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  1. 1. DNA, RNA, Genetic code, and biosynthesis of proteins
  2. 2. DNA vs. RNA <ul><li>1. Number of strands </li></ul><ul><li>2. Nucleotide composition </li></ul><ul><li>3. Synthesis </li></ul><ul><li>4. Location </li></ul><ul><li>5. Functions </li></ul>
  3. 3. The Gene <ul><li>A gene is traditionally defined as a unit of heredity </li></ul><ul><li>It’s a DNA segment that encodes a single polypeptide chain (1 gene-one-polypeptide hypothesis </li></ul><ul><li>Careful! : Alternative sequences encoding same polypeptide/same gene can produse different polypeptides. </li></ul>
  4. 4. Gene operation <ul><li>Regulatory region </li></ul><ul><li>Coding/structural region </li></ul>
  5. 5. How is genetic information passed from DNA to a protein? <ul><li>Key qns </li></ul><ul><li>How is genetic information passed on from generation to generation, or just cell to cell? </li></ul><ul><li>How can a “group of letters&quot; determine what proteins are made in the cell and direct the cell's activities? </li></ul>
  6. 6. <ul><li>According to the central dogma of molecular genetics, the function of DNA is to </li></ul><ul><ul><li>store information and pass it on to RNA, </li></ul></ul><ul><li>while the function of RNA is </li></ul><ul><ul><li>to read, decode and use the information received from DNA to make proteins. </li></ul></ul>
  7. 7. Central Dogma of Molecular Biology <ul><ul><ul><li>DNA mRNA </li></ul></ul></ul><ul><li>Permanent </li></ul><ul><li>Genetic </li></ul><ul><li>archive </li></ul><ul><li>Same in </li></ul><ul><li>all cells </li></ul>transcription translation rev transcription replication Transient Carrier of specific information Different in different cells <ul><li>Transient expression </li></ul><ul><li>of information </li></ul><ul><li>for action in cell </li></ul><ul><li>Different in </li></ul><ul><li>different cells </li></ul><ul><ul><ul><li>Protein </li></ul></ul></ul>
  8. 8. DNA Replication <ul><li>Is the process by which a replica, or identical copy, of DNA is made. </li></ul><ul><li>Replication occurs every time a cell divides so that information can be preserved and handed down to offspring. </li></ul>
  9. 9. mRNA transcription <ul><li>Is the process by which the genetic messages contained in DNA are &quot;read&quot; or transcribed. </li></ul><ul><li>The product of transcription, known as messenger RNA (mRNA), leaves the cell nucleus and carries the message to the sites of protein synthesis. </li></ul>
  10. 10. Translation <ul><li>Is the process by which the genetic messages carried by mRNA are decoded and used to build proteins. </li></ul>
  11. 11. Challenge! <ul><li>For instance, if you eat a piece of beef and the cells of your pancreas need to secrete a digestive enzyme, then the one gene for that enzyme will be transcribed from DNA to mRNA and then to the digestive enzyme to digest the piece of beef. </li></ul><ul><li>Do we need DNA to replicate for this? </li></ul>
  12. 12. <ul><li>NO!! </li></ul><ul><li>The only reason a cell has for replicating its DNA is if it's going to divide </li></ul>
  13. 13. Replication process <ul><li>DNA replication begins with a partial unwinding of the double helix at an area known as the replication fork ( under DNA helicase ) . </li></ul><ul><li>As the two DNA strands separate (&quot;unzip&quot;) and the bases are exposed, the enzyme DNA polymerase moves into position at the point where synthesis will begin </li></ul>
  14. 14. <ul><li>The start point for DNA polymerase is a short segment of RNA known as an RNA primer . </li></ul><ul><li>The primer is &quot;laid down&quot; complementary to the DNA template by an enzyme known as RNA polymerase or Primase </li></ul><ul><li>The DNA polymerase (once it has reached its starting point as indicated by the primer) then adds nucleotides one by one in an exactly complementary manner, A to T and G to C </li></ul>Replication process
  15. 15. <ul><li>DNA polymerase is described as being &quot;template dependent&quot; in that it will &quot;read&quot; the sequence of bases on the template strand and then &quot;synthesize&quot; the complementary strand </li></ul><ul><ul><li>DNA polymerase catalyzes the formation of the hydrogen bonds between each arriving nucleotide and the nucleotides on the template strand. </li></ul></ul><ul><ul><li>DNA polymerase also catalyzes the reaction between the 5' phosphate on an incoming nucleotide and the free 3' OH on the growing polynucleotide </li></ul></ul>Replication process
  16. 17. <ul><li>The last step is for an enzyme to come along and remove the existing RNA primers and then fill in the gaps with DNA. </li></ul>Replication process
  17. 18. Transcription <ul><li>Types of RNA </li></ul><ul><ul><li>Ribosomal RNAs - </li></ul></ul><ul><ul><li>Exist outside the nucleus in the cytoplasm of a cell in structures called ribosomes . </li></ul></ul><ul><ul><li>Ribosomes are small, granular structures where protein synthesis takes place. </li></ul></ul><ul><ul><li>Each ribosome is a complex consisting of about 60% ribosomal RNA (rRNA) and 40% protein </li></ul></ul>
  18. 19. <ul><li>2. Transfer RNAs - </li></ul><ul><ul><li>The function of transfer RNAs (tRNA) is to deliver amino acids one by one to protein chains growing at ribosomes. </li></ul></ul><ul><li>3. Messenger RNAs - </li></ul><ul><ul><li>Are the nucleic acids that &quot;record&quot; information from DNA in the cell nucleus and carry it to the ribosomes and are known as messenger RNAs (mRNA). </li></ul></ul>Transcription
  19. 20. <ul><li>mRNA is synthesized by the transcription of a portion of one strand of DNA (the active strand) to produce a complementary single RNA strand </li></ul><ul><li>Synthesis of mRNA takes place in the nucleus under the enzyme RNA polymerase II </li></ul>RNA Transcription ( Nucleus)
  20. 21. <ul><li>Transcription is initiated by signals from cytoplasm. </li></ul><ul><li>One strand of the DNA is activated (or suppressing factor removed!) </li></ul><ul><ul><li>induction of local unwinding of the DNA helix </li></ul></ul><ul><ul><li>RNA polymerase II binds at DNA promoter region for transcription initiation </li></ul></ul>Synthesis of mRNA
  21. 22. Synthesis of mRNA
  22. 23. Synthesis of mRNA
  23. 24. Synthesis of mRNA
  24. 25. Synthesis of mRNA
  25. 26. <ul><li>Post-transcriptional mRNA processing </li></ul><ul><ul><li>Poly adenylation (3’ end)-up to 200 bases in eukaryotes </li></ul></ul><ul><ul><li>Methylation (5’ end)-a cap </li></ul></ul><ul><ul><li>Splicing-removal of introns </li></ul></ul><ul><li>mRNA leaves nucleus and moves to cytoplasm </li></ul>Synthesis of mRNA
  26. 27. Protein synthesis <ul><li>The information (message) in the mRNA is decoded into an amino acid sequence of a polypepetide </li></ul>
  27. 28. Genetic code <ul><li>DNA transfers information to mRNA in the form of a code defined by a sequence of nucleotides bases. </li></ul><ul><li>During protein synthesis, ribosomes move along the mRNA molecule and &quot;read&quot; its sequence (three nucleotides at a time) called codons, from the 5' end to the 3' end. </li></ul><ul><li>Each amino acid is specified by the mRNA's codon, and then pairs with a sequence of three complementary nucleotides carried by a particular tRNA (anticodon). </li></ul>
  28. 29. Genetic code <ul><li>Since RNA is constructed from four types of nucleotides. </li></ul><ul><li>There are 64 possible triplet sequences or codons (4x4x4). </li></ul><ul><li>Three of these possible codons specify the termination of the polypeptide chain. </li></ul><ul><li>They are called &quot; stop codons &quot;. That leaves 61 codons to specify only 20 different amino acids. </li></ul>
  29. 30. Characteristics of the genetic code <ul><li>Degenerate : 64 codons </li></ul><ul><ul><li>(AUG  initiator) </li></ul></ul><ul><ul><li>AGA, AGG, UGA  terminator </li></ul></ul><ul><ul><ul><li>Ther remains 61 coding codons </li></ul></ul></ul><ul><li>Unambigous : every codon codes for a single aa </li></ul><ul><li>Non overlapping </li></ul><ul><li>Non punctuated </li></ul><ul><li>Universal </li></ul>
  30. 31. Central Dogma of Molecular Biology <ul><ul><ul><li>DNA mRNA </li></ul></ul></ul><ul><li>Permanent </li></ul><ul><li>Genetic </li></ul><ul><li>archive </li></ul><ul><li>Same in </li></ul><ul><li>all cells </li></ul>transcription translation rev transcription <ul><ul><ul><li>Protein </li></ul></ul></ul>
  31. 32. <ul><li>Protein synthesis is achieved by the interaction of mainly the following molecules (nucleus and cytoplasm) </li></ul><ul><ul><li>DNA </li></ul></ul><ul><ul><li>RNA: mRNA and tRNA </li></ul></ul><ul><ul><li>Several enzymes </li></ul></ul><ul><ul><li>Ribosomes </li></ul></ul><ul><ul><li>Amino acids </li></ul></ul>Key Components in the Process of Protein Synthesis
  32. 33. <ul><li>DNA : </li></ul><ul><ul><ul><li>Stores all the genetic information of an organism. Same in all cells of an organism </li></ul></ul></ul><ul><ul><ul><li>Determines which protein to make and when to make it. </li></ul></ul></ul><ul><ul><ul><li>Only a gene that is to be expressed is transcribed to mRNA </li></ul></ul></ul>Key Components in the Process of Protein Synthesis
  33. 34. Key Components in the Process of Protein Synthesis <ul><li>RNAs: mRNA </li></ul><ul><ul><ul><li>mRNA carries genetic information from DNA segment (a gene), to the translation site, i.e. ribosome in the cytoplasm. </li></ul></ul></ul><ul><ul><ul><li>mRNA for a gene is transiently present in the cell when that gene is expressed, therefore different in different cells </li></ul></ul></ul>
  34. 35. <ul><ul><li>tRNA brings amino acids to the translation site, the ribosome. </li></ul></ul><ul><ul><li>Enzymes </li></ul></ul><ul><ul><ul><li>RNA polymerase- formation of RNA from a DNA template </li></ul></ul></ul><ul><ul><ul><li>DNA polymerase -DNA replication </li></ul></ul></ul><ul><ul><ul><li>Reverse transcriptase- formation of DNA using an RNA template </li></ul></ul></ul><ul><ul><ul><li>Synthetases- catalyse covalent links by activation of the tRNA. There is (close to) one synthetase per amino acid </li></ul></ul></ul>Key Components in the Process of Protein Synthesis
  35. 36. <ul><li>Ribosomes </li></ul><ul><ul><li>They provide the sites for protein synthesis </li></ul></ul><ul><li>Amino acids </li></ul><ul><ul><li>These are the building blocks of proteins. They are sequentially joined together to form polypeptide chains </li></ul></ul>Key Components in the Process of Protein Synthesis
  36. 37. <ul><li>The basic steps of translation are </li></ul><ul><ul><li>Initiation </li></ul></ul><ul><ul><li>Elongation </li></ul></ul><ul><ul><li>Termination </li></ul></ul>Translation (cytoplasm)
  37. 38. Translation <ul><li>Initiation </li></ul><ul><ul><li>Special tRNAi, charged with methionine (Met-tRNAi) is used to initiate translation (AUG) </li></ul></ul><ul><ul><li>Eukaryotic Initiation Factor 2 (eIF-2) plus GTP binds Met-tRNAi, the complex enters the P site of the 40S ribosome . </li></ul></ul><ul><ul><li>eIF-3 facilitates the binding of ‘Met-tRNAi-40S ribosome complex’ to mRNA by migrating the complex along mRNA to AUG start codon (ATP used for energy) </li></ul></ul>
  38. 39. <ul><li>Initiation </li></ul><ul><ul><li>eIF-6 brings 60S ribosome to Met-tRNAi-40S-mRNA complex , and eIFs-2 and 3 are released </li></ul></ul><ul><ul><li>When 60S-40S joining is complete, all eIFs dissociate </li></ul></ul><ul><ul><li>Met-tRNAi-40S-60S-mRNA complex (polysome) left </li></ul></ul>Translation
  39. 40. <ul><li>Elongation </li></ul><ul><ul><li>Each amino acid has its activating enzyme that activates and catalyses its transfer to a molecule of aminoacyl-tRNA. </li></ul></ul><ul><ul><li>tRNA binds amino acid (by covalent bonds) in presence of Aminoacyl-tRNA synthetase, to yield a reactive Aminoacyl-tRNA </li></ul></ul>Translation
  40. 41. <ul><li>Elongation </li></ul><ul><ul><li>Aminoacyl-tRNA then moves to the polysome </li></ul></ul><ul><ul><li>Anticodon of the Aminoacyl-tRNA recognizes and binds the mRNA codon (attached onto the polysome) by H-bonds </li></ul></ul>Translation
  41. 42. <ul><li>Elongation </li></ul><ul><ul><li>Amino acids are transferred from the tRNA to the growing chain under Peptidyl transferase enzyme </li></ul></ul><ul><ul><li>The Polysome moves along mRNA, 5’-3’ synthesizing polypeptide chain, 1 amino acid per charged tRNA, N- to C-terminal. </li></ul></ul><ul><ul><li>Amino acid order is directed by the nucleotide sequence on mRNA </li></ul></ul>Translation
  42. 44. <ul><li>Termination </li></ul><ul><li>Process not fully understood </li></ul><ul><ul><li>Stop (nonsense) codons </li></ul></ul><ul><ul><ul><li>UAG (amber), UAA (ochre), UGA (opal) recognized by a eukaryotic release factor (eRF) </li></ul></ul></ul><ul><ul><li>Polypeptide released from last tRNA </li></ul></ul><ul><ul><li>Expulsion of tRNA, dissociation of ribosome, as separate subunits </li></ul></ul>Translation
  43. 49. Post-ribosomal processing <ul><li>In specialized tissues in eukaryotes </li></ul><ul><li>Examples: </li></ul><ul><ul><li>hydroxylation of proline and lysine in collagen </li></ul></ul><ul><ul><li>addition of carbohydrate groups  glycoproteins </li></ul></ul><ul><ul><li>removal of a peptide: activation of insulin and proteolytic enzymes </li></ul></ul>
  44. 50. The Protein Molecule Sickle cell mutataion Heme Iron atom Sickle cell mutataion Heme Iron atom Hemoglobin
  45. 52. Final <ul><li>The process is extremely fast </li></ul><ul><li>The process is complex </li></ul><ul><li>The process is very crucial to life </li></ul>
  46. 53. Recombinant DNA Technology and Gene Cloning
  47. 54. Introduction <ul><li>Recombinant DNA </li></ul><ul><ul><li>Is DNA that has been made artificially by joining two/more DNAs from different sources. </li></ul></ul><ul><ul><li>Its gene product is called a recombinant protein </li></ul></ul><ul><li>2. Recombinant DNA Technology </li></ul><ul><li>Is the technology/technique of joining 2 or more DNA segments to obtain a single recombinant DNA molecule </li></ul>
  48. 55. Background and Use <ul><li>Recombinant DNA technology came into being in the 1970s </li></ul><ul><li>It is a powerful toll that has enabled science to manipulate DNA/genes to suit human requirements </li></ul><ul><ul><li>Express a desired gene to get a recombinant protein for study </li></ul></ul><ul><ul><li>Create gene libraries </li></ul></ul>
  49. 56. Tools for Recombinant DNA Technology <ul><li>The technology requires that DNA is cut , and joined with other fragments of DNA to get the new (Recombinant) DNA </li></ul><ul><li>Cutting : Restriction (endonucleases) enzymes </li></ul><ul><li>Joining : Ligases </li></ul>
  50. 57. <ul><li>A restriction enzyme recognizes and cuts DNA only at a particular sequence of nucleotides. </li></ul><ul><li>E.g the bacterium Hemophilus aegypticus produces an enzyme named Hae III that cuts DNA wherever it encounters the sequence 5'GGCC3' 3'CCGG5' </li></ul>1. Restriction enzymes
  51. 58. <ul><li>The cut is made between the adjacent G and C. </li></ul><ul><li>5'GGCC3‘ </li></ul><ul><li>3'CCGG5 </li></ul>Restriction enzymes
  52. 59. Restriction enzymes <ul><li>They cut DNA at specific short sequences </li></ul>
  53. 60. <ul><li>HaeIII and AluI cut straight across the double helix producing &quot;blunt&quot; ends. </li></ul><ul><li>However, many restriction enzymes cut in an offset fashion. </li></ul><ul><li>The ends of the cut have an overhanging piece of single-stranded DNA. These are called &quot;sticky ends&quot; because they are able to form base pairs with any DNA molecule that contains the complementary sticky end. </li></ul>Restriction digestion
  54. 61. <ul><li>Any other source of DNA treated with the same enzyme will produce such molecules. </li></ul><ul><li>Mixed together, these molecules can join with each other by the base pairing between their sticky ends. </li></ul><ul><li>The union can be made permanent by another enzyme, DNA ligase , that forms covalent bonds along the backbone of each strand. </li></ul>
  55. 62. <ul><li>The result is a molecule of recombinant DNA ( rDNA ). </li></ul>
  56. 64. Some general features of Restriction enzymes/digestion <ul><li>1. They split both DNA strands </li></ul><ul><li>2. Recognize ‘specific’ sequences, </li></ul><ul><li>3. the sequence recognized is usually 4-8 np </li></ul><ul><li>4. Found in prokaryotes to cleave foreign DNA </li></ul><ul><li>5. Most cleave to produce symmetrical fragments </li></ul>
  57. 65. 2. Cloning vectors <ul><li>Are DNA stuff used for insertion and cloning of target genes </li></ul><ul><li>Many types exist, but bacterial plasmids have many advantages over the others! </li></ul>
  58. 66. Plasmids <ul><li>Circular, exrachromosomal DNA in prokaryotes </li></ul><ul><li>Have own origin of replication, single </li></ul><ul><li>Mainly for drug resistance in bacteria </li></ul>
  59. 67. <ul><li>Are small (a few thousand base pairs) </li></ul><ul><li>Usually carry only one or a few genes </li></ul>Plasmids
  60. 68. <ul><li>Plasmids used in genetic engineering are called vectors. </li></ul><ul><li>They are used to transfer genes from one organism to another and typically contain a genetic marker conferring a phenotype that can be selected for or against. </li></ul>
  61. 72. Transformation <ul><li>Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. </li></ul><ul><li>Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes. </li></ul>
  62. 73. Detection of transformed E.coli
  63. 74. An overview of applications of recombinant DNA technology <ul><li>Name of the Project: To produce human lactogen in large amounts in the laboratory </li></ul><ul><li>Steps: </li></ul><ul><li>1. know the gene that encode the lactogen protein in man </li></ul><ul><li>2.Have a suitable cloning vector </li></ul><ul><li>3. Digest the cloning vector and human DNA with same Restriction enzyme </li></ul><ul><li>4. insert the gene into the cloning vector and ligate by DNA ligase </li></ul><ul><li>5. Introduce the recombinant cloning vector into E.coli </li></ul>
  64. 75. <ul><li>6. culture E.coli </li></ul><ul><li>NB the E.coli will now produce among others the human lactogen protein! </li></ul><ul><li>For the whole process to be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. </li></ul><ul><li>Producing many identical copies of the same recombinant molecule is called cloning . Cloning can be done in vitro , by a process called the polymerase chain reaction ( PCR ). </li></ul>
  65. 76. In vitro cloning of DNA: The Polymerase Chain Reaction <ul><li>Next? </li></ul>