Biochem synthesis of rna(june.23.2010)


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Biochem synthesis of rna(june.23.2010)

  1. 1. Synthesis of RNA
  2. 2. Synthesis of RNA from a NDA template is called transcription. Genes are transcribed by enzymes called RNA polymerases that generate a single-stranded RNA.
  3. 3. Primary transcript The RNA strand that is initially generated. Direction The DNA template is copied in the 3’ to 3’ direction. The RNA transcript is synthesized in the 5’ to 3’ direction. RNA polymerases The DNA template is copied in the 3’ to 3’ direction. The RNA transcript is synthesized in the 5’ to 3’ direction. They can initiate the synthesis of the new strands in the absence of a primer.
  4. 4. Action of RNA polymerases
  5. 5. Synthesize RNA from DNA. 5’- 3’ direction.
  6. 6. <ul><li>Limits of DNA polymerase III </li></ul><ul><ul><li>can only build onto 3  end of an existing DNA strand </li></ul></ul>Leading & Lagging strands Leading strand Lagging strand Okazaki fragments <ul><li>Leading strand </li></ul><ul><ul><li>continuous synthesis </li></ul></ul><ul><li>Lagging strand </li></ul><ul><ul><li>Okazaki fragments </li></ul></ul><ul><ul><li>joined by ligase </li></ul></ul><ul><ul><ul><li>“ spot welder” enzyme </li></ul></ul></ul>DNA polymerase III   growing replication fork 5  5  5  5  3  3  3  5  3  5  3  3  ligase Okazaki 3  5 
  7. 7. Recognize the startpoint for transcription of each gene.
  8. 8. Sensitive to signals that reflect the need for the gene product and control the frequency of transcription.
  9. 9. Types of RNA polymerases
  10. 10. Eukaryote cells Polymerase I produces most of the rRNAs Polymerase II produces most of the mRNAs Polymerase III produces small RNAs such as tRNA and 5 S RNA. All have the same mechanism of action. Recognize different type of promoters.
  11. 11. Prokaryote cells Bacterial cells have a single RNA polymerase to generate all of the different types of RNA (mRNA, rRNA, and tRNA). Tuberculosis Rifampicin Rifampicin Bacterial RNA polymerase
  12. 13. Alert Do Not Eat Death Cap
  13. 14. Amanita phalloides (the death cap) These mushrooms contain toxin α -amanitin . Block the action of RNA polymerase II . 40-90% die within a few days.
  14. 16. Recognition of DNA by RNA polymerases
  15. 17. RNA polymerase must recognize which genes to transcribe. Transcribed genes are only a small fraction of the total DNA. The genes that are transcribed differ from one type of cell to another Change with changes in physiologic conditions.
  16. 18. The signals in DNA that RNA polymerase recognizes are called promoters . Promoters are sequences in DNA that determine the startpoint and the frequency of transcription. Often composed of smaller sequences called boxes or elements.
  17. 19. A schematic view of a eukaryotic gene, and steps required to produce a protein product.
  18. 20. Adenine- and Thymine-rich consensus In E.coli TATAAT is the consensus sequence TATA or Pribnow box. General Transcription Factor (GTF) binds to TATA box Facilitate the binding of RNA pol II
  19. 21. Eukaryotic gene promoter consensus sequences.
  20. 22. A. Eukaryotic general transcription factors (CTF, SP1, TFIID) bind to consensus sequences found in promoters for  RNA polymerase II . Additional general transcription factors (TFIIs) are required for assembly of the initiation complex and recruitment of polymerase. B. Enhancer stimulation of RNA polymerase II .
  21. 23. Prokaryote
  22. 24. Transcribed region contains Introns and Exons. Intron= Do not contain coding sequence for proteins. Exons= Carrying the coding sequence for proteins. The first RNA form produced is heterogeneous nuclear RNA.
  23. 25. Modification of hnRNA A cap is added at the 5’ end (cap sidt) Poly-A tail added to the 3” end. Introns are removed by a process called splicing to produce the mature mRNA.
  24. 27. Rho-independent termination of transcription. A. DNA template sequence generates a self-complementary sequence in the RNA. B. Hairpin structure formed by the RNA. “N” represents a non-complementary base.
  25. 28. <ul><li>Messenger RNA is transcribed from the template strand of a gene. </li></ul><ul><li>RNA polymerase separates the DNA strands at the appropriate point and bonds the RNA nucleotides as they base-pair along the DNA template. </li></ul><ul><li>Like DNA polymerases, RNA polymerases can add nucleotides only to the 3’ end of the growing polymer. </li></ul><ul><ul><li>Genes are read 3’->5’, creating a 5’->3’ RNA molecule. </li></ul></ul>1. Transcription is the DNA-directed synthesis of RNA Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  26. 29. <ul><li>Specific sequences of nucleotides along the DNA mark where gene transcription begins and ends. </li></ul><ul><ul><li>RNA polymerase attaches and initiates transcription at the promotor , “upstream” of the information contained in the gene, the transcription unit . </li></ul></ul><ul><ul><li>The terminator signals the end of transcription. </li></ul></ul><ul><li>Bacteria have a single type of RNA polymerase that synthesizes all RNA molecules. </li></ul><ul><li>In contrast, eukaryotes have three RNA polymerases (I, II, and III) in their nuclei. </li></ul><ul><ul><li>RNA polymerase II is used for mRNA synthesis. </li></ul></ul>
  27. 30. <ul><li>Transcription can be separated into three stages: initiation, elongation, and termination . </li></ul>
  28. 31. <ul><li>The presence of a promoter sequence determines which strand of the DNA helix is the template. </li></ul><ul><ul><li>Within the promoter is the starting point for the transcription of a gene. </li></ul></ul><ul><ul><li>The promoter also includes a binding site for RNA polymerase several dozen nucleotides upstream of the start point. </li></ul></ul><ul><ul><li>In prokaryotes, RNA polymerase can recognize and bind directly to the promoter region. </li></ul></ul>
  29. 32. <ul><li>In eukaryotes, proteins called transcription factors recognize the promoter region, especially a TATA box , and bind to the promoter. </li></ul><ul><li>After they have bound to the promoter, RNA polymerase binds to transcription factors to create a transcription initiation complex . </li></ul><ul><li>RNA polymerase then starts transcription. </li></ul>
  30. 33. <ul><li>As RNA polymerase moves along the DNA, it untwists the double helix, 10 to 20 bases at time. </li></ul><ul><li>The enzyme adds nucleotides to the 3’ end of the growing strand. </li></ul><ul><li>Behind the point of RNA synthesis, the double helix re-forms and the RNA molecule peels away. </li></ul>
  31. 34. <ul><li>A single gene can be transcribed simultaneously by several RNA polymerases at a time. </li></ul><ul><li>A growing strand of RNA trails off from each polymerase. </li></ul><ul><ul><li>The length of each new strand reflects how far along the template the enzyme has traveled from the start point. </li></ul></ul><ul><li>The congregation of many polymerase molecules simultaneously transcribing a single gene increases the amount of mRNA transcribed from it. </li></ul><ul><li>This helps the cell make the encoded protein in large amounts. </li></ul>
  32. 35. <ul><li>Transcription proceeds until after the RNA polymerase transcribes a terminator sequence in the DNA. </li></ul><ul><ul><li>In prokaryotes, RNA polymerase stops transcription right at the end of the terminator. </li></ul></ul><ul><ul><ul><li>Both the RNA and DNA is then released. </li></ul></ul></ul><ul><ul><li>In eukaryotes, the polymerase continues for hundreds of nucleotides past the terminator sequence, AAUAAA. </li></ul></ul><ul><ul><ul><li>At a point about 10 to 35 nucleotides past this sequence, the pre-mRNA is cut from the enzyme. </li></ul></ul></ul>
  33. 36. 2. Eukaryotic cells modify RNA after transcription <ul><li>Enzymes in the eukaryotic nucleus modify pre-mRNA before the genetic messages are dispatched to the cytoplasm. </li></ul><ul><li>At the 5’ end of the pre-mRNA molecule, a modified form of guanine is added, the 5’ cap . </li></ul><ul><ul><li>This helps protect mRNA from hydrolytic enzymes. </li></ul></ul><ul><ul><li>It also functions as an “attach here” signal for ribosomes. </li></ul></ul>
  34. 37. <ul><li>At the 3’ end, an enzyme adds 50 to 250 adenine nucleotides, the poly(A) tail . </li></ul><ul><ul><li>In addition to inhibiting hydrolysis and facilitating ribosome attachment, the poly(A) tail also seems to facilitate the export of mRNA from the nucleus. </li></ul></ul><ul><li>The mRNA molecule also includes nontranslated leader and trailer segments. </li></ul>
  35. 38. <ul><li>The most remarkable stage of RNA processing occurs during the removal of a large portion of the RNA molecule during RNA splicing . </li></ul><ul><li>Most eukaryotic genes and their RNA transcripts have long noncoding stretches of nucleotides. </li></ul><ul><ul><li>Noncoding segments, introns , lie between coding regions. </li></ul></ul><ul><ul><li>The final mRNA transcript includes coding regions, exons , that are translated into amino acid sequences, plus the leader and trailer sequences. </li></ul></ul>
  36. 39. <ul><li>RNA splicing removes introns and joins exons to create an mRNA molecule with a continuous coding sequence. </li></ul>
  37. 40. <ul><li>This splicing is accomplished by a spliceosome . </li></ul><ul><ul><li>spliceosomes consist of a variety of proteins and several small nuclear ribonucleoproteins ( snRNPs ). </li></ul></ul><ul><ul><li>Each snRNP has several protein molecules and a small nuclear RNA molecule ( snRNA ). </li></ul></ul><ul><ul><ul><li>Each is about 150 nucleotides long. </li></ul></ul></ul>
  38. 41. (1) Pre-mRNA combines with snRNPs and other proteins to form a spliceosome. (2) Within the spliceosome, snRNA base-pairs with nucleotides at the ends of the intron. (3) The RNA transcript is cut to release the intron, and the exons are spliced together; the spliceosome then comes apart, releasing mRNA, which now contains only exons.
  39. 42. <ul><li>In this process, the snRNA acts as a ribozyme, an RNA molecule that functions as an enzyme. </li></ul><ul><li>Like pre-mRNA, other kinds of primary transcripts may also be spliced, but by diverse mechanisms that do not involve spliceosomes. </li></ul><ul><li>In a few cases, intron RNA can catalyze its own excision without proteins or extra RNA molecules. </li></ul><ul><li>The discovery of ribozymes rendered obsolete the statement, “All biological catalysts are proteins.” </li></ul>
  40. 43. <ul><li>RNA splicing appears to have several functions. </li></ul><ul><ul><li>First, at least some introns contain sequences that control gene activity in some way. </li></ul></ul><ul><ul><li>Splicing itself may regulate the passage of mRNA from the nucleus to the cytoplasm. </li></ul></ul><ul><ul><li>One clear benefit of split genes is to enable a one gene to encode for more than one polypeptide. </li></ul></ul><ul><li>Alternative RNA splicing gives rise to two or more different polypeptides, depending on which segments are treated as exons. </li></ul><ul><ul><li>Early results of the Human Genome Project indicate that this phenomenon may be common in humans. </li></ul></ul>
  41. 44. <ul><li>Split genes may also facilitate the evolution of new proteins. </li></ul><ul><li>Proteins often have a modular architecture with discrete structural and functional regions called domains . </li></ul><ul><li>In many cases, different exons code for different domains of a protein. </li></ul>
  42. 45. <ul><li>The presence of introns increases the probability of potentially beneficial crossing over between genes. </li></ul><ul><ul><li>Introns increase the opportunity for recombination between two alleles of a gene. </li></ul></ul><ul><ul><ul><li>This raises the probability that a crossover will switch one version of an exon for another version found on the homologous chromosome. </li></ul></ul></ul><ul><ul><li>There may also be occasional mixing and matching of exons between completely different genes. </li></ul></ul><ul><ul><li>Either way, exon shuffling could lead to new proteins through novel combinations of functions. </li></ul></ul>
  43. 46. Ribosomes <ul><li>2 subunits, separate in cytoplasm until they join to begin translation </li></ul><ul><ul><li>Large </li></ul></ul><ul><ul><li>Small </li></ul></ul><ul><li>Contain 3 binding sites </li></ul><ul><ul><li>E </li></ul></ul><ul><ul><li>P </li></ul></ul><ul><ul><li>A </li></ul></ul>
  44. 47. Translation - Initiation A E Large subunit P Small subunit fMet UAC GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  45. 48. Translation - Elongation A E Ribosome P UCU Arg Aminoacyl tRNA Phe Leu Met Ser Gly Polypeptide CCA GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  46. 49. Translation - Elongation Aminoacyl tRNA A E Ribosome P Phe Leu Met Ser Gly Polypeptide Arg UCU CCA GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  47. 50. Protein Synthesis ANYTHING ACID AMINE C O OH C N H H H C HO H C H O C N H H H C H H C H O OH C N H H H C HO H Serine C H O OH C N H H H C H H Alanine H C O OH C R N H H Amino Acid H 2 O
  48. 51. Translation - Elongation A E Ribosome P CCA Arg UCU Phe Leu Met Ser Gly Polypeptide GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  49. 52. Translation - Elongation A E Ribosome P Aminoacyl tRNA CGA Ala CCA Arg UCU Phe Leu Met Ser Gly Polypeptide GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  50. 53. Translation - Elongation A E Ribosome P CCA Arg UCU Phe Leu Met Ser Gly Polypeptide CGA Ala GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5’ mRNA 3’
  51. 54. Transcription And Translation In Prokaryotes 3’ 5’ 5’ 3’ Ribosome Ribosome 5’ mRNA RNA Pol.
  52. 55. Transcription vs. Translation Review <ul><li>Transcription </li></ul><ul><li>Process by which genetic information encoded in DNA is copied onto messenger RNA </li></ul><ul><li>Occurs in the nucleus </li></ul><ul><li>DNA mRNA </li></ul><ul><li>Translation </li></ul><ul><li>Process by which information encoded in mRNA is used to assemble a protein at a ribosome </li></ul><ul><li>Occurs on a Ribosome </li></ul><ul><li>mRNA protein </li></ul>
  53. 56. Reverse transcriptase