Conservation of codon optimality within families

Alistair Martin, Charlotte Deane

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?!

Question - Experimental “Oddities”

Synonymous switches have an effect:

● Can cause exons to be skipped

● Can cause a reduction in activity

● Can cause misfolding

Answer - Cotranslational folding

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)

Conservation of codon optimality within families

Alistair Martin, Charlotte Deane

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.

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: 43Structural Domains: 454

Database Stats

Scoring a SCOP family (1)Protein Sequencepdb-1 (HUMAN) V F T V E V K N Y Gpdb-2 (ECALL) V Y N V Y V R - N Gpdb-3 (HUMAN) K Y K A E W R A V Gpdb-4 (YEAST) - - - - D V P G D R

mRNA Sequencepdb-1 (HUMAN) ACU GUU GAA GUC AAA AAC UAC GGApdb-2 (ECALL) AAU GUA UAU GUU CGA --- AAC GGApdb-3 (HUMAN) AAG GCC GAG UGG CGU GCU GUG GGCpdb-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

Scoring a SCOP family (2)mRNA Sequencepdb-1 (HUMAN) ACU GUU GAA GUC AAA AAC UAC GGApdb-2 (ECALL) AAU GUA UAU GUU CGA --- AAC GGApdb-3 (HUMAN) AAG GCC GAG UGG CGU GCU GUG GGCpdb-4 (YEAST) --- --- GAU GUG CCA UGU GAC AGG

Translation Scorespdb-1 (HUMAN) 0.3 0.9 0.1 0.6 0.4 0.1 0.8 0.6pdb-2 (ECALL) 0.5 0.8 0.4 0.9 0.5 --- 0.6 0.5pdb-3 (HUMAN) 0.6 0.6 0.1 0.6 0.9 0.2 0.1 0.1pdb-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.

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 Scorespdb-1 (HUMAN) 0.3 0.9 0.1 0.6 0.4 0.1 0.8 0.6pdb-2 (ECALL) 0.5 0.8 0.4 0.9 0.5 --- 0.6 0.5pdb-3 (HUMAN) 0.6 0.6 0.1 0.6 0.9 0.2 0.1 0.1pdb-4 (YEAST) --- --- 0.2 0.7 0.4 0.1 0.7 0.5

Optimality Scorespdb-1 (HUMAN) 0 +1 -1 0 0 -1 +1 0pdb-2 (ECALL) 0 +1 0 +1 0 -- 0 0pdb-3 (HUMAN) 0 0 -1 0 +1 -1 -1 -1pdb-4 (YEAST) -- -- -1 0 0 -1 0 0

Organism specific thresholds determine which codons are optimal (+1) , nonoptimal (-1), or neither (0).

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 Scorespdb-1 (HUMAN) 0 +1 -1 0 0 -1 +1 0pdb-2 (ECALL) 0 +1 0 +1 0 -- 0 0pdb-3 (HUMAN) 0 0 -1 0 +1 -1 -1 -1pdb-4 (YEAST) -- -- -1 0 0 -1 0 0

Conservation Scores

SCOP family specific thresholds determine optimal (red) and nonoptimal (blue) conserved codons.

Scoring a fold family - Summary

StructuralAlignment

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.

Scoring a fold family - Result

Is there any conservation?

How many SCOP families have more conserved residues than expected by chance?

OptimalityAssignmentThresholds

Looking forward

● Remove signal from conserved residues

● Correlation to structural features

● Update the CSandS database

● Investigate the ribosome tunnel

● Subgroup analysis - renaturation, chaperone

Questions?