terminate transcription. • The Rho protein binds to the RNA transcript at the upstream site which is 70-80 nucleotides long and rich in C residues. Upon binding, the Rho moves along the RNA in the 3' direction. If movement of the polymerase is slow, the Rho will catch up and terminate the transcription at the downstream termination site. Rho has ATPase activity which can induce release of the polymerase from DNA.
  155. 155. Reverse transcription Scheme of reverse transcription • Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into DNA in order to see a cell's genome. • The main enzyme responsible for this type of transcription 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. • An associated enzyme, ribonuclease H, digests the RNA strand and reverse transcriptase synthesises a complementary strand of DNA to form a double helix DNA structure. • This cDNA is integrated into the host cell's genome via another enzyme (integrase) causing the host cell to generate viral proteins which reassemble into new viral particles. Subsequently, the host cell undergoes programmed cell death (apoptosis).
  156. 156. Eukaryotic RNA polymerases
  157. 157. 1. The mechanism of eukaryotic transcription is similar to that in prokaryotes. 2. A lot more proteins are associated with the eukaryotic transcription machinery, which results in the much more complicated transcription. 3. Three eukaryotic polymerases transcribe different sets of genes. 4. In addition, eukaryotic cells contain additional RNA Pols in mitochondria and chloraplasts. Main Features of eukaryotic transcription
  158. 158. Type Location Substrate RNA Pol I Nucleoli Most rRNAs gene RNA Pol II Nucleo-plasm All protein-coding genes and some snRNA genes RNA Pol III Nucleo-plasm tRNAs, 5S rRNA, U6 snRNA and other small RNAs Three eukaryotic polymerases
  159. 159. RNA polymerase subunits Each eukaryotic polymerase contains 12 or more subunits. – the two largest subunits are similar to each other and to the b’ and b subunits of E. coli RNA Pol. – There is one other subunit in all three RNA Pol homologous to alfa subunit of E. coli RNA Pol. – Five additional subunits are common to all three polymerases. – Each RNA Pol contain additional four or seven specific subunit.
  160. 160. RNA polymerase activities 1. Transcription mechanism is similar to that of E. coli polymerase (How?) 2. Different from bacterial polymerasae, they require accessory factors for DNA binding.
  161. 161. The CTD of RNA pol II 1. The C-terminus of RNA Pol II contains a stretch of seven amino acids that is repeated 52 times in mouse and 26 times in yeast RNA pol II. 2. The heptapeptide sequence ( Seven amino acids) is: Tyr-Ser-Pro-Thr-Ser- Pro-Ser 3. This repeated sequence is known as carboxyl terminal domain (CTD) 4. The CTD sequence may be phosphorylated at the serines and
  162. 162. 5. The CTD is unphosphorylated at transcription initiation, and phosphorylation occurs during transcription elongation as the RNA Pol II leaves the promoter. 6. Because it transcribes all eukaryotic protein-coding gene, RNA Pol II is the most important RNA polymerase for the study of differential gene expression. The CTD is an important target for differential activation of transcription elongation.
  163. 163. RNA Pol II 1. located in nucleoplasm 2. catalyzing the synthesis of the mRNA precursors for all protein- coding genes. 3. RNA Pol Ⅱ-transcribed pre- mRNAs are processed through cap addition, poly(A) tail addition and splicing.
  164. 164. Promoters • Eukaryotic genes, like their prokaryotic counterparts, require promoters for transcription initiation. Each of the three types of polymerase has distinct promoters. •RNA polymerase I transcribes from a single type of promoter, present only in rRNA genes, that encompasses the initiation site. In some genes, RNA polymerase III responds to promoters located in the normal, upstream position; in other genes, it responds to promoters imbedded in the genes, downstream of the initiation site.
  165. 165.  Promoters for RNA polymerase II can be simple or complex. As is the case for prokaryotes, promoters are always on the same molecule of DNA as the gene they regulate.  Most promoters contain a sequence called the TATA box around 25-35 bp upstream from the start site of transcription. It has a 7 bp consensus sequence 5’-TATA(A/T)A(A/T)-3’.
  166. 166. •TATA box acts in a similar way to an E. coli promoter –10 sequence to position the RNA Pol II for correct transcription initiation.
  167. 167. Some eukaryotic genes contain an initiator element instead of a TATA box. The initiator element is located around the transcription start site. Other genes have neither a TATA box nor an initiator element, and usually are transcribed at very low rates.
  168. 168. Enhancers Sequence elements which can activate transcription from thousands of base pairs upstream or downstream.
  169. 169. • Exert strong activation of transcription of a linked gene from the correct start site. • activate transcription when placed in either orientation with respect to linked genes Able to function over long distances of more than 1 kb whether from an upstream or downstream position relative to the start site. • Exert preferential stimulation of the closets of two tandem promoters General characteristics of Enhancers
  170. 170. The TATA-Box-Binding Protein Initiates the Assembly of the Active Transcription Complex  Promotors constitute only part of the eukaryotic gene expression. Transcription factors that bind to these elements also are required. For example, RNA polymerase II is guided to the start site by a set of transcription factors known collectively as TFII (TF stands for transcription factor, and II refers to RNA polymerase II). Individual TFII factors are called TFIIA, TFIIB, and so on. Initiation begins with the binding of TFIID to the TATA box
  171. 171. 1. TFIID: Multiprotein Complex, including TBP, other proteins are known as TAFIIs. TBP is the only protein binds to TATA box
  172. 172. TBP: 1. a general transcription factor bound to DNA at the TATA box. 2. a general transcription required by all 3 RNA pol.
  173. 173. 2. TFIIA • binds to TFIID • stabilizes TFIID-DNA complex
  174. 174. 3. TFIIB & RNA Pol binding • binds to TFIID •Binds to RNA Pol with TFIIF
  175. 175. 4-1 TFIIE binding •Necessary for transcription
  176. 176. 4-2 TFIIJ, TFIIH binding •Necessary for transcriptio n
  177. 177. 5. phosphorylation of the polymerase CTD by TFIIH Formation of a processive RNA polymerase complex and allows the RNA Pol to leave the promoter region.
  178. 178. Initiation of RNA synthesis For RNAP II (protein-coding genes), initiation requires several transcription factors that assist binding to promoter sites. Promoters sites recognized by RNAP II (and associated protein factors) are several conserved elements that are located upstream from the transcription start point (the +1 base).
  179. 179. Elongation of RNA via RNAP II  Elongation of the RNA chain is similar to that in prokaryotes except that a 7-methyl guanosine (7-MG) cap is added to the 5’ end when the growing RNA chain is fairly short (20-30 bases in length).  The 7-MG cap is “attached” by an unusual 5’-5’ triphosphate linkage and serves to protect the growing RNA from degradation by nucleases. This “capping” is part of RNA processing in eukaryotes.
  180. 180. Termination of RNA synthesis 1- Transcription by RNAP II (for protein-coding genes) is not really terminated, in the sense that transcription continues for 1,000 - 2,000 bases after or downstream from the site that ultimately will become the 3’ end of the mature transcript. 2- Termination of transcription via RNAPI and RNAP III is via response to discrete termination signals.
  181. 181. In general: (a) The “functional” transcript actually results from endonucleolytic cleavage of the primary transcript. (b) Cleavage occurs 10-30 bases downstream from the conserved sequence AAUAAA. (c) After cleavage, an enzyme [poly(A) polymerase] adds about 200 adenine (A) bases to the 3’ends. This is called polyadenylation or the addition of poly-A tails. **The function of poly-A tails is to increase stability of the transcript and to assist in transport of the mRNA from the nucleus to the cytoplasm. This is another part of RNA processing is eukaryotes.
  182. 182. Thanks
  183. 183. Introduction  In molecular biology and genetics, splicing is a modification of the nascent pre-mRNA taking place after or concurrently with its transcription, in which introns are removed and exons are joined. This is needed for the typical eukaryotic messenger RNA before it can be used to produce a correct protein through translation. For many eukaryotic introns, splicing is done in a series of reactions which are catalyzed by the spliceosome, a complex of small nuclear ribonucleoproteins (snRNPs), but there are also self-splicing introns
  184. 184. Over view The protein coding genes of eukaryotes typically contain regions of DNA that serves no coding functions. Non coding regions called introns, interrupt the coding regions called exons. When the genes is transcribed to RNA, both the coding and non coding regions are copied. However eukaryotic cell having a mechanism of removing introns from RNA, in a process called RNA splicing, a newly transcribed RNA molecule is cut at the intron – exon boundaries, its intron are discarded. And its exon are joined together. RNA splicing occur within the nucleus before RNA migrates to the cytoplasm. In the cytoplasm, ribosome translate the RNA- now containing uninterrupted coding information- in to protein.
  185. 185. mRNA processing and splicing  pre-mRNA –The nuclear transcript that is processed by modification and splicing to give an mRNA.  RNA splicing – The process of excising introns from RNA and connecting the exons into a continuous mRNA.
  186. 186. Eukaryotic mRNA is modified, processed, and transported
  187. 187. The 5′ End of Eukaryotic mRNA Is Capped  A 5′ cap is formed by adding a G to the terminal base of the transcript via a 5′–5′ link.  The capping process takes place during the transcription, which may be important for transcription reinitiation. Eukaryotic mRNA has a methylated 5’ cap
  188. 188. The 5′ End of Eukaryotic mRNA Is Capped  The 5′ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated.  The cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation.
  189. 189. The 3′ Ends of mRNAs Are Generated by Cleavage and Polyadenylation • The sequence AAUAAA is a signal for cleavage to generate a 3′ end of mRNA that is polyadenylated. • The reaction requires a protein complex that contains a specificity factor, an endonuclease, and poly(A) polymerase. • The specificity factor and endonuclease cleave RNA downstream of AAUAAA. The 3’ end of mRNA is generated by cleavage
  190. 190. The 3′ Ends of mRNAs Are Generated by Cleavage and Polyadenylation  The specificity factor and poly(A) polymerase add ~200 A residues processively to the 3′ end.  The poly(A) tail controls mRNA stability and influences translation. There is a single 3’ end-processing complex
  191. 191. Pre-mRNA Splicing Proceeds through a Lariat  Splicing requires the 5′ and 3′ splice sites and a branch site just upstream of the 3′ splice site.  A lariat is formed when the intron is cleaved at the 5′ splice site, and the 5′ end is joined to a 2′ position at an A at the branch site in the intron.
  192. 192. Pre-mRNA Splicing Proceeds through a Lariat  The intron is released as a lariat when it is cleaved at the 3′ splice site, and the left and right exons are then ligated together. Splicing proceeds through a lariat
  193. 193. snRNAs Are Required for Splicing  small cytoplasmic RNAs (scRNA) – RNAs that are present in the cytoplasm (and sometimes are also found in the nucleus).  small nuclear RNA (snRNA) – One of many small RNA species confined to the nucleus; several of them are involved in splicing or other RNA processing reactions.  small nucleolar RNA (snoRNA) – A small nuclear RNA that is localized in the nucleolus.
  194. 194. snRNA Proteins Are Required for Splicing  The five snRNPs involved in splicing are U1, U2, U5, U4, and U6.  Together with some additional proteins, the snRNPs form the spliceosome.
  195. 195. tRNA Splicing Involves Cutting and Rejoining in Separate Reactions  RNA polymerase III terminates transcription in a poly(U)4 sequence embedded in a GC-rich sequence.  tRNA splicing occurs by successive cleavage and ligation reactions. tRNA splicing recognized a specific structure
  196. 196.  An endonuclease cleaves the tRNA precursors at both ends of the intron.  Release of the intron generates two half-tRNAs with unusual ends that contain 5′ hydroxyl and 2′–3′ cyclic phosphate. tRNA splicing has separate cleavage and ligation stages
  197. 197. Production of rRNA Requires Cleavage Events and Involves Small RNAs  RNA polymerase I terminates transcription at an 18-base terminator sequence.  The large and small rRNAs are released by cleavage from a common precursor rRNA; the 5S rRNA is separately transcribed. Generation of mature eukaryotic rRNAs
  198. 198.  Protein synthesis is based on the sequence of mRNA, which is made up of nucleotides while proteins are made up of amino acids. There must be a specific relationship between the nucleotide sequence and amino acid sequence. This relationship is the so called genetic code, which was deciphered by Marshall Nirenberg and his colleagues in early 1960s. It turns out that three nucleotides (a codon) code for one amino acid, as shown in the following figure. The Genetic Code
  199. 199. The standard genetic code. Synthesis of a peptide always starts from methionine (Met), coded by AUG. The stop codon (UAA, UAG or UGA) signals the end of a peptide. This table applies to mRNA sequences. For DNA, U (uracil) should be replaced by T (thymine). In a DNA molecule, the sequence from an initiating codon (ATG) to a stop codon (TAA, TAG or TGA) is called an open reading frame (ORF), which is likely (but not always) to encode a protein or polypeptide.
  200. 200. Order in the Genetic Code  The genetic code is not randomly assigned. If an amino acid is coded by several codons, they often share the same sequence in the first two positions and differ in the third position. Such assignment is accomplished by the design of wobble position.
  201. 201. The Ribosome • Ribosomes are the sites of protein synthesis in both prokaryotic and eukaryotic cell. • 70S for bacterial ribosome and 80S for eukaryotic cell. • Both prokaryotic and eukaryotic ribosomes are composed of two distinct subunits, each containing characteristic proteins and rRNAs.
  202. 202. Lec 9 molecular Biology Dr. Dlnya Asad
  203. 203. This lecture is to describe in detail the process of protein synthesis, whereby a messenger RNA is translated by the ribosome in the cytoplasmic compartment. The parallels and differences between eukaryote and prokaryote translation will be considered. As well as constituting a central component of the machinery of the cell. Overview
  204. 204. • Proteins are synthesized from mRNA templates by a process that has been highly conserved throughout evolution. • All mRNAs are read in the 5´ to 3´ direction, and poly peptide chains are synthesized from the amino to the carboxy terminus. • Each amino acid is specified by three bases (a codon ) in the mRNA, according to a nearly universal genetic code. • The basic mechanics of protein synthesis are also the same in all cells.
  205. 205. • Translation is carried out on ribosomes, with tRNAs serving as adaptors between the mRNA template and the amino acids being incorporated into protein. • Protein synthesis thus involves interactions between three types of RNA molecules (mRNA templates, tRNAs, and rRNAs), as well as various proteins that are required for translation.
  206. 206. • Consists of approximately 70 to 80 nucleotides. • Cloverleaf structures • All tRNAs have the sequence CCA at their 3´ terminus, and amino acids are covalently attached to the ribose of the terminal adenosine. • The mRNA template is then recognized by the anticodon loop, located at the other end of the folded tRNA, which binds to the appropriate codon by complementary base pairing. tRNA
  207. 207. The incorporation of the correctly encoded amino acids into proteins depends on the attachment of each amino acid to an appropriate tRNA by the action of the enzyme aminoacyl tRNA synthetases.
  208. 208. The reaction proceeds in two steps. First, the amino acids is activated by reaction with ATP to form an aminoacyl AMP synthetase intermediate. The activated amino acids is then joined to the 3´ terminus of the tRNA.
  209. 209. • After being attached to tRNA, an amino acids is aligned on the mRNA template by complementary base pairing between the mRNA codon and the anticodon of the tRNA. • Codon-anticodon base pairing is somewhat less stringent than the standard A-U and G-C base pairing, leading to the nonstandard base pairing (wobble), and then making the genetic code redundant (redundancy of the genetic code), because Inosine located in the tRNA anticodon loop can base-pair with either C, U, or A in the third position on mRNA.
  210. 210. The Organization of mRNAs and the Initiation of Translation Both prokaryotic and eukaryotic mRNAs contain untranslated regions (UTRs) at their 5´ and 3´ ends. Eukaryotic mRNAs also contain 5´ 7-methylguanosine (m7G) caps and 3´ poly-A tails.
  211. 211. • Prokaryotic mRNAs are frequently polycistronic: They encode multiple proteins, each of which is translated from an independent start site. Eukaryotic mRNAs are usually monocistronic, encoding only a single protein.
  212. 212. • In both prokaryotic and eukaryotic cells, translation always initiates with the amino acid methionine, usually encoded by AUG. Alternative initiation codons, such as GUG, are used occasionally in bacteria (GUG normally encodes valine). • In most bacteria, protein synthesis is initiated with a modified methionine residue (N-formylmethionine), whereas unmodified methionines initiate protein synthesis in eukaryotes (except in mitochondria and chloroplasts, whose ribosomes resemble those of bacteria).
  213. 213. The signals that identify initiation codons are different in prokaryotic and eukaryotic cells • Initiation codons in bacterial mRNAs are preceded by a specific sequence called a Shine-Delgarno sequence that aligns the mRNA on the ribosome for translation by base- pairing with a complementary sequence near the 3´ terminus of 16S rRNA. • Ribosomes recognize most eukaryotic mRNAs by binding to the 7-methylguanosine cap at their 5´ terminus . • The ribosomes then scan downstream of the 5´ cap until they encounter an AUG initiation codon.
  214. 214. Translation is generally divided into three stages:  Initiation  Elongation  Termination.
  215. 215. • In both prokaryotes and eukaryotes the first step of the initiation stage is the binding of a specific initiator methionyl tRNA and the mRNA to the small ribosomal subunit. • The large ribosomal subunit then joins the complex, forming a functional ribosome on which elongation of the polypeptide chain proceeds. • A number of specific non-ribosomal proteins are also required for the various stages of the translation process.
  216. 216. • The first translation step in bacteria is the binding of three initiation factors (IF-1, IF-2, and IF-3) to the 30S ribosomal subunit. • The mRNA and initiator N-formylmethionyl tRNA then join the complex. • A 50S ribosomal subunit associate with the complex. • The result is the formation of a 70S initiation complex (with mRNA and initiator tRNA bound to the ribosome) that is ready to begin peptide bond formation during the elongation stage of translation.
  217. 217. • Initiation in eukaryotes is more complicated and requires at least ten proteins, which are designated eIFs (eukaryotic initiation factors). The factors bind to the 40S ribosomal subunit, and associates with the initiator methionyl tRNA . • The mRNA is recognized by initiation factors via mRNA 5´ cap and poly-A tail at the 3’ end then brought to the 40S ribosomal subunit. • The 40S ribosomal subunit, in association with the bound methionyl tRNA and eIFs, then scans the mRNA to identify the AUG initiation codon. When the AUG codon is reached, a 60S subunit binds to the 40S subunit to form the 80S initiation complex of eukaryotic cells.
  218. 218. Elongation of the polypeptide chain. The ribosome has three sites for tRNA binding, designated the P (peptidyl), A (aminoacyl), and E (exit) sites. The initiator methionyl tRNA is bound at the P site. The first step in elongation is the binding of the next aminoacyl tRNA to the A site by pairing with the second codon of the mRNA. The aminoacyl tRNA is escorted to the ribosome by an elongation factor.
  219. 219. Once elongation factor has left the ribosome, a peptide bond can be formed between the initiator methionyl tRNA at the P site and the second aminoacyl tRNA at the A site. This reaction is catalyzed by the large ribosomal subunit, with the rRNA playing a critical role. The result is the transfer of methionine to the aminoacyl tRNA at the A site of the ribosome, forming a peptidyl tRNA at this position and leaving the uncharged initiator tRNA at the P site. The next step in elongation is translocation, the ribosome moves three nucleotides along the mRNA, positioning the next codon in an empty A site. This step translocates the peptidyl tRNA from the A site to the P site, and the uncharged tRNA from the P site to the E site. The ribosome is then left with a peptidyl tRNA bound at the P site, and an empty A site. The binding of a new aminoacyl tRNA to the A site then induces the release of the uncharged tRNA from the E site, leaving the ribosome ready for insertion of the next amino acid in the growing polypeptide chain.
  220. 220. • Elongation of the polypeptide chain continues until a stop codon (UAA, UAG, or UGA) is translocated into the A site of the ribosome. Cells do not contain tRNAs with anticodons complementary to these termination signals; instead, they have release factors that recognize the signals and terminate protein synthesis. The release factors bind to a termination codon at the A site and stimulate hydrolysis of the bond between the tRNA and the polypeptide chain at the P site, resulting in release of the completed polypeptide from the ribosome. The tRNA is then released, and the ribosomal subunits and the mRNA template dissociate
  221. 221. 1 Codon recognition Amino acid Anticodon A siteP site Polypeptide 2 Peptide bond formation 3 Translocation New peptide bond mRNA movement mRNA Stop codon
  222. 222. • Messenger RNAs can be translated simultaneously by several ribosomes in both prokaryotic and eukaryotic cells. • Thus, mRNAs are usually translated by a series of ribosomes, spaced at intervals of about 100 to 200 nucleotides • The group of ribosomes bound to an mRNA molecule is called a polyribosome, or polysome.
  223. 223. Thanks
  224. 224. Why regulate gene expression? It takes a lot of energy to make RNA and protein. Therefore some genes active all the time because their products are in constant demand. Others are turned off most of the time and are only switched on when their products are needed.
  225. 225. Gene Control in Prokaryotes  One way in which prokaryotes control gene expression is to group functionally related genes together so that they can be regulated together.  This grouping is called an operon (The clustered genes are transcribed together from one promoter giving a polycistronic messenger RNA).
  226. 226. Gene Control in Prokaryotes The prokaryotic genes organized in to operons. An operon can be defined as a cluster gene that encode the proteins necessary to perform coordinated function. Genes of the same operon have related functions within the cell and are turned on (expressed) and off together (suppressed). The first operon discovered was the lac operon so named because its products are involved in lactose breakdown.
  227. 227. An operon consists of:  a promoter (binding site for RNA polymerase)  a repressor binding site called an operator that overlaps the promoter.  structural genes
  228. 228. Operator Repressor proteins encoded by repressor genes, are synthesized to regulate gene expression. They bind to the operator site to block transcription by RNA polymerase. Promoter The promoter sequences are recognized by RNA polymerase, When RNA polymerase binds to the promoter, transcription occurs.
  229. 229. Activators The activity of RNA polymerase is also regulated by interaction with accessory proteins called activators The presence of the activator removes repression and transcription occurs
  230. 230. Two major modes of transcriptional regulation function in bacteria (E. coli) to control the expression of operons: Repression Induction Both mechanisms involve repressor proteins. Induction happen in operons that produce gene products needed for the utilization of energy. Repression regulate operons that produce gene products necessary for the synthesis of small biomolecules such as amino acids.
  231. 231. Inducible system Also called Positive control The effector molecule interacts with the repressor protein such that it cannot bind to the operator. With inducible systems, the binding of the effector molecule to the repressor greatly reduces the affinity of the repressor for the operator as a result the repressor is released and transcription proceeds. A classic example of an inducible (catabolite-mediated) operon is the lac operon, responsible for obtaining energy from galactosides such as lactose.
  232. 232. Repressible system Also called Negative control The effector molecule interacts with the repressor protein such that it can bind to the operator . With repressible systems, the binding of the effector molecule to the repressor greatly increases the affinity of repressor for the operator, the repressor binds and stops transcription. For the trp operon , the addition of tryptophan (the effector molecule) to the E. coli environment shuts off the system because the repressors binds at the operator.
  233. 233. In addition to negative control mediated by a repressor, expression from a repressible operons is attenuated by sequences within the transcribed RNA. A classic example of a repressible (and attenuated) operon is the trp operon, responsible for the biosynthesis of tryptophan.
  234. 234. Structure of the lac Operon The lac operon have three structural genes: Z, , Y and A The z gene codes for β-galactosidase , responsible for the hydrolysis of the disaccharide, lactose into its monomeric units, galactose and glucose. The y gene codes for permease, which increases permeability of the cell to galactosides. The a gene encodes a transacetylase. In addition to the structural genes the lac operon also has regulatory genes: Promoter: Binding site for RNA polymerase Operator: Binding site of repressor
  235. 235. expression The control of the lac operon occurs by both positive and negative control mechanisms. Negative control of the lac operon What happens to lac operon when glucose is present and lactose is absent? During normal growth on a glucose-based medium (lacking lactose), the lac repressor is bound to the operator region of the lac operon, preventing transcription.
  236. 236. What happens when glucose is absent and lactose is present? The few molecules of lac operon enzymes present will produce a few molecules of allolactose from lactose. Allolactose is the inducer of the lac operon. The inducer binds to the repressor causing a conformational shift that causes the repressor to release the operator. With the repressor removed, the RNA polymerase can now bind the promoter and transcribe the operon.
  237. 237. Positive Control of the lac operon What happens when both glucose and lactose levels are high? Since the inducer is present, the lac operon will be transcribed but the rate of transcription is very slow (almost repressed) because glucose levels are high and therefore cAMP levels are low. The repression of the lac operon under these conditions is termed catabolite repression and is a result of the low levels of cAMP that results from an adequate glucose supply. This repression is maintained until the glucose supply is exhausted.
  238. 238. What happens when glucose levels start dropping in the presence of lactose? As the level of glucose in the medium falls, the level of cAMP increases. Simultaneously the inducer (allolactose) is also binds to the lac repressor (since lactose is present). The net result is an increase in transcription from the operon. The ability of cAMP to activate (increase) expression from the lac operon results from an interaction of cAMP with a protein termed CRP (for cAMP receptor protein). The protein is also called CAP (for catabolite activator protein).
  239. 239. The cAMP-CAP complex binds to a region of the lac operon just upstream of the promoter. This binding stimulates RNA polymerase activity 20-to-50-fold. (Repression of the lac operon is relieved in the presence of glucose if excess cAMP is added.) cAMP is therefore an activator of the lac operon. This type of regulation by an activator is positive in contrast to the negative control exerted by repressors.
  240. 240. trp operon  The trp operon encodes the genes for the synthesis of tryptophan.  As with all operons, the trp operon consists of the promoter, operator and the structural genes.  It is also subject to negative control by a repressor  In this system, unlike the lac operon, the gene for the repressor is not adjacent to the promoter, but rather is located in another part of the E. coli genome.  Another difference is that the operator resides entirely within the promoter  Unlike an inducible system, the repressible operon is usually turned on.
  241. 241. Structure of the trp operon The operon consists of:  Five structural genes that code for the three enzymes required to convert chorismic acid into tryptophan.  A gene (trpL) which functions in attenuation. Operator promoter
  242. 242. Gene Gene Function P/O: Promoter; operator sequence is found in the promoter trp L Leader sequence; containing attenuator (A) sequence the leader trp E: Gene for anthranilate synthetase subunit trp D : Gene for anthranilate synthetase subunit trp C: Gene for glycerolphosphate synthetase trp B: Gene for tryptophan synthetase subunit trp A: Gene for tryptophan synthetase subunit
  243. 243. Negative control of trp operon The affinity of the trp repressor for binding the operator region is enhanced when it binds tryptophan, blocking further transcription of the operon and, as a result, the synthesis of the three enzymes will decline, hence tryptophan is a co-repressor, this means that when tryptophan is absent expression of the trp operon occurs. the rate of expression of the trp operon is graded in response to the level of tryptophan in the cell.
  244. 244. Attenuation of the trp operon  Expression of trp operon is reduced by the addition of trytophan. Tryptophan synthesis also controlled by two other components: 1.tRNA, specifically tryptophanyl-tRNA, tRNATrp (tRNATrp charged with tryptophan). 2.the trpL gene
  245. 245.  Trp L gene found between operator and trp E gene, thus the attenuator region is composed of sequences found within the transcribed RNA of the operon.  It is involved in controlling transcription from the operon after RNA polymerase has initiated synthesis of the proteins.  The leader sequence (trp L) contains 14 amino acids including tandem tryptophan codons (2 codons).
  246. 246. How does leader sequence affect transcription of the trp operon? It contains two consecutive trp codons and therefore serves to measure the tryptophan supply in the cell. If the supply is inadequate, small amount of tRNA will be charged and the leader peptide will be translated without problem. If the supply is good, large amount of tRNA will be charged, and translation will stall at the trp codons. How? 
  247. 247. The trpL mRNA consists of four region can adopt a number of different conformations. It contains several self-complementary regions which can form a variety of stem-loop structures Different stem-loops can form depending on the level of tryptophan in the cell and hence the level of charged trp- tRNAs determine the position of ribosome on the leader polypeptide as well as determining the rate of translation.
  248. 248.  In the case of the trpL mRNA, when the cellular levels of tryptophan are high, the levels of the tryptophan tRNA are also high.  Immediately after transcription, the ribosome follows right behind RNA polymerase until it is halted by a stop codon prior to the region 2 which prevent stem loop formation between 2 & 3 and permits formation of the terminator stemloop (3 & 4) which will cause RNA polymerase to dissociate when reach the UUUUU rich region in the end of the 4 region terminating transcription.
  249. 249. How is the terminator stem-loop formed?  Because of the quick translation of domain 1, domain 2 becomes associated with the ribosome complex.  Then domain 3 binds with domain 4, and transcription is attenuated because of this stem loop formation.  The stem loop formed by binding of domains 3 and 4 is found near a region rich in uracil and acts as the transcriptional terminator loop.  Consequently, RNA polymerase is dislodged from the template.
  250. 250. trp Operon Transcription Under Low Levels of Tryptophan  Under low cellular levels of tryptophan, the translation of the short peptide on domain 1 is slow .  As a result domain 2 does not become associated with the ribosome.  Rather domain 2 of the leader mRNA associates with domain 3 of the leader mRNA.  This stem loop structure is the anti-terminator. Its formation prevents formation of the terminator.
  251. 251.  This structure permits the continued transcription of the operon. Then the trpE-A genes are translated, and the biosynthesis of tryptophan occurs  Domain 4 is called the attenuator because its presence is required to reduce (attenuate) mRNA transcription in the presence of high levels of tryptophan.
  252. 252. • Review Articles: • Regulation of RNA polymerase I transcription in the nucleolus - Genes and Develop., 2003. • Roles of the heat shock transcription factors in regulation of the heat shock response and beyond - FASEB J., 2001. • Translational Control of Viral Gene Expression in Eukaryotes - Microbiology and Molecular Biology Reviews, 2000. • References: • Molecular Biology of the Cell, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, Garland Science, 2007. • Molecular cell biology, 1986, Darnell, Lodish, and Baltimore.

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