1. Transposons Chapter 21 高雄醫學大學 生物醫學暨環境生物學系 張學偉 助理教授 Email: changhw@kmu.edu.tw 分機 : 2691changhw@kmu.edu.tw

2. 2 1. Acquisition of new sequences- horizontal transfer of genetic material between genomes by extrachromosomal elements 2. Rearrangements of existing sequences- transfer within the genome 21.1 Introduction Evolution of genomes via:

3. 3 1. Acquisition of new sequences- horizontal transfer of genetic material between genomes by extra- chromosomal elements a) Bacteria- plasmids move by conjugation (F plasmid, Hfr) b) Phages – spread by infection (transduction) c) Both can transfer host genome with its own replicon

4. 4 2. Rearrangements of existing sequences- transfer within the genome a) Unequal recombination- mispairing in homologous recombination b) Nonreciprocal recombination- results in duplication of loci; one copy- original function, the other- evolves c) Transposable elements (transposons)

5. 5 Figure 21.1

6. 6 Transposons (transposable elements) definition “discrete sequences in the genome that are mobile- able to transport themselves to other locations within the genome.”

7. 7 Basic concept for Transposons 1. Move directly from one site in the genome to another (do not need other vectors) 2. not rely on any relationship between sequences at the donor and recipient sites- Non-homologous recombination. 3. Internal counterpart to vectors that move sequences between genomes (phages & plasmids). May provide a major source of mutations in the genome. 4. Two classes: a) DNA transposon; b) Retroviruses & retroposons 5. Transposons found in both prokaryotes and eukaryotes.

8. 8 Bacterial vs. Eukaryotic Tranaposons Bacterial transposons carry genes that transpose themselves. Eukaryotes Although many are defective (lost ability to transpose independently) and rely on the enzymes from a few functional transposons. Many number and variety of transposons included.

9. 9 Transposable elements can promote rearrangement of the genome directly or indirectly: directly (Tn itself) 1.cause deletions or inversions or 2.lead to movement of host sequences to new locations. indirectly 1.serve as substrates for cellular recombination systems as “portable regions of homology” 2.two copies-sometimes on different chromosomes in eukaryotes- provide sites for reciprocal recombination. 3.These types of exchanges lead to deletions, insertions, inversions, or translocations.

10. 10 Natural selection view for Transposons Neither advantage nor disadvantage on the phenotype. But the selfish DNA concerned only with its own propagation (parasite to the genome; yet-selective advantage) Is an independent entity that residues in the genome.

11. 11 Good comment for Transposons Any transposition event conferring a selective advantage, e.g., genetic rearrangement. It will lead to preferential survival

12. 12 Other features of Transposon 1. Transposons are DNA elements that transpose to different places on the DNA. 2. Transposition is the movement of the transposon. 3. It requires special protein factors particularly to cut and ligate the DNA  transposase 4. NO homology is required between the transposon and the target sequence. 5. First identified in bacterial operon- spontaneous silencing.

13. 13 21.2 Insertion Sequences Are Simple Transposition Modules An insertion sequence is a transposon that codes for the enzyme(s) needed for transposition flanked by short inverted terminal repeats (IR, ITR). is closely related rather than identical at the ends of transposon are autonomous unit- sponsors its own transposition Recognition of the ends (IR)- critical in transposition; point mutations abort it KEY CONCEPT

14. 14 IR (inverted terminal repeat) The target site is  random, hotspots, or preferred (bent DNA; consensus sequence; inactive region).  duplicated during insertion forms DR (direct repeat). The length of the DR is: –characteristic for any particular transposon Figure 21.2 IRDR KEY CONCEPT

15. 15 Rate comparison 1. Rate of transposition: ~10 -3 -10 -4 per element per generation 2. Rate of spontaneous mutation: ~10 -5 -10 -7 per generation 3. Rate of reversion (by precise excision of the IS element) ~10 -6 -10 -10 per generation ~10 3 times less frequent than insertion

16. 16 21.3 Composite Transposons Have IS Modules Composite transposons have a central region flanked by an IS element at each end. Central region carry other genes (antibiotic resistance or other markers). IS elements only carry enzymes needed for transposition (transposase, resolvase). IS-L IS-R KEY CONCEPT

17. 17 Either one or both of the IS elements of a composite transposon may transposition. A functional IS module can transpose either itself (Fig.21.2) or the entire transposon An active IS element at either end may also transpose independently. Figure 21.3 DR IR NOT identical but close related KEY CONCEPT

18. 18 Composite transposons evolved 1. Composite transposons evolved when two originally independent units associated with the central region - IS element transposed to a recipient site close to a donor site 2. Two identical modules may remain identical or diverge 3. Lack of selective pressure of both modules to remain active

19. 19 Figure 21.04: IS elements can mobilize other sequences. Inside-out With marker  high freq. under selection > IS10 Transposition frequency declines with distance between IS10.  length-depend factor determines the size of common composite Tn. Mobility frequency: IS10 > Tn10

20. 20 Summary on Transposons 1. The smallest transposons are called insertion sequences (IS elements). 2. IS elements have inverted repeats at either end and a transposase gene in between; they do NOT have any resistance or other markers. 3. Transpoase is responsible for: (1) creating a target site (random, or consensus seq); (2) recognize the ends (IS) of the transposon. 4. Two IS elements flanking a marker gene(s) form a composite transposon.

21. 21 21.4 Transposition Occurs by Both Replicative and Nonreplicative Mechanisms Figure 21.5 ATGCA TACGT corrected All transposons use a common mechanism 1 2 3 The stagger between the cuts determines the length of the direct repeats. The target repeat is characteristic of each transposon; reflects the geometry of the cutting enzyme KEY CONCEPT

22. 22 The order of events and exact nature of the connections between transposon and target DNA determine whether transposition is: –Replicative & Nonreplicative (cut-and-paste) The use of staggered ends is common to all transpositions: three types Replicative [R] TnA Nonreplicative [N] IS, Tn10 and Tn5 Conservative [C] (nonreplicative) Some Tn use only one type Others multiple types: R and N/C  IS1, IS903, R or N/C  phage Mu KEY CONCEPT

23. 23 Animation

24. 24 Figure 21.6 Replicative Transposon is duplicated; a copy of the original element is made at a recipient site (TnA); donor keeps original copy Transposition- an increase in the number of Tn copies ENZs: transposase (acts on the ends of original Tn) and resolvase (acts on the duplicated copies)

25. 25 Figure 21.7 Nonreplicative transposition Nonreplicative a) Transposon moves from one site to another and is conserved; breaks in donor required host repair system. b) IS, Tn10 and Tn5 use this mechanism; no Tn copy increase c) ENZs: only transposase

26. 26 Figure 21.08: Movement conserves bonds. Conservative (nonreplicative) a) Tn excised from donor and inserted in target – every nucleotide bond is conserved like in lambda integration (Site-Specific Recombination) large elements (episomes?) b) ENZs: transposase (related to integrase family) P.851 Epiosome: is a plasmid able to integrate into bacterial DNA

27. 27 21.5 Transposons Cause Rearrangement of DNA Homologous recombination between multiple copies of a transposon causes rearrangement of host DNA. [deletion, inversion] Homologous recombination between the repeats of a transposon may lead to precise or imprecise excision. KEY CONCEPT

28. 28 Figure 21.09: Direct repeats recombine to excise material. Recombination using cellular enzymes Reciprocal recombination between two copies of the transposon Lost from the cell Single copy on chromosome IS1L IS1R recombination between two elements in same orientation (DR) Tn inserts a copy at a second site near its original location Step 1 Step 2

29. 29 Figure 21.10: Inverted repeat recombination inverts material. (A) (B) (A) (B) (A) Excisions not supported by Tn’s: Precise excision - removes transposon & one copy of duplicated sequence; rare Tn10= ~10 -9 ; recombination between 5-9 bp duplicated target sequences Imprecise excision - leaves a remnant of the transposon; Tn10= ~10 -6. sufficient to prevent target gene reactivation. recombination between 24 bp IS-modules of a composite Transposon Recombination using cellular enzymes  not relies on Tn- coded function P854 P859 recombination between two elements in opposite orientation (IR)

30. 30 21.6 Common Intermediates for Transposition Transposition starts by forming a strand transfer complex. –The transposon is connected to the target site through one strand at each end. (1 x 2) KEY CONCEPT Both replicative and non-replicative transposition use a common mechanism: IS elements, prokaryotic & eukaryotic transposons, and bacteriophage Mu, retroviral DNA and the first stages of immunoglobulin recombination use the similar mechanism.

31. 31 2. Transposon nicked at both ends; target nicked at both strands 3. Nicked ends joined crosswise; covalent connection between the transposon and the target 1. Synapsis stage- two ends of transposon are brought together [shown after cleavage but actually occurs previously] Joining transposon to its target- common pathway

32. 32 The Mu transposase forms the complex by: 1.synapsing the ends of Mu DNA 2.followed by nicking 3.then a strand transfer RX Figure 21.12 KEY CONCEPT 1 2 3 Replicative transposition follows if the complex is replicated. Nonreplicative transposition follows if it is repaired. Animation show the detail Next step differs and determines the type of transposition: MuA tetramer

33. 33 Figure 21.12 1 2 A Mu transposon passes through 3 stable stages: - MuA binds to ends as tetramer forming a synapsis. - MuA subunits act in trans to cut next to R1 and L1 (coordinately; two active sites to manipulate DNA). - MuA acts in trans to cut the target site DNA and mediate in trans strand transfer Mu integrates by nonreplicative transposition; during lytic cycle- number of copies amplified by replicative transposition MuA also binds to internal site- needed for complex formation but not strand cleavage 3 cuts in trans transfers in trans

34. 34 Figure 21.12 1 2 3 In strand transfer complex transposon is connected to the target site through one strand at each end cuts in trans transfers in trans

35. 35 Figure 21.12 1 2 3 The MuB protein chooses targets; Mu Tn moves >10-15 kb away from original site (target immunity). MuB binds to the MuA-Mu DNA complex; MuA causes MuB to hydrolize ATP; MuB released from the donor DNA. MuB binds nonspecifically to the target DNA and stimulates the recombination activity of MuA in transposition complex. MuA clears MuB from the donor - gives preference for transposition to the target.

36. 36 21.7 Replicative Transposition Proceeds through a Cointegrate Replication of a strand transfer complex generates a cointegrate: –A fusion of the donor and target replicons. The cointegrate has two copies of the transposon. –They lie between the original replicons. Figure 21.13 by resolvase (homolgous recombination) KEY CONCEPT

37. 37 Recombination between the transposon copies regenerates the original replicons, but the recipient has gained a copy of the transposon. The recombination reaction is catalyzed by a resolvase coded by the transposon. KEY CONCEPT

38. 38 Figure 21.14: Mu transposition uses a crossover intermediate.

39. 39 21.8 Nonreplicative Transposition Proceeds by Breakage and Reunion Nonreplicative transposition results if: –a crossover structure is nicked on the unbroken pair of donor strands and –the target strands on either side of the transposon are ligated Figure 21.15 KEY CONCEPT donor target

40. 40 Two pathways for nonreplicative transposition differ according to whether: –the first pair of transposon strands are joined to the target before the second pair are cut (Tn5), or –whether all four strands are cut before joining to the target (Tn10) KEY CONCEPT

41. 41 Figure 21.16: Transposition can use cleavage and ligation. Two pathways for nonreplicative transposition differ according to : –whether all four strands are cut before joining to the target (Tn10) or –whether the first pair of transposon strands are joined to the target before the second pair are cut (Tn5), KEY CONCEPT

42. 42 Figure 21.17: Tn5 is cleaved from flanking DNA. Two pathways for nonreplicative transposition differ according to : –whether all four strands are cut before joining to the target (Tn10) or –whether the first pair of transposon strands are joined to the target before the second pair are cut (Tn5), 1. nicking 2.interstrand Rx 3 4. cleavage

43. 43 Figure 21.18: Transposon ends are joined. dimer Tn 5 and Tn 10 transposases both function as dimers.

44. 44 21.9 TnA Transposition Requires Transposase and Resolvase Replicative transposition of TnA requires: –a transposase to form the cointegrate structure –a resolvase to release the two replicons The action of the resolvase resembles lambda Int protein. It belongs to the general family of topoisomerase-like site-specific recombination (SSR) reactions. –They pass through an intermediate in which the protein is covalently bound to the DNA. KEY CONCEPT

45. 45 Figure 21.19: TnA transposon organization is conserved. Transposase Resolvase Repressor (mutant ↗ Tn freq) Internal res site feedback (38bp) Replicative Tn, Non-IS-dependent, DR (~5bp) generated at target sites. Control regions Limiting factor in transposition Dual role Res 1. Can be replaced by RecA-mediated general recombination, but less efficient. 2. 15-20 bp of res site are identical to att. 3. But protein mechanism is different: res- intramolecular resolution. Att- intermolecular resolution TnA features

46. 46 21.10 Transposition of Tn10 Has Multiple Controls Figure 21.20: Tn10 has two promoters. IS10L inactive Promoter strong weak RNA stable OUT RNA function as an antisense RNA. One copy  no effect 5 copies  significant Multicopy inhibition reduces the rate of transposition of any one copy of a transposon when other copies of the same transposon are introduced into the genome KEY CONCEPT

47. 47 Figure 21.21 Tn must maintain min freq to survive, but too great freq may damage the host cells. Multiple mechanisms affect the rate of transposition. KEY CONCEPT

48. 48 21.11 Controlling Elements in Maize Cause Breakage and Rearrangements Transposition in maize was discovered because of the effects of chromosome breaks. –The breaks were generated by transposition of “controlling elements (Transposon)- Ds element.” KEY CONCEPT Figure 21.22: Transpositions are clonally inherited.

49. 49 The break generates one chromosome that has: –a centromere –a broken end –one acentric fragment The acentric fragment is lost during mitosis; –detected by the disappearance of dominant alleles in a heterozygote. Figure 21.23. A break at controlling element causes loss of an acentric fragment KEY CONCEPT (site for chr. breakage) dominant recessive

50. 50 Fusion between the broken ends of the chromosome generates dicentric chromosomes. –These undergo further cycles of breakage and fusion. The breakage-fusion-bridge cycle is responsible for the occurrence of somatic variegation. Figure 21.24 Ds provides a site to initiate the chromatid breakage-fusion-bridge cycle. KEY CONCEPT

51. 51 Figure 21.25 21.12 Controlling Elements Form Families of Transposons The numbers, types, and locations of the control elements are characteristic for each maize strain. KEY CONCEPT Each family of controlling elements in maize has two classes: autonomous and nonautonomous Note! Ds = Dt Common feature Specific feature

52. 52 Mutator transposon is one of the simplest elements MuDR (autonomous element) code for genes: mudrA codes for MURA transposase mudrB codes for nonessential accessory protein In MuDR, de-methylation of the terminal repeats increases transposase experssion.

53. 53 Figure 21.26: The Ac element gas five exons that code for a transposase; Ds element have internal deletions. Ac element Ds elements have internal deletions. Ds alone could not induce the breakage. (need Ac) Transposition of Ac/Ds occurs by a non-replicative mechanism. Phage using Ac/Ds results in DNA methylation change. May code for a repressor of transposition transposase That inactivate the trans- acting transposase Ds is an incomplete version of Ac itself

54. 54 Autonomous controlling elements code for proteins that enable them to transpose. Nonautonomous controlling elements have mutations that eliminate their capacity to catalyze transposition (internal sequences). –They can transpose when an autonomous element provides the necessary proteins via trans-acting transpoase. Autonomous controlling elements have changes of phase (reversible), when their properties alter as a result of changes in the state of methylation. KEY CONCEPT [note! Ds is coded for deleted form of transpoase; Ac is complete functional; that is why always written in Ac/Ds system]

55. 55 Transposable elements in eukaryotes: Barbara McClintock (1902-1992) Cold Spring Harbor Laboratory, NY Nobel Prize in Physiology and Medicine 1983 “for her discovery of mobil genetic elements” Studied transposable elements in corn (Zea mays) 1940s-1950s (formerly identified as mutator genes by Marcus Rhoades 1930s) Nonautonomous DNA tn (Ds) require the activator (Ac) to be in the same cells.

56. 56 21.13 Spm Elements Influence Gene Expression Spm elements affect gene expression at their sites of insertion, when the TnpA protein binds to its target sites at the ends of the transposon. Spm elements are inactivated by methylation. KEY CONCEPT

57. 57 Figure 21.27: Spm/En has two genes. tnpA a spliced 2500-bp mRNA (Exon1-11); tnpB 6000-bp mRNA (containing ORF1+ORF2) defective dSpm

58. 58 21.14 The Role of Transposable Elements in Hybrid Dysgenesis P elements are transposons that are carried in P strains of Drosophila melanogaster (fly), but not in M strains. When a P male is crossed with an M female, transposition is activated. [Note! M male x P female, transposition is inactivated]  hybrid dysgenesis KEY CONCEPT p853

59. 59 The insertion of P elements at new sites in these crosses: –inactivates many genes –makes the cross infertile Figure 21.28 Hybrid dysgenesis is asymmetrical = infertile Dysgenesis is principally a phenomenon of the germ cells. P-specific sequences can induce dysgenesis by insertional inactivation. P-specific seq are many (30-50 copies) and locate in different chr., but not in M strain.

60. 60 21.15 P Elements Are Activated in the Germline P elements are activated in the germline of P male x M female crosses. This is because a tissue- specific splicing event removes one intron (somatic expression). –This generates the coding sequence for the transposase. Figure 21.29 Pelement has four exons; the first three are spliced together in somatic expression; all four are spliced together in germline expression KEY CONCEPT 31bp IR Generate DR of target DNA (~8bp)

61. 61 The P element also produces a repressor of transposition. –It is inherited maternally in the cytoplasm. Figure 21.30 The presence of the repressor explains why M male x P female crosses remain fertile. KEY CONCEPT No repressor

62. Retroviruses and Retroposons Chapter 22 高雄醫學大學 生物醫學暨環境生物學系 張學偉 助理教授 Email: changhw@kmu.edu.tw 分機 : 2691changhw@kmu.edu.tw Retro-transposons

63. 63 22.1 Introduction Figure 22.1 Reproductive cycles (continuous) of retroviruses and retroposons. Retroposons are confined to an intracellular cycle. Single strand Retrovirus - Transposition involved RNA intermediate is unique to eukaryotes. RNA  DNA  RNA Retroposon - Transposition through RNA intermediate. (similar) Itself no transposition activity but with active element sequence [with help from retrovirus] Like a lysogenic bacteriphage

64. 64 22.2 The Retrovirus Life Cycle Involves Transposition-Like Events A retrovirus has two copies of its genome of single-stranded RNA. An integrated provirus is a double- stranded DNA sequence. KEY CONCEPT

65. 65 A retrovirus generates a provirus by reverse transcription of the retroviral genome. Figure 22.2 integrase Long terminal repeat

66. 66 22.3 Retroviral Genes Codes for Polyproteins Figure 22.3 A typical retrovirus has three genes 5-cap polyA gap-pol Env proteins gag & pol in different frame: gap >> gag-pol Continued R seqment

67. 67 Gag and Pol proteins are translated from a full- length transcript of the genome. Translation of Pol requires a frameshift by the ribosome. Env is translated from a separate mRNA that is generated by splicing. Each of the three protein products is processed by proteases to give multiple proteins. KEY CONCEPT

68. 68 Figure 22.04: HIV buds from the membrane. Photo courtesy of Matthew A. Gonda, Ph.D., Chief Executive Officer, International Medical Innovations, Inc. Viron- physical virus particles p.866 Viroid- small infected nucleic acids without protein coats. 比較 A process that is reversed during infection

69. 69 22.4 Viral DNA Is Generated by Reverse Transcription Retroviruses are called Plus (+) strand viruses because the viral RNA itself codes for the protein products. Complementary DNA of Virus RNA called minus (-) strand DNA.  another strand in duplex DNA called plus (+) strand DNA. RNase H degrade the RNA part of RNA-DNA hybrid.

70. 70 22.4 Viral DNA Is Generated by Reverse Transcription A short sequence (R) is direct repeated at each end of the viral RNA. –The 5’ and 3’ ends are R- U5 and U3-R, respectively. Figure 22.5 Retrovirial RNA ends in direct repeat (R), the free linear DNA ends in LTR and the provirus ends in LTRs that are shortened by two bases each. R segment KEY CONCEPT

71. 71 Figure 22.6-a Reverse transcriptase starts synthesis when a tRNA primer binds to a site 100 to 200 bases from the 5’ end. KEY CONCEPT (+) (-)

72. 72 Figure 22.6-b When the enzyme reaches the end, the 5’-terminal bases of RNA are degraded. –This exposes the 3’ end of the DNA product. KEY CONCEPT The exposed 3’ end base pairs with the 3’ terminus of another RNA genome. Synthesis continues, generating a product in which the 5’ and 3’ regions are repeated. –This gives each end the structure U3-R-U5. (-)

73. 73 Similar strand switching events occur when reverse transcriptase uses the DNA product to generate a complementary strand. Figure 22.7 Synthesis of plus-strand DNA requires a second jump. KEY CONCEPT DNA (-) RNA DNA (-) DNA (+)

74. 74 Strand switching is an example of the copy choice mechanism of recombination. Figure 22.8 KEY CONCEPT DNA (-) RNA (+) copy choice A type of recombination used by RNA virus, in which the RNA polymerase switches from one template to another during synthesis p869

75. 75 Linear DNA is inserted directly into the host chromosome by the retroviral integrase enzyme. Figure 22.9 KEY CONCEPT Two base pairs of DNA are lost from each end of the retroviral sequence during the integration reaction. The organization of proviral DNA in a chromosome is the same as a transposon. –The provirus is flanked by short direct repeats (DR) of a sequence at the target site. Cp. Fig22.5 22.5 Viral DNA Integrates into the Chromosome at radom sites.

76. 76 Part of Fig. 22.5 Left LTR Response for initiating transcription of provirus Right LTR Sometimes (rarely) sponsor transcription of host seq near integration site. LTR also carries an enhancer that acts on cellular and viral seq. U3 carries promoter

77. 77 22.6 Retroviruses May Transduce Cellular Sequences Figure 22.10: Replicative-defective transforming viruses have a cellular seq substituted for parial viral seq. From spliced RNA copies of cellular seq. c-onc Onc = Oncogenesis, transform ability c-onc usually interrupted by introns v-onc is un-interrupted

78. 78 Figure 22.11 Transforming retroviruses are generated by a recombination event: A cellular RNA sequence replaces part of the retroviral RNA. Helper virus

79. 79 22.7 Yeast Ty Elements Resemble Retroviruses Figure 22.12: Ty elements have two genes. Ty = Transposon yeast interspersed repeat DNA Same transpose mechanism to retrovirus Freq < bacterial Tn Two major classes: Ty1 (30 copies) Ty917 (6 copies) ps: , 100 copies, considerable heterogeneity

80. 80 Ty elements are classic retroposons, with a reverse transcriptase activity. –They transpose via an RNA intermediate. Figure 22.13 KEY CONCEPT Ty elements does not give rise to infectious particles, but virus- like particles (VLPs) accumulate within the cells.

81. 81 Endogenous retroviruses Ty transposons have a similar organization to endogenous retroviruses. endogenous retroviruses are retroviruses derived from ancient infections of germ cells in humans, mammals and other vertebrates; as such their proviruses are passed on to the next generation and now remain in the genome. Most retroviruses (such as HIV-1) infect somatic cells, but some can also infect germline cells (cells that make eggs and sperm) http://en.wikipedia.org/wiki/Endogenous_retrovirus KEY CONCEPT

82. 82 copia is a retroposon that is abundant in D. melanogaster. Figure 22.15 22.8 Many Transpable Elements Reside in D. melanogaster.

83. 83 22.9 Retroposons Fall into Three Classes Retroposons of the viral superfamily are transposons that mobilize via an RNA that does not form an infectious particle. Figure 22.16 KEY CONCEPT 12 3 LTR

84. 84 Some retroposons directly resemble retroviruses in their use of LTRs. - Others do not have LTRs. Other elements can be found that were generated by an RNA- mediated transposition event; –But they do not themselves code for enzymes that can catalyze transposition. –[Just need help] Figure 22.17 KEY CONCEPT Plant contain another type of small mobile element, called MITE (miniature inverted- repeat transposable element) No relationship to SINE, LINE

85. 85 Transposons and retroposons constitute almost half of the human genome. Figure 22.18 KEY CONCEPT Only one SINE have been active in the human lineage: the common Alu element

86. 86

87. 87 22.10 The Alu Family Has Many Widely Dispersed Members A major part of repetitive DNA in mammalian genomes consists of repeats of a single family: –organized like transposons –derived from RNA polymerase III transcripts KEY CONCEPT Individual members of the Alu family are related rather than identical. Alu sequence is related to 7SL RNA, a compartment of the signal recognition particle.

88. 88 22.11 Processed Pseudogenes Originated as Substrates for Transposition A processed pseudogene is derived from an mRNA sequence by reverse transcription. Figure 22.19 KEY CONCEPT No intron DR RNA polmerase II

89. 89 Evidence: 1. many of the poly-A retroposons that have been detected by large-scale genomic sequencing are truncated elelments.  most of these are missing region from 5’end.  lost the ability to transpose. 2. Processed pseudogenes  not expressed by cell due to lack of promoter, intron or truncate near 5’end. (many cellular gene had been truncated at 5’end)  these pseudogenes are often flanked by short repeat  this is structure of LINE-promoted transposition of cellular mRNA.

90. 90 processed pseudogenes do not carry any information used to transposition. do not carry out reverse transcription of RNA. A dead ends of evolution. Active LINE element provides most of the RTase activity Acts for transposition on: (1) its own (2) SINE For generating processed pseudogenes. Summary

91. 91 22.12 LINES Use an Endonuclease to Generate a Priming End LINES do not have LTRs. They require the retroposon to code for an endonuclease that generates a nick to prime reverse transcription. Figure 22.20 DNA-binding protein RT-aseendonuclease KEY CONCEPT 5’ 3’

92. 92 Note Although transposition of cellular RNA can occur, it is a rare event. LINE-encoded protein (ORF1&2) bind immediately to their own RNA during translation  show highly preference to its own RNA rather than the cellular RNA.

93. 93 Figure 22.21: LINES proteins are cis-acting. Reverse transcription often does not proceed fully to the end, so the copy is inactive. Original from RNA pol II lacks of promoter are necessary inactive.

94. 94 Figure 22.22: Autonomous act on nonautonomous elements. For transposition to survive, they must occur in the germline.