Crick/Clark/Student Petition

Evolution of Genetic Coding

Pieczenik- Theory of Genotypic SelectionCoding ConstraintsPalindromesInternal TerminatorsBase pairing- Nussinov,Pieczenik,Griggs,Kleitman AlgoritmGU base pairing

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

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.

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?

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

tRNA Competing for Translation

Combinatorial Constraint Not Imposed on mRNAThe 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

Combinatorial Sequence Constraints on Ribosome

Binding Sites

First DNA Sequence- ΦX 174 Gene G Ribosome Binding Site

ATG.TTTCAGACTTT- Palindrome Mirror Image

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

Combinatorial RNA Ligation- Problem

How does one code 1-2 million proteins with only 19.000-30,000 coding sequences?

Combinatorial RNA Ligation- Background

miRNA are 22 base RNA strands cleaved from hairpin structuresmiRNA are known to suppress translation of mammalian mRNAmiRNA catalyze the cleavage of plant mRNA.Cleavage reactions are reversible as ligation reactions

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

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

Hypothesis

miRNA catalyzes the combinatorial ligation of 2 independent mRNA

This creates a completely new coding sequence which means a novel protein

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

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

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

Negative Selection vs Positive Selection for

miRNA

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.

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

Short Protein-Protein BLAST Searches

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

Peptide Sequences Coded by Complement of miRNAs

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.

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.

Experimental Tests

Michaelis-Menton Equation for miRNA Ligation

Vo = Vmax [mRNA1+mRNA2]

Kmi + [mRNA1+mRNA2]

Kmi=MM constant for miRNA ligation

Symmetry of Antibody=Antigen

Universes4-5 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

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

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

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

Polio Pathogenicity

Sequence of Non-Pathogenic Competitive HIV Strain- Donor (A)and

Recipient (C)

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

Infectious Hypovirulence

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

miRNA mimics

Crick/Clark

Crick/Clark Submitted Nature Petition Gets George Pavlakis Out of Prison.

George Pavlakis continues work on HIV vaccine