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Crick/Clark/Student Petition

Crick/Clark/Student Petition. Evolution of Genetic Coding. Pieczenik- Theory of Genotypic Selection Coding Constraints Palindromes Internal Terminators Base pairing- Nussinov,Pieczenik,Griggs,Kleitman Algoritm GU base pairing. Evolution of Genetic Coding.

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Crick/Clark/Student Petition

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  1. Crick/Clark/Student Petition

  2. Evolution of Genetic Coding • Pieczenik- Theory of Genotypic Selection • Coding Constraints • Palindromes • Internal Terminators • Base pairing- Nussinov,Pieczenik,Griggs,Kleitman Algoritm • GU base pairing

  3. Evolution of Genetic Coding • Crick, Brenner, Klug, Pieczenik Model of tRNA-mRNA interaction and Evolution of the code. • Pieczenik Hypothesis of Combinatorial RNA Ligation • Mechanism for evolution of mRNA sequences

  4. Selective Constraints on Combinatorial Possibilities • All combinations are made a priori • Selection under constraints are made a posteriori • Selection can be for physical chemical constraints and/or for informational coding constraints.

  5. Problems • 1) Are Nucleotide Sequences Random or are there Rules of Harmony? • 2) What are the Constraints on Nucleotide and Protein Sequences? • 3) What are the Constraints on mRNA imposed by Ribosome Binding Sites? • 4) What are the Constraints on mRNA imposed by translation by tRNA? • 5) What are the Constraints on mRNA imposed by miRNA combinatorial ligation? • 6) What are the Constraints imposed on antibody-antigen interactions and their codings? • 7) What are the Constraints imposed on the lipid combinatorial?

  6. Constraint on mRNA imposed by translation by tRNA • Common Uracil 5’ to the anti-codon and Pu 3 ‘ to anti-codon creates • PuNPy constraint on mRNA if there is a flip of anti-codon in translation. • PuNPy, PuNPy,PuNPy

  7. tRNA Competing for Translation

  8. Combinatorial Constraint Not Imposed on mRNA • The inverse non existent G 5’ to the anti-codon and U 3’ to the anti-codon • Creates PyNPu constraint on mRNA, if there is a flip of anti-codon in translation • PyNPu,PyNPu,PyNPu

  9. Combinatorial Sequence Constraints on Ribosome Binding Sites • First DNA Sequence- ΦX 174 Gene G Ribosome Binding Site • ATG.TTTCAGACTTT- Palindrome Mirror Image

  10. Two Combinatorial mRNA Sequence Constraints on RBS • Gene V- f1 bacteriophage –RBS • Palindrome • Internal Terminator • Combined Into One Sequence • fMet.Ile.Lys.Val.Glu.Ile.Lys

  11. Combinatorial RNA Ligation- Problem • How does one code 1-2 million proteins with only 19.000-30,000 coding sequences?

  12. Combinatorial RNA Ligation- Background • miRNA are 22 base RNA strands cleaved from hairpin structures • miRNA are known to suppress translation of mammalian mRNA • miRNA catalyze the cleavage of plant mRNA. • Cleavage reactions are reversible as ligation reactions

  13. miRNA are conserved across species • miRNA can base pair with mRNA forming double helix • 22 bp is exactly 2 full turns of the A form RNA helix- Rosalind Franklin/Hugh Robertson. • Around 321 miRNA exist in most organisms

  14. miRNA as adaptors and ligase • Most miRNA have a splice site, AG / GU, directly in the middle of sequence • Rnase III, discovered by Hugh Robertson, and Dicer are enzymes that cleave the A-form RNA helix

  15. Hypothesis • miRNA catalyzes the combinatorial ligation of 2 independent mRNA • This creates a completely new coding sequence which means a novel protein

  16. Each of the mRNA will contain one 11 bp seq. complementary to the halves of the miRNA • This creates an RNA triplex where half of the miRNA is hybridized with the 11 bp complement in each mRNA • The miRNA is thought to bring the 2 mRNA and 2 H2O into close contact

  17. Because the mRNA must be complementary to the miRNA sequence the ligation is sequence specific. GU base pairing is allowed. • Now any coding can be paired with any other coding to give an entirely novel sequence • This allows for (20k)^2 / 321= 1.25 mil. proteins

  18. Negative Selection vs Positive Selection for miRNA • Sequences that contain the entire complement to the miRNA compete with other mRNA to form a helix • These sequences are negatively selected against because the helix will prevent translation of the sequence and Dicer recognizes the helix and destroys it-Silencing

  19. Sequence Search • BLAST is a program that compares a query to known sequences • Difficulty in using BLAST because on requires combinatorial matches of GU base pairing in addition to GC base pairing. BLAST is not suited for this type of search.

  20. Short Protein-Protein BLAST Searches • However, protein-protein matches eliminate this problem initially. • What is found is that sequences that are coded by antiparallel complements of miRNA, which is what would be created in the new ligated message, do appear much more frequently than once in the protein data base of known protein sequences

  21. The combinations are then translated For example, miRNA let-7 (6) in the 5′ to 3′ has one open reading frame and three open reading frames in its antiparallel complement in the 5′ to 3′ direction. The antiparallel complement would correspond to coding sequences which would appear in mRNA which are ligated with the mechanism postulated. into protein sequence in all six frames

  22. Three phases of the antiparallel complement of let-7 are (i) Asn.Tyr.Thr.Thr.Tyr.Tyr.Leu; (ii) Thr. Ile.Gln.Pro.Thr.Thr.Ser; and (iii) Leu.Tyr.Asn. Leu.Leu.Pro.His/Gln These sequences are found in several proteins e.g. splicing factor U2Af, bromodomain-containing protein (stimulates transcription activity), testis-determining factor, coiled-coil domain-containing protein 3 precursor, DNA-directed RNA polymerase I subunit 2, inter alia.in-containing protein (stimulates transcription activity), testis-determining factor, coiled-coil domain-containing protein 3 precursor, DNA-directed RNA polymerase I subunit 2, inter alia. Peptide Sequences Coded by Complement of miRNAs

  23. Experimental Tests • Create triplex RNAs with miRNA sequences and complementary mRNA sequences and demonstrate ligation of the two mRNA sequences, generating a ligated mRNA and the miRNA unreacted. • Develop an miRNA dependant mRNA ligating system and translation system.

  24. Michaelis-Menton Equation for miRNA Ligation • Vo = Vmax [mRNA1+mRNA2] Kmi + [mRNA1+mRNA2] • Kmi=MM constant for miRNA ligation

  25. 4-5 amino acids define monoclonal antibody binding specificity = 160,000-3.2 million 20^4.66=1.15million 20^5=3.2 million Combinatorial of V, J, D Antibody segments = 2-3 million Symmetry of Antibody=Antigen Universes

  26. Combinatorial Lipids • Tiacly Glycerides • N^3/2 + N^2/2 = unique stereoisomers • N = number of saturated and unsaturated fatty acid chains • Selection for 37 degree fluidity creates a ratio of 1:1 saturated to unsaturated fatty acid chains

  27. Theory of Genotypic Selection • Direct Genotypic Selection, historical or present, on nucleic acids for replication, transcription, processing and translation. • A Posteriori Imposes Sequence and Amino Acid Constraints on A priori all possible Nucleic Acid Sequences

  28. GU Base Pairing Constraint • tRNA-mRNA interactions- Crick, Brenner, Klug, Pieczenik Model • Hairpin Structures-Nussinov, Pieczenik, Grigg, Kleitman Base Pairing Algorithm-miRNA, IRIS, RNA editing, polio pathogenicity C to U at 472

  29. Polio Pathogenicity

  30. Sequence of Non-Pathogenic Competitive HIV Strain- Donor (A)and Recipient (C)

  31. Infectious Hypovirulence • Non-Pathogenic Donor Strain Evolved from Healthy HIV sero-positive Gabonese, through Mexico unto San Francisco Where it Was Identified in Long Term Asymptomatic HIV positive with Healthy HIV positive Partner. • Frequency 22/10,000= 1/500 • 4 asymptomatics in HIV patient pop of 2,000 • 1 with healthy HIV partner

  32. miRNA mimics • HIV, Hep C, RNA virus may mimic miRNA Combinatorial Ligation Adaptor Functions Creating new combinations and suppressing proper cellular miRNA Combinatorial Ligation Functions • Stable Hairpin and miRNA duplex both 22 nucleotides long on average • Similar Target Region Size of 22 for pathogenicity may either be hairpin or miRNA ligation mimicking site.

  33. Crick/Clark • Crick/Clark Submitted Nature Petition Gets George Pavlakis Out of Prison. • George Pavlakis continues work on HIV vaccine

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