1. Different mechanisms of reading the isoleucine codon in three domains of life Dr. Debabrata Mandal Assistant professor, Dept. of Biotechnology National Institute of Pharmaceutical Education and Research (NIPER)-Hajipur 6 th World Congress on Biotechnology (New Delhi) 5-7 Th October, 2015 1

2. Translation…… Role of mRNA, tRNA and ribosome  Translation is the process where nucleic acid message (mRNA) is converted to protein in presence of tRNA and ribosome  Codon… a triplet of 3 bases (combination of A, U, G, C) codes for an amino acid which is read by tRNA on ribosome  Each codon is read by a specific tRNA which is aminoacylated by an amino acid Genetic code mRNA : tRNA : ribosome complex 2

3. aa Secondary and tertiary structure of tRNA Anticodon Numerical nomenclature of tRNA 3436 5’3’ C A U G U A 5’3’ 34 (wobble base) Met 3

4. Almost all tRNAs are chemically modified…. why?  tRNA is the most highly modified class of RNA species, and modifications are found in tRNAs from all organisms that have been examined.  It was observed that ~13% of bases are modified with ~57 different type of modifications (642 tRNAs from 77 organisms)  Modifications provide proper folding and protect tRNAs from degradation by RNases  Modifications in and around the anticodon helps in proper ribosome binding and correct translation  Modification of wobble base helps in Codon Degeneracy– one tRNA reading multiple codon tRNA modification in yeast 4

5. Codon degeneracy -- number of tRNA species is fewer than the 61 sense codons, suggesting that some tRNAs read more than one codon. Due to modification at wobble base (base 34) one tRNA can decode more than one codon. Anticodon, wobble base and modifications List of wobble modifications across three domains of life 5’3’ U*A C A U G 5’3’ G U G 5’ 3’ U U G 5’3’ C U G 5’3’ U*---mcmo 5 U Val 5

6. General overview of the talk  Role of tRNA modifications in reading the genetic code  Identification of a new tRNA modification (a modification that changes the identity and property of a tRNA)  Why this modification is essential for survival (Bacteria, archaea and Eukaryotes?  How a tRNA modification enzyme can be used as an antimicrobial target ? 6

7. Genetic code and isoleucine tRNAs of bacteria ATP Lysine TilS CC*  Modification of C to C*(lysidine) changes Met tRNA to Ile tRNA.  tRNA 2 Ile reads exclusively Ile codon AUA but not the Met codon AUG 7

8. Mechanism of reading the Isoleucine codon AUA in three domains of life 8

9. ModificationEnzyme Bacteria Lysidine TilS Archaea ? ? Conversion of tRNA (met) to tRNA (Ile) by wobble modification in bacteria Almost all archaea like bacteria have 3 Met tRNAs--- one for start codon (initiator met tRNA) and 2 others for internal AUG codons (elongator met tRNA) One of elongator met tRNA is converted to Ile tRNA by lysidine modification--- Discovered in 1988 in bacteria Our model organism– Haloarcula marismortui (a halophile ) – grows at 4.2-4.5 M NaCl genomic database available from 2004 9

10. (Met) (Ile) tRNA 12 = tRNA Met tRNA 34 = tRNA Ile Two met tRNAs of H. marismortui P P RNA 2008 OH - Hma Hma MetRS IleRS OH - Hma Hma MetRS IleRS 10

11. P 0--- no tRNA 1--- T7 transcript 2--- Hma total tRNA Primer extension of tRNA 34 ≡ tRNA 2 Ile Modification at position 34 blocks reverse transcriptase and gives a truncated product 11

12. Purification of tRNA 2 Ile and tRNA 1 Ile Target tRNA tRNA 2 Ile = 0.1% of total tRNA (1 µg out of 1000 µg) Streptavidin-sepharose + Biotin labeled primer ---- purification of a single tRNA from total tRNA pool Ethidium bromide Northern 1 2 3 1---- total tRNA 2---- tRNA 2 Ile 3---- tRNA 1 Ile This method of purification can be used for purification of any tRNA from any source 12

13. mRNA-dependent decoding by tRNA 2 Ile & tRNA 1 Ile in presence of ribosome tRNA 2 Ile tRNA 1 Ile G A U 5’3’ tRNA 1 Ile C U A G U A 5’3’ C*A U 5’3’ tRNA 2 Ile A U A G U A 5’3’ Ribosome + mRNA + radiolabeled tRNA 13

14. Analysis of the modified C*34 by Mass spectrometry RNase T1 fragment– 5’-GCUC*AU[t 6 A]ACCGp-3’ RNase A fragment----5′-C*AUp-3’ m/z values indicate an extra + ve charge and an additional mass of 112 Da, due to modification of C34 C C* H ATP, Enzyme? 14

15. Lysidine vs Agmatidine Similar yet different strategies for reading AUA codon in Bacteria and Archaea PNAS 2010 Tils Tias 15

16. PNAS 2010 ModificationEnzyme Bacteria Lysidine TilS Archaea Agmatidine ? Lysidine vs Agmatidine -changing the identity and property a of tRNA by wobble modification 16

17. ModificationEnzyme Bacteria Lysidine TilS Archaea Agmatidine TiaS TiaS--- tRNA 2 Ile 2-agmatinylcytidine (agmatidine) synthetase TilS--- tRNA 2 Ile 2-lysidinylcytidine (lysidine) synthetase 17

18.  Why a modified C (not U) base pairs with A (mutagenesis and functional analysis of the mutant tRNA) (Mutagenesis and analysis using H. volcanii as a host)  How agmatidine helps in reading the AUA codon and rejects the AUG codon (Analysis of the crystal structure of the tRNA 2 Ile :70S ribosome : mRNA complex (In collaboration with V. Ramakrishnan Lab) How and why a modified C (not U) base pairs with A??????? 5’3’ C A U Wt A U A 5’ 3’ * 5’3’ U A U Mutant A U A 5’ 3’ C34 U34 mutation G U A 5’ 3’ G U A 5’ 3’ 18

19.  Crystal structure of tRNA 2 Ile :70S ribosome : mRNA complex--- A single hydrogen bond between agmatidine (agm 2 C34) of the tRNA with base A of the mRNA AUA How modified C base pairs with A? In collaboration with Ramakrishnan Lab 3.2 Å Resolution  This interaction is further stabilized by another potential hydrogen bond between the agmatidine side chain and the mRNA phosphate backbone 5’3’ C A U A U A 5’ 3’ * √ 19

20. How modified C does not base pairs with G?  Agmatidine modification ensures that it does not read the AUG codon and, therefore, mistranslation does not happen  The modification ensures that it reads the AUA codon only Nature Str. & Mol Biol. 2013 5’3’ C A U G U A 5’ 3’ * X 20

21. Why a modified C (not U) base pairs with A ?  Overexpression of mutant in H. volcanii.  Analysis of aminoacylation of mutant and wt tRNA  Codon reading properties of the mutant tRNA 5’3’ C A U Wt A U A 5’ 3’ * 5’3’ U A U Mutant A U A 5’ 3’ C34 U34 mutation 21

22. Codon reading properties of the Wt and mutant tRNA Mutant tRNA can mistranslate Methionine (AUG) instead of Isoleucine (AUA)--- a possible reason why UAU anticodon is not used instead of C*AU. RNA 2014 22

23. Summary and future  Prokaryotes, eukarotes and archaea have evolved different strategies for reading the isoleucine codon AUA  Archaeal tRNA Ile have a tRNA modification that is different from proakaryotes and eukaryotes------ Lysidine v/s agmatidine.  Modification is essential for survival as it maintains strict codon specificity ------ prevents global mistranslation  Similarities in structure of Agmatidine and Lysidine… bacteria and archaea have developed similar strategies for reading the AUA codon…. An example of convergent evolution  Parasites (like Leishmania and Trypanosoma) Isoleucine tRNAs lack Lysidine/Agmatidine modification and still can discriminate AUA vs. AUG codon….How?  Tils (enzyme responsible for Lysidine modification) is essential for bacteria…. Could be a potential antimicrobial target 23

24. Acknowledgement Supervisor Prof. Uttam L. RajBhandary Lab members Caroline kohrer Gayathri Srinivasan Collaborators Prof. Patrick Limbach, University of Cincinnati Prof. Venkatraman Ramakrishnan, MRC laboratory of molecular Biology, Cambridge, UK Prof Peter Dedon, MIT (Biological Engineering) 24

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27. In vitro codon reading properties of tRNA N U A G U A 5’3’ G A U 3H -Ile1 Filter Wash Count N U A G U A 5’3’ C*A U 3H -Ile2 Filter Wash Count N=C, A or G C*= modified C 27

28. Partial alkali hydrolysis and RNase T1 digestion of tRNA 2 Ile of H. marismortui G31 G30 T1 Alkali C*34 A positively charged modification and/or High Molecular weight addition to C34 28

29. In vivo In vivo and In vitro aminoacylation pattern of Wt and mutant tRNA The mutant tRNA is poorly aminoacylated --- one possible reason why UAU anticodon is not used instead of C*AU. The mutant is, however, aminoacylated by E. coli IleRS 29

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31. Wt Mut (X) Analysis of the U34 base of mutant tRNA by partial alkali digestion and radiolabeling B C Gel purification of labeled fragments having X34 ( agmatidine for wt) or U34 (for mutant) base at the 5’-end Digestion with nuclease P1 to mono-nucleosides and analysis by TLC 31

32. U34(mutant) 1D 2D C*34(wt) Analysis of 32 P-labeled base 34 of wt and mutant tRNA by TLC Wt Mutant pA pC pG pU The U34 Base in mutant tRNA is modified pA pC pG pU 5’3’ C*AU Wt C34 U34 mutation 5’3’ U AU Enzyme? 5’3’ U AU * Mut Ile 32

33. (1) 165 Da--- loss of 59 Da ( loss of CH 5 N 3 –Guanido group) 17 Da (NH 3 ) 59 Da (CH 5 N 3 ) Collision induced dissociation (CID) of base ion (MW 224.0) (2) 207 Da— loss of 17 Da ( loss of NH 3 ) 33

34. C4N3H40C4N3H40 modification Loss of C2- Oxygen Addition of 5C, 4N C 9 H 18 N 7 + H ATP Enzyme (?) + H2OH2O (MW 131.0) Identification of modified base at position C34 decarboxylation Agmatidine (agmatine modified cytidine) 34

35. Agmatine modified Cytidine and Agmatine standard give similar peaks after Collision induced dissociation. CID of agmatine modified cytidine (224 Da) CID of agmatine standard (131 Da) Mass spectral analysis of agmatine modified cytidine and agmatine standard 35

36. C*-- lysidine C*-- agmatidine Agmatidine side chain N does not have free electron pair- positivite charge is delocalized on three N atoms Agmatidine side chain is different from lysidine 36

37. Analysis of the modified U34 by Mass spectrometry 5- cyanometyl uridine 5- cyanometyl deoxy uridine  Mass spec analysis indicates a 39 dalton additional mass  Fragmentation compared with 5-cyanomethyl deoxyuridine gives similar pattern 37

38. 5’3’ C*AU Wt C34 U34 mutation 5’3’ U AU Enzyme? 5’3’ U AU * Mut Ile Cyanomethyl Uridine--- cnm 5 U IleRS Mutation of C34 to U34----- Mutant tRNA gets deaminoacylated----- because if it is aminoacylated it can mistranslate Met instead of Ile during translation 38

39.  Generation of enzymes from extremophiles for biotechnology industry  Design small molecule inhibitors of TilS ( or TiaS) as potential antibacterial target Proposed research plan  Knock-out strain of tils/tias. Cross-talk between Tils & Tias  Replacement of C*AU-tRNA with the UAU-tRNA at the chromosome. 39

40. TilS as an antibacterial target  An essential enzyme and conserved in, almost, all bacteria. No eukaryotic counterpart.  Cytosolic enzyme and therefore easy to target an inhibitor against TilS.  Analogs of Lysine/arginine can be tested for inhibitors.  Blocking the modification makes inactive tRNA, inhibits translation and promotes cell death. Why Tils is a potential antimicrobial target ? 40

41. Inhibition of TilS by small molecule analogues of Lysine/Arginine 5’3’ C A U Tils ATP 5’3’ C A U * IleRS 5’3’ C A U Ile Normal translation and cell growth 5’3’ C A U Tils+ Inhb( ) ATP 5’3’ C A U 5’3’ C A U IleRS 5’3’ C A U Inhibition of translation and cell death IleRS 5’3’ C A U Inhibition of translation and cell death (1) (2) Analysis of tRNA 2 Ile for aminoacylation by northern hybridization 41

42. Archaeal tRNA Ile2 Bacteria 5’3’ C A U * Bacterial tRNA Ile2 Archaea 5’3’ C A U * TilSTiaS C * Lysidine or Agmatidine TiaS TilS  whether archaeal tRNA will be modified by TilS or Bacterial tRNA by TiaS  positive and negative determinant of TiaS/TilS in the tRNA  random mutagenesis of Tils/Tias for incorporation of multiple substrates  Replacement of C*AU-tRNA with the UAU-tRNA at the chromosome Cross-talk of Tils vs Tias ? ? ? 42

43. Biotechnological use of enzymes from extremophiles 43

44. (isolation from natural resources/ recombinant enzymes) Application in biotechnology Extremophiles as a source of novel enzymes 44

45. SEM analysis after 4 hrs of treatment Cells + marketed oil Cells + Vyome oil (VB-001) 45

46. VB-001 causes more damage to the cells compared to marketed oil 46

47. TEM analysis Cells + marketed oil Cells + Vyome oil (VB-001) 47

48. Anti-dandruff shampoo with Zinc pyrithione Current challenges Our strategy Poor drug depositionLipid-coated/ micronized Frequent applicationMore efficacy/ less application High relapse rates Less relapse Itching and dryness Shinny and greasy / less itching ZPT powder Reduced size Lipid- coated/micronized Retention on Goat skin (Franz Diffusion cell)—Ex vivo In vitro time kill (dose- and time- kinetics) Ex vivo time kill with goat skin(dose- and time- kinetics) 48

49. Ex vivo fungal kill with retained zinc pyrithione Goat skin is applied with different shampoo formulations 5 min application followed by rinse-off Skin with retained ZPT is infected with fungus and grown for 24 hrs 49

50. Pig skin culture for fungal infection Pig skin model for skin SD Vyome formulation is more efficacious than marketed formulation 50

51. Aspirations…..  Study gene expression profile for fungus after treatment of different actives  Look for skin biomarkers of fungal infection  Microscopic & histological studies for comparison with human skin  Establish Goat-skin ex-vivo model for multiple product development 51

52. Archaea  Third domain of life---Have similarities to both bacteria and eukaryotes (discovered in 1977)  Archaea like bacteria have no cell nucleus or membrane bound organelles  Archaeal mechanism of transcription and translation is similar to eukaryotes  Many archaeal organisms can survive extreme conditions ( hot springs, salt lakes, glaciers etc) and can use variety of energy sources Good model organism for comparative study of the bacterial and eukaryotic genetics and metabolism 52

53. Thank You 53

54. Zinc Pyrithione Piroctone olamine 54

55. Clinical data VB-001 55

56. In Vitro model for fungal kill study with VB-001 56

57. Modification at position 34 in tRNA 2 Ile is NOT lysidine Analysis of tRNA 2 Ile after reaction with free –NH 2 group reacting biotin probe RNA 2008 57

58. Partial alkali hydrolysis and RNase T1 digestion of tRNA 2 Ile of H. marismortui G31 G30 T1 Alkali C*34 A positively charged modification and/or High Molecular weight addition to C34 58

59. Archaea  Third domain of life---Have similarities to both bacteria and eukaryotes (discovered in 1977)  Archaea like bacteria have no cell nucleus or membrane bound organelles  Archaeal mechanism of transcription and translation is similar to eukaryotes  Many archaeal organisms can survive extreme conditions ( hot springs, salt lakes, glaciers etc) and can use variety of energy sources Good model organism for comparative study of the bacterial and eukaryotic genetics and metabolism 59

60. Archaea Crenarchaeaota: most thermophilic and psychrophilic archaea are found in this group. Euryarcheota: methanogens, halophiles, thermophiles. Korarcheota: found in hot springs. None have been grown in pure culture. 60

61. Euryarcheota Found in dead sea Grows at 4.2-4.5 M NaCl Genomic database available from 2004 Extremely halophilic microorganisms makes the Great Salt Lake into a deep red color that is visible from space Microbe 2006 Our model organism--- Haloarcula Marismortui 61

62. Major Ile codons--- AUC, AUU ( decoded by tRNA 1 Ile ) Rare Ile codon------ AUA No tRNA annotated for codon AUA in H. marismortui G A U C U A 5’3’ 5’ U U A 5’3’ tRNA 1 Ile 5’3’ N A U A U A 5’3’ tRNA 2 Ile ? What is the mechanism for reading the Ile codon AUA in archaea ? Codon Usage table 62

63. 5’3’ N A U A U A 5’3’  No TilS or homologue is found ( lysidine modification like bacteria absent)  No Inosine-34 containing tRNA  a new tRNA or a tRNA (from the known tRNA database) with unknown modification is there to decode AUA codon  Two elongator methionine tRNA with CAU anticodon found ( similar to bacteria where one tRNA is converted to Ile tRNA by wobble modification)  One of the elongator methionine tRNA could be a potential Ile tRNA N = not lysidine or inosine Which tRNA reads AUA codon in H. marismortui ? 63

64. Anti-dandruff shampoo with Zinc pyrithione Current challenges Our strategy Poor drug depositionLipid-coated/ micronized Frequent applicationMore efficacy/ less application High relapse rates Less relapse Itching and dryness Shinny and greasy / less itching ZPT powder Reduced size Lipid- coated/micronized Retention on Goat skin (Franz Diffusion cell)—Ex vivo In vitro time kill (dose- and time- kinetics) Ex vivo time kill with goat skin(dose- and time- kinetics) 64

65. (SH1) (SH2) (VSH1), (VSH2) (VSH2) (SH1) (SH2) (VSH1) Time kill and Growth kinetics--- Dynamic Model Dilution+Growth Irreversible damage caused by excipients/stabilizers ---- Growth kinetics different ---- in vitro model to show the relapse 65

66. Franz diffusion cell---- for skin retention Goat skin is applied with different shampoo formulations 5 min application followed by rinse-off 20% better retention than marketed shampoo 66

67. Ex vivo fungal kill with retained zinc pyrithione Goat skin is applied with different shampoo formulations 5 min application followed by rinse-off Skin with retained ZPT is infected with fungus and grown for 24 hrs 20% extra retention does not provide significant difference in efficacy 67

68. Causative agent(s): Malassezia species Trichophyton species Candida albicans Treatment options (topical): Nizral (2% Ketoconazole cream) Johnson & Johnson, Xolegel (2% Ketoconazole gel) Aqua Pharma Current challenges in topical therapy: Long treatment duration Recurrence of infection Hypothesisof Vyome formulation Higher penetration & retention due to nano-size Cumulative accumulation of active in epidermis leads to less recurrence Application for less no of days Causative agent(s): Malassezia species Trichophyton species Candida albicans Treatment options (topical): Nizral (2% Ketoconazole cream) Johnson & Johnson, Xolegel (2% Ketoconazole gel) Aqua Pharma Current challenges in topical therapy: Long treatment duration Recurrence of infection Hypothesisof Vyome formulation Higher penetration & retention due to nano-size Cumulative accumulation of active in epidermis leads to less recurrence Application for less no of days It is an inflammatory skin disorder presents with scaly, flaky, itchy and red skin Despite multiple treatment options continues to remain a challenge to practicing clinicians and dermatologists It is an inflammatory skin disorder presents with scaly, flaky, itchy and red skin Despite multiple treatment options continues to remain a challenge to practicing clinicians and dermatologists General Introduction on Topical fungal infection, current therapeutics & challenges Infected Skin Vyome therapeutics Standard topical agents Improved formulation development 68

69. Pig skin culture for fungal infection Pig skin model for skin SD Vyome formulation is more efficacious than marketed formulation 69

70. Aspirations…..  Animal model for fungal infection----if, ex vivo why not in vivo ?  Study gene expression profile for fungus after treatment of different actives  Look for skin biomarkers of fungal infection  Microscopic & histological studies 70

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72. Thank You 72

73. Ph. D.--------- 1999-2005 Human RBCs ---Identification of active cell death machinery Pro-caspase-3 Caspase-3 Oxidative stress - Inhibitor + Inhibitor FEBS Letters --2002 73

74. Identification of Caspase-3 target in Human RBC Band-3 an anion exchanger protein (Cl-/HCO3- exchange) is found to be target, In vitro Cleavage sites are found to be critical for membrane integrity Apoptosis induced in HEK-293 cells expressing Band-3 – Cleavage, in vivo Physiological relevance? Aged RBCs are more apoptotic Young Old Aged RBCs have more ROS, cleaved Caspase-3 and therefore fragmented Band-3 JBC-- 2003 74

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