1. Jena Institute of Molecular Biotechnology Swetlana Nikolajewa, Andreas Beyer, Maik Friedel, Jens Hollunder, Thomas Wilhelm Institute of Molecular Biotechnology, Jena Germany

2. Overview: Purine-Pyrimidine Patterns Part 1 New Classification Scheme of the Genetic code Part 2 Type II Restriction Enzyme Binding Sites

3. Overview: Genetic Code Part 1. The purine-pyrimidine scheme of the genetic codes shows amino-acids patterns and regularities of codons symmetry characteristics possible predecessors of our contemporary quaternary triplet code explanation for the number (22) of tRNA genes in mammalian mitochondrial genome

4. TC PuRines vs. PYrimidines A G

5. Purine pairs with Pyrimidine 3 H Bonds 2 H Bonds

6. 3 nucleobases (triplets) of A, G, C, U code for 20 AAs 64 possible codons (4x4x4=4 3 )  3 termination codons: UGA, UAG, UAA  Met (AUG) codon is also the start codon 2nd base UCAG 1st base U UUU Phe UUC Phe UUA Leu UUG Leu UCU Ser UCC Ser UCA Ser UCG Ser UAU Tyr UAC Tyr UAA Stop UAG Stop UGU Cys UGC Cys UGA Stop UGG Trp UCAGUCAG 3rd base C CUU Leu CUC Leu CUA Leu CUG Leu CCU Pro CCC Pro CCA Pro CCG Pro CAU His CAC His CAA Gln CAG Gln CGU Arg CGC Arg CGA Arg CGG Arg UCAGUCAG A AUU Ile AUC Ile AUA Ile AUG Met ACU Thr ACC Thr ACA Thr ACG Thr AAU Asn AAC Asn AAA Lys AAG Lys AGU Ser AGC Ser AGA Arg AGG Arg UCAGUCAG G GUU Val GUC Val GUA Val GUG Val GCU Ala GCC Ala GCA Ala GCG Ala GAU Asp GAC Asp GAA Glu GAG Glu GGU Gly GGC Gly GGA Gly GGG Gly UCAGUCAG The Common Genetic Code Table The Common Genetic Code Table contains 64 fields…

7. Purine-Pyrimidine Classification Scheme of the Genetic Code binary representation of nucleobases purines : A, G → 1 pyrimidines: C, U → 0 C G binds via 3 hydrogen bonds in the complementary base pairing A U binds via 2 hydrogen bonds in the complementary base pairing 2 3 = 8 different binary triplets 000, 001, …,111 each of these has again 8 possibilities, for instance:  000 stands for three pyrimidines: CCC, CCU, UUC, …, UUU  111 stands for three purines: GGG, GGA, GAA, …, AAA

8. Codon Strong codons 6 H bonds Mixed codons 5 H bonds Mixed codons 5 H bonds Weak codons 4 H bonds Pro CC (A/G) Proline Ala GC (C/U) Alanine Ala GC (A/G) Alanine Leu CU (A/G) Leucine Thr AC (C/U) Threonine Thr AC (A/G) Threonine Ser UC (C/U) Serine Val GU (C/U) Valine Val GU (A/G) Valine Phe UU (C/U) Phenylalanine Ile AU (C/U) Isoleucine Ile/Met AU (A/G) Isoleucine/Methionine 000 001 100 101101 Arg CG (C/U) Arginine Cys UG (C/U) Cystein His CA (C/U) Histidine Tyr UA (C/U) Tyrosine 010010 Arg CG (A/G) Arginine Stop/Trp UG (A/G) Tryptophan Gln CA (A/G) Glutamine Stop UA (A/G) 011 Gly GG (C/U) Glycine Asp G A (C/U) Asparatic acid Asn AA (C/U) Asparagine 110 Gly GG (A/G) Glycine Glu G A (A/G) Glutamatic acid 111 Leu CU (C/U) Leucine Leu UU (A/G) Leucine Ser UC (A/G) Serine Ser AG (C/U) Serine Arg AG (A/G) Arginine Pro CC (C/U) Proline Lys AA (A/G) Lysine Purine-Pyrimidine Table of the Genetic Code …the new scheme contains the same information in only 32 fields.

9. Codon Strong 6 hydrogen bonds Mixed 5 hydrogen bonds Mixed 5 hydrogen bonds Weak 4 hydrogen bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Ser UC (C/U) Val GU (C/U) Val GU (A/G) Phe UU (C/U) Ile AU (C/U) Ile/Met AU (A/G) 000 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop/Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asparatic acid Asn AA (C/U) Asparagine 110 Gly GG (A/G) Glu GA (A/G) Glutamatic acid 111 Leu CU (C/U) Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) Arg AG (A/G) Pro CC (C/U) Lys AA (A/G) Lysine Amino Acid Patterns: Polar Requirement of NCN and NUN Codons C. R. Woese, G. J. Olsen, M. Ibba, D. Söll Aminoacyl-tRNA Synthetases, the Genetic Code, and the Evolutionary Process. MMBR 2000(64) 202-236

10. Codon Strong 6 H-bonds Mixed 5 H-bonds Mixed 5 H-bonds Weak 4 H- bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Ser UC (C/U) Val GU (C/U) Val GU (A/G) Phe UU (C/U) Ile AU (C/U) Ile/Met AU (A/G) 000 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop/Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu CU (C/U) Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) Arg AG (A/G) Pro CC (C/U) Lys AA (A/G) Kyte&Doolittle, 1982, http://biology-pages.infohttp://biology-pages.info Amino Acid Patterns: Hydrophobicity

11. Codon Strong 6 H-bonds Mixed 5 H-bonds Mixed 5 H-bonds Weak 4 H-bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Val GU (C/U) Val GU (A/G) Ile AU (C/U) Ile/Met AU (A/G) 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop/Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) Arg AG (A/G) Lys AA (A/G) Codon-Anticodon Symmetry Ser UC (C/U) Phe UU (C/U) 000 Leu CU (C/U) Pro CC (C/U)

12. Codon Strong 6 H-bonds Mixed 5 H- bonds Mixed 5 H-bonds Weak 4 H-bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Ser UC (C/U) Val GU (C/U) Val GU (A/G) Phe UU (C/U) Ile AU (C/U) Ile/Met AU (A/G) 000 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop/Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu CU (C/U) Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) Arg AG (A/G) Pro CC (C/U) Lys AA (A/G) Point Symmetry D. Halitsky Extending the (Hexa-)Rhombic Dodecahedral Model of the Genetic Code: the Code's Four 6-fold Degeneracies and the Ten Orthogonal Projections of the 5-cube as 3-cube. Computer Systems Technology 2004

13. Codon Strong 6 H-bonds Mixed 5 H- bonds Mixed 5 H-bonds Weak 4 H-bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Ser UC (C/U) Val GU (C/U) Val GU (A/G) Phe UU (C/U) Ile AU (C/U) Ile/Met AU (A/G) 000 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop/Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu CU (C/U) Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) Arg AG (A/G) Pro CC (C/U) Lys AA (A/G) Codon-Reverse Codon (XYZ↔ZYX) Symmetry

14. AUC CUA GAU Asp UAG STOP Asp Codon-Reverse Codon (XYZ↔ZYX) Symmetry Stop AUC

15. Evolution of the Genetic Code binary doublet: 4 1 =4 fields 00 0101010101010101 1010101010101010 11 00* 01*01*01*01*01*01*01*01* 10*10*10*10*10*10*10*10* 11* quaternary doublet code: 4 2 =16 fields our contemporary code is the quaternary triplet code: 4 3 =64 fields 00 0101 1010 11 CGU, UAC,…

16. Codon Strong 6 H bonds Mixed 5 H bonds Mixed 5 H bonds Weak 4 H bonds Pro CC (A/G) Proline Ala GC (C/U) Alanine Ala GC (A/G) Alanine Leu CU (A/G) Leucine Thr AC (C/U) Threonine Thr AC (A/G) Threonine Ser UC (C/U) Serine Val GU (C/U) Valine Val GU (A/G) Valine Phe UU (C/U) Phenylalanine Ile AU (C/U) Isoleucine Ile/Met AU (A/G) Isoleucine/Methionine 000 001 100 101101 Arg CG (C/U) Arginine Cys UG (C/U) Cystein His CA (C/U) Histidine Tyr UA (C/U) Tyrosine 010010 Arg CG (A/G) Arginine Stop/Trp UG (A/G) Tryptophan Gln CA (A/G) Glutamine Stop UA (A/G) 011 Gly GG (C/U) Glycine Asp GA (C/U) Asparatic acid Asn AA (C/U) Asparagine 110 Gly GG (A/G) Glycine Glu GA (A/G) Glutamatic acid 111 Leu CU (C/U) Leucine Leu UU (A/G) Leucine Ser UC (A/G) Serine Ser AG (C/U) Serine Arg AG (A/G) Arginine Pro CC (C/U) Proline Lys AA (A/G) Lysine Evolution: Scenario 1 00 0101010101010101 1010101010101010 11

17. Codon Strong 6 H bonds Mixed 5 H bonds Mixed 5 H bonds Weak 4 H bonds Pro CC (A/G) Proline Ala GC (C/U) Alanine Ala GC (A/G) Alanine Leu CU (A/G) Leucine Thr AC (C/U) Threonine Thr AC (A/G) Threonine Ser UC (C/U) Serine Val GU (C/U) Valine Val GU (A/G) Valine Phe UU (C/U) Phenylalanine Ile AU (C/U) Isoleucine Ile/Met AU (A/G) Isoleucine/Methionine 000 001 100 101101 Arg CG (C/U) Arginine Cys UG (C/U) Cystein His CA (C/U) Histidine Tyr UA (C/U) Tyrosine 010010 Arg CG (A/G) Arginine Stop/Trp UG (A/G) Tryptophan Gln CA (A/G) Glutamine Stop UA (A/G) 011 Gly GG (C/U) Glycine Asp GA (C/U) Asparatic acid Asn AA (C/U) Asparagine 110 Gly GG (A/G) Glycine Glu GA (A/G) Glutamatic acid 111 Leu CU (C/U) Leucine Leu UU (A/G) Leucine Ser UC (A/G) Serine Ser AG (C/U) Serine Arg AG (A/G) Arginine Pro CC (C/U) Proline Lys AA (A/G) Lysine Evolution: Scenario 2 00 0101010101010101 1010101010101010 11

18. Codon Strong 6 H-bonds Mixed 5 H-bonds Mixed 5 H-bonds Weak 4 H-bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) 1/2 Thr AC (C/U) Thr AC (A/G) Val GU (C/U) Val GU (A/G) Ile AU (C/U) Ile/Met AU (A/G) 5/0 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Stop /Trp UG (A/G) 9/0 Gln CA (A/G) Stop UA (A/G) 2/4 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu UU (A/G) 1/0 Ser UC (A/G) 1/0 Ser AG (C/U) Arg AG (A/G) 6/6 Lys AA (A/G) 3/0 Deviations from the Standard Code Ser UC (C/U) Phe UU (C/U) 000 Leu CU (C/U) 1/1 Pro CC (C/U) http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi

19. Mitochondrial genomes have several surprising features genetic code of mitochondria only 22 tRNAs are required for mammalian mitochondrial protein synthesis ?

20. Codon Strong 6 H bonds Mixed 5 H bonds Mixed 5 H bonds Weak 4 H bonds Pro CC (A/G) Ala GC (C/U) Ala GC (A/G) Leu CU (A/G) Thr AC (C/U) Thr AC (A/G) Val GU (C/U) Val GU (A/G) Ile AU (C/U) Met/Met AU (A/G) 001 100 101101 Arg CG (C/U) Cys UG (C/U) His CA (C/U) Tyr UA (C/U) 010010 Arg CG (A/G) Trp /Trp UG (A/G) Gln CA (A/G) Stop UA (A/G) 011 Gly GG (C/U) Asp GA (C/U) Asn AA (C/U) 110 Gly GG (A/G) Glu GA (A/G) 111 Leu UU (A/G) Ser UC (A/G) Ser AG (C/U) STOP AG (A/G) Lys AA (A/G) The Mammalian Mitochondrial Genetic Code Ser UC (C/U) Phe UU (C/U) 000 Leu CU (C/U) Pro CC (C/U) http://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi

21. Codon Strong 6 H bonds Mixed 5 H bonds Mixed 5 H bonds Weak 4 H bonds tRNA Ile AU (C/U) tRNA Met AU (A/G) 001 100 101101 tRNA Cys UG (C/U) tRNA His CA (C/U) tRNA Tyr UA (C/U) 010010 tRNA Trp UG (A/G) tRNA Gln CA (A/G) STOP UA (A/G) 011 tRNA Asp GA (C/U) tRNA Asn AA (C/U) 110 tRNA Glu GA (A/G) 111 tRNA Leu 2 UU (A/G) tRNA Ser 2 AG (C/U) STOP AG (A/G) tRNA Lys AA (A/G) The Mammalian Mitochondrial Code 8 tRNAs for family codons + 14 tRNAs for non-family codons = 22 tRNA Ser 1 UC tRNA Phe UU (C/U) 000 tRNA Leu 1 CU tRNA Pro CC tRNA Ala GC tRNA Arg CG tRNA Gly GG tRNA Thr AC tRNA Val GU http://mamit-trna.u-strasbg.fr/2DStructures.html

22. Part 2. Common Patterns in Type II Restriction Enzyme Binding Sites

23. G A A T T C Restriction Enzyme (Endonuclease) Restriction enzymes recognize short specific DNA sequences enable bacteria to destroy foreign DNA are useful tools in biotechnology G A A T T C The most well studied class of REs is type II, which cleave DNA within their recognition sequences Many recognition sequences are palindromic

24. Are REase similar in the binding sites? Restriction Enzyme Source Recognition Sequence Pur (1)–pyr (0) pattern AluI Arthrobacter luteus AG ↓ CT HaeIII Haemophilus aegyptius GG ↓ CC BamHI Bacillus amyloliquefaciens G ↓ GA TCC HindIII Haemophilus influenzae A ↓ AG CTT EcoRI Escherichia coli G ↓ AA TTC 11↓00 1↓11 000 11↓00 1↓11 000 Examples from Kimball‘s Biology Pages

25. How significant is the Pattern RR/YY (11/00)? Frequencies of  dinucleotides  trinucleotides  tetranucleotides coded in three possible coding scheme :  R vs Y (G, A vs C, T)  K vs M (G, T vs C, A)  S vs W (G, C vs A, T) Type II 3726 In the symmetrical set the most significant dinucleotides are RR (or 11) (p-value <10 -63 ) and YY (or 00) (p-value <10 -29 ) In the asymmetric set RRR, YYY and YYYY are even more significant, but RR and YY also stand out. Symmetrical (98%) recognition sequences Asymmetrical (2%) recognition sequences

26. Why is the Motif RR..YY preferred? specific geometrical properties  minimal slide values  strong tilt in the negative direction  positive roll  low stacking energy Figure 1 Example of an interaction between an H-bond donor cluster (resulting from two adjacent purines AA) and an H-bond acceptor. Dinucleotides RR..YY are characterized by: stronger H-bond donor and acceptor clusters

27. Outlook Looking for binary patterns in the genomes Additional information Thank you for your attention ! http://www.imb-jena.de/tsb

28. Codon Strong 6 hydrogen bonds Mixed 5 hydrogen bonds Mixed 5 hydrogen bonds Weak 4 hydrogen bonds Pro CC (A/G) Proline Ala GC (C/U) Alanine Ala GC (A/G) Alanine Leu CU (A/G) Leucine Thr AC (C/U) Threonine Thr AC (A/G) Threonine Ser UC (C/U) Serine Val GU (C/U) Valine Val GU (A/G) Valine Phe UU (C/U) Phenylalanine Ile AU (C/U) Isoleucine Ile/Met AU (A/G) Isoleucine/Methionine 000 001 100 101101 Arg CG (C/U) Arginine Cys UG (C/U) Cystein His CA (C/U) Histidine Tyr UA (C/U) Tyrosine 010010 Arg CG (A/G) Arginine Stop/Trp UG (A/G) Tryptophan Gln CA (A/G) Glutamine Stop UA (A/G) 011 Gly GG (C/U) Glycine Asp GA (C/U) Asparatic acid Asn AA (C/U) Asparagine 110 Gly GG (A/G) Glycine Glu GA (A/G) Glutamatic acid 111 Leu CU (C/U) Leucine Leu UU (A/G) Leucine Ser UC (A/G) Serine Ser AG (C/U) Serine Arg AG (A/G) Arginine Pro CC (C/U) Proline Lys AA (A/G) Lysine Purine-Pyrimidine Scheme of the Genetic Code