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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
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
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