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Conservation of codon optimality

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Group presentation that contains:
- outlining the basics of translation
- experimental evidence that shows proteins from synonymous mRNA sequences differ
- hypothesis for how synonymous codons effect the resulting protein structure
- the methodology I use to test for the conservation of codon choice within related proteins

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Conservation of codon optimality

  1. 1. Conservation of codon optimality within families Alistair Martin, Charlotte Deane
  2. 2. Renaturation “The original structure of some proteins can be regenerated upon removal of the denaturing agent and restoration of conditions favouring the native state. Proteins subject to this process, called renaturation, include serum albumin from blood, hemoglobin (the oxygen-carrying pigment of red blood cells), and the enzyme ribonuclease” - Encyclopedia Britannica All the information is contained in the protein sequence! Who cares about degeneracy?!
  3. 3. Question - Experimental “Oddities” Synonymous switches have an effect: ● Can cause exons to be skipped ● Can cause a reduction in activity ● Can cause misfolding
  4. 4. Answer - Cotranslational folding
  5. 5. Prior Work “N-terminal regions are generally translated slower than C-terminal regions” - Saunders & Deane (2010 ) “the first 5-10 codons of protein-coding genes are often codons that are less frequently used in the rest of the genome” - Bentele et al. (2013) “cell cycle-regulated genes expressed in different phases display different codon preferences” - Morgenstern et al. (2012)
  6. 6. Conservation of codon optimality within families Alistair Martin, Charlotte Deane
  7. 7. Starting point - CSandS (2010) Mapping of mRNA seq to protein seq ● 4000+ matches ● High quality ● Human curated ● Structural Information ● Taxa Information ● Bad documentation Saunders R, Deane CM, Nucleic Acids Res., 2010, 38(19), 6719-28.
  8. 8. Modifying the database Added ● SCOP Families (SCOP 1.75B) ● tRNA gene copy # (GtRNAdb) ● SCOP family structural alignment (MAMMOTH-Mult) Removed ● Enforce 40% seq id ● NMR experiments ● Minimum of 7 in SCOP family ● Organisms without tRNA data ● Misaligned families SCOP families: 43 Structural Domains: 454
  9. 9. Database Stats
  10. 10. Scoring a SCOP family (1) Protein Sequence pdb-1 (HUMAN) V F T V E V K N Y G pdb-2 (ECALL) V Y N V Y V R - N G pdb-3 (HUMAN) K Y K A E W R A V G pdb-4 (YEAST) - - - - D V P G D R mRNA Sequence pdb-1 (HUMAN) ACU GUU GAA GUC AAA AAC UAC GGA pdb-2 (ECALL) AAU GUA UAU GUU CGA --- AAC GGA pdb-3 (HUMAN) AAG GCC GAG UGG CGU GCU GUG GGC pdb-4 (YEAST) --- --- GAU GUG CCA UGU GAC AGG Structural alignment produced by MAMMOTH-mult on SCOP family domain fragments Known mRNA sequence mapped onto alignment Mapping mRNA One to one matching of codons to amino acids. 100% coverage by mRNA sequence Codon > amino acid if any difference
  11. 11. Scoring a SCOP family (2) mRNA Sequence pdb-1 (HUMAN) ACU GUU GAA GUC AAA AAC UAC GGA pdb-2 (ECALL) AAU GUA UAU GUU CGA --- AAC GGA pdb-3 (HUMAN) AAG GCC GAG UGG CGU GCU GUG GGC pdb-4 (YEAST) --- --- GAU GUG CCA UGU GAC AGG Translation Scores pdb-1 (HUMAN) 0.3 0.9 0.1 0.6 0.4 0.1 0.8 0.6 pdb-2 (ECALL) 0.5 0.8 0.4 0.9 0.5 --- 0.6 0.5 pdb-3 (HUMAN) 0.6 0.6 0.1 0.6 0.9 0.2 0.1 0.1 pdb-4 (YEAST) --- --- 0.2 0.7 0.4 0.1 0.7 0.5 Organism specific translation speed scores given to each codon. Profile is then smoothed. Translation Speed Scores Using the tRNA Adaptation Index (tAI). This is determined by : - tRNA gene copy number - Simple Crick’s wobble pairing Other scoring systems exist.
  12. 12. Scoring a SCOP family (3) Optimality Thresholds Determined using the organism specific open reading frames within database. Manually specified thresholds. Issues with organisms present in low frequency. Translation Scores pdb-1 (HUMAN) 0.3 0.9 0.1 0.6 0.4 0.1 0.8 0.6 pdb-2 (ECALL) 0.5 0.8 0.4 0.9 0.5 --- 0.6 0.5 pdb-3 (HUMAN) 0.6 0.6 0.1 0.6 0.9 0.2 0.1 0.1 pdb-4 (YEAST) --- --- 0.2 0.7 0.4 0.1 0.7 0.5 Optimality Scores pdb-1 (HUMAN) 0 +1 -1 0 0 -1 +1 0 pdb-2 (ECALL) 0 +1 0 +1 0 -- 0 0 pdb-3 (HUMAN) 0 0 -1 0 +1 -1 -1 -1 pdb-4 (YEAST) -- -- -1 0 0 -1 0 0 Organism specific thresholds determine which codons are optimal (+1) , nonoptimal (-1), or neither (0).
  13. 13. Scoring a SCOP family (4) Conservation Scores Simple codon-wise average of optimality scores. Must have at least 5 codons in an aligned column. Randomisation of optimality scores produces SCOP family specific specified thresholds (5%). Optimality Scores pdb-1 (HUMAN) 0 +1 -1 0 0 -1 +1 0 pdb-2 (ECALL) 0 +1 0 +1 0 -- 0 0 pdb-3 (HUMAN) 0 0 -1 0 +1 -1 -1 -1 pdb-4 (YEAST) -- -- -1 0 0 -1 0 0 Conservation Scores SCOP family specific thresholds determine optimal (red) and nonoptimal (blue) conserved codons.
  14. 14. Scoring a fold family - Summary Structural Alignment Conserved Codons 1. Map mRNA Seq. 2. Attribute translation speed scores to each Codon. 3. Assign optimal, non- optimal or neither to each codon. 4. Determine conservation scores for each column.
  15. 15. Scoring a fold family - Result
  16. 16. Is there any conservation? How many SCOP families have more conserved residues than expected by chance? Optimality Assignment Thresholds
  17. 17. Looking forward ● Remove signal from conserved residues ● Correlation to structural features ● Update the CSandS database ● Investigate the ribosome tunnel ● Subgroup analysis - renaturation, chaperone
  18. 18. Questions?

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