The Genetic Code & tRNA

Translation Overview

• translation is the mRNA guided synthesis of proteins and performed by ribosomes

• translation occurs after the codons (triplet of nucleotides coding for an amino acid) are read in a successive non-overlapping manner by a tRNA

• among the 3 possible reading frames, typically only one will be used in order to translate a given mRNA to synthesize a polypeptide

  • there’s a signal that indicates where to stop and start translation

Codon

• a triplet of nucleotides on the mRNA coding for an amino acid

• there are 64 codons coding for 20 amino acids and 1 stop signal (proteins are typically composed of 20 a.a’s)

• b/c there are more combinations (64) than coded elements (21), the genetic code is considered to be redundant (degenerate)

• therefore, several amino acids are encoded by multiple codons

• some tRNAs can recognize more than 1 codon

tRNA Structure: 2D

• tRNA are small molecules (70-90 nts)

• their secondary structure is represented by a cloverleaf

• the anticodon arm (bottom) is located opposite to the amino acid arm (top)

• there are several modified bases (e.g. inosine) often present in the Wobble position, dihydrouridine in the D arm and pseudouridine

tRNA Structure: 3D

• the 3D tertiary structure resembles a twisted L w/ the anti-codon and amino acid positions locate in both extremities

tRNA Structure & Processing

• tRNA are transcribed by RNA pol III and are then processed (base modification, cleavage, splicing, 3’ end editing)

• all tRNAs have the same CCA sequence at the 3’ end that is added post-transcriptionally

tRNA Processing: Adenosine Deaminase (ADAT2/3)

• the tRNA adenosine deaminase (ADAT2/3) modifies adenosine bases on some tRNA anti-codon sequences to create inosine (I)

• Inosine can form atypical base pairing with A, C, and U

Anti-codon

• the anti-codon binds to the codon in an anti-parallel manner

  • the first nucleotide of the codon binds to the 3rd nucleotide of the anti-codon

• the first 2 nucleotides of the codon will form strong W-C base pairing w/ the anti-codon

Wobble Base

• the first nucleotide of the anti-codon forms a weaker interaction in the Wobble position w/ the third nucleotide of the codon

• non-conventional base pairing can occur

  • e.g. the inosine minor base in the anti-codon can interact w/ A, U, C in the 3rd position of the codon sequence

  • e.g. U can interact w/ A and G

• this allows a more rapid dissociation of tRNA from the mRNA (a higher affinity would significantly reduce the translation rate)

• it also reduces the number of required tRNA

• while a minimum of 32 tRNAs are required, cells typically express between 40-50 different tRNAs to recognize the 61 diff codons

Wobble Base Figure

The Codon Flexibility: Genetic Code Variations

• whereas the genetic code is considered “universal”, there are exceptions

• the genetic code is slightly different in mitochondria

  • mitochondria contains its own genome that encodes a small number of proteins

  • e.g. the UGA stop codon encodes for a tryptophan (Trp) when translated from the mitochondria

Codon Flexibility: Translational Frameshifts

• in most cases, only one reading frame encodes for a protein

• there are exceptions where a second reading frame can be used (e.g. viruses)

  • in this case, the secondary structure of the mRNA can induce a slipping or skipping of the ribosome (ribosome shifts by 1 nucleotide while translating to read a completely diff set of codons → diff protein)

• advantage: using one mRNA to encode multiple proteins

Codon Flexibility: Translational Frameshifts Example

• in 95% of the cases, the Rous sarcoma Gag protein is normally stopped after the Leu residue

• in 5% of cases, the ribosome will slip -1 nucleotide after the UUA codon to place the AUA codon in aminoacyl site and to translate the Gal-Pol fusion protein, that is later cleaved into two polypeptide

Codon Flexibility: mRNA Editing

• mRN editing can alter the protein sequence → more diversity in the # of proteins that can be generated by a limited number of genes

• editing is mediated by adenosine deaminase that can generate Inosine and cytidine deaminase (C → U), such as APOBEC (apoB mRNA editing catalytic peptide)

• APOBEC1 edits a specific codon at position 2153 to convert a Gln (CAA) to a stop codon (UAA)

  • generates a shorter isoform of the apoB protein

The Codon Usage Bias

• while the same codons encode for the same a.a’s, the codon usage is different from species to spcecies

  • e.g. 22% of Arg codons are AGA in the human genome, while 1% of the Arg codons are AGA in E. coli. Accordingly, there are more tRNA w/ the GCG anti-codon compared to UCU in E. coli

• in many fast-dividing organisms, there is a strong correlation b/w codon usage and protein abundance, as translation of an abundant protein will be favored by the higher concentrations of corresponding tRNAs in the cell

• there’s a correlation between frequency of a given codon and tRNA abundance.

• Genes for which numerous low frequency codons are expressed at lower levels

The Codon Adaptation Index (CAI)

• CAI is a method for analyzing codon usage bias

• CAI measures the deviation of a given protein coding gene sequence w/ respect to the codon usage bias in the genome

• CAI can be used to predict expression level of a protein based on its DNA sequence (especially in fast growing organism like the yeast S. cerivisaie)

• the codons used in highly abundant proteins are more represented in the genome (~high CAI)

• between 0 and 1

Mitochondria

• mitochondrial genome has 22 tRNAs