The genetic code connects the sequence of nucleic acids in DNA (RNA) to the sequence of amino acids in proteins. It enables the use of information stored in nucleic acid sequences.
Characteristics:
It consists of triplets.
It is nonoverlapping and read sequentially.
It does not include “punctuation”.
It has 5’-to-3’ directionality.
It is degenerate.
Not all codons specify amino acids; there are three stop codons: UAA, UAG, and UGA.
Many amino acids are represented by more than one codon.
Universality and Exceptions
The genetic code is nearly universal, allowing for the production of insulin in bacteria.
Small differences exist:
Ciliated protozoa: one stop codon (UGA) only.
Mitochondria: four stop codons (works because they have their own tRNAs).
Using the Genetic Code
Genetic code table is used to translate mRNA sequences into amino acid sequences.
Key Players in Translation
Transfer RNAs (tRNAs) and Aminoacyl-tRNA synthetases link mRNA and amino acids.
Aminoacyl-tRNA synthetases add amino acids to tRNAs.
General Properties of tRNAs
tRNAs are single-stranded RNAs approximately 70-90 nucleotides in length.
In three dimensions, tRNAs are L-shaped.
Secondary structure includes five unpaired regions, including the acceptor stem (with 3’ CCA terminus) and the anticodon loop.
Mature tRNAs include many modified bases that facilitate or limit base pairing, such as inosine, methylated cytosine, and dihydrouridine.
tRNA-mRNA Interactions
Each codon on an mRNA base pairs with the complementary anticodon on tRNA.
Direction of translation is 5’-to-3’ along an mRNA.
tRNA nomenclature: tRNA is named based on its anticodon (or amino acid), with the sequence given in 5’-to-3’ direction in the tRNA sequence (e.g., tRNATrp which is equivalent to tRNACCA).
Wobble
There are 61 codons encoding amino acids, but fewer than 61 tRNAs, implying that some tRNAs can read more than one codon.
The phenomenon is known as wobble.
Last two bases of anticodon (first two of codon) pair precisely.
The first base of anticodon sets the number of codons it can read:
C or A: one codon.
U or G: two codons.
I: three codons.
Example: tRNAIGC (encoding alanine) reads GCU, GCC, and GCA.
Preparing tRNAs for Translation
Before tRNAs can be used in translation, they must be connected to the correct amino acid.
This addition needs to be thermodynamically favorable.
The form used in translation is aminoacyl-tRNA (charged tRNA), featuring an ester linkage to the 3’ CCA arms of tRNA, through the 2’ or 3’-OH of ribose.
It is the activated form of an amino acid.
Addition of AAs is catalyzed by aminoacyl-tRNA synthetases, with one synthetase for each AA.
Steps in production of charged tRNAs:
Activation of amino acid.
Transfer of amino acid to tRNA.
Proofreading of newly-added tRNA.
Activating and Adding Amino Acids
Before it can be added to a tRNA, an amino acid must be activated.
The amino acid reacts with ATP to form an aminoacyl adenylate, producing pyrophosphate.
The aminoacyl adenylate (AMP) is then transferred to the appropriate tRNA, with the free energy of transfer is near zero.
Both activation and transfer are catalyzed by aminoacyl-tRNA synthetase.
Net cost of both steps is equivalent of two ATP.
Selecting the Right Amino Acid
Aminoacyl-tRNA synthetases must be highly selective for their amino acids.
The shape of, and specific interactions within, the active site are critical.
Example: Addition of threonine
Challenge is to prevent addition of similar amino acids like valine (lacks additional favorable interactions).
Hydroxyl on threonine interacts with coordinated zinc ion and hydrogen-bonds with an aspartate in the synthetase.
Serine also contains a hydroxyl and its similarity leads to misincorporation in one of 5000 additions. Incorrect addition is dealt with by editing site.
Serine is small enough to enter, but threonine is not.
Synthetases often use double sieve principle: Active side excludes oversized AAs; editing site cleaves undersized AAs.
Selecting the Right tRNA
Aminoacyl-tRNA synthetases must bring together the correct AAs and the correct tRNAs.
Anticodon loop is important, but so are other regions.
Regions with modified bases are often used in recognition.
Synthetase tRNA and Amino Acid Recognition
Multiple sources of error can result in the addition of the incorrect amino acid to a peptide:
Incorrect tRNA-amino acid coupling
Incorrect tRNA-mRNA interactions
Errors must be infrequent enough to produce functional protein.
Actual error rate in peptide synthesis is 10^{-5} to 10^{-4}.
Codon Bias
Degeneracy in the genetic code may confer some advantages, providing a “buffer” against mutations.
Selections among synonymous codons may allow additional translational control, as abundances of different tRNAs are non-uniform.
Differences in translation rates among synonymous codons may be significant.
Altering the Genetic Code
An understanding of the genetic code enables its systematic modification.
Addition of custom amino acids (fluorescence, labels, linkers).
Elimination of synonymous codons to create viral resistance.
Ribosomes
Ribosomes are the complexes that make use of charged tRNAs.
The E. coli ribosome includes two subunits:
50S (large) subunit:
Proteins – 34 total (L1-L34)
RNA – 23S, 5S
30S (small) subunit:
Proteins – 21 total (S1-S21)
RNA – 16S
Ribosomes are ribozymes, with rRNAs accounting for two-thirds of their mass.
Ribosomes are abundant (up to 100,000 copies per cell).
Co-transcriptional Translation (E. coli)
The direction of translation matches the direction of mRNA production (5’-to-3’).
If ribosomes have access to mRNA, it can be translated co-transcriptionally.
Key Concepts
General properties of the genetic code
What are the most important features of the genetic code?
What is degeneracy and what purpose might it serve?
What general features do all tRNAs have in common?
What steps are necessary for the charging of a tRNA with an amino acids?
What part of a tRNA interacts with mRNA?
tRNAs
Is the genetic code universal?
Aminoacyl-tRNA synthetases and charging tRNA
What could happen if a mitochondrial protein were translated in the cytosol (or vice versa)?
Understand conventions for writing the sequence of tRNA anticodons and mRNA codons
What is wobble? What bases (at what position in the anticodon) make it possible?
What is the energetic cost of activating an amino acid?
What is the role of pyrophosphate in the charging process?
How do synthetases recognize the correct amino acid?
How do synthetases recognize the correct tRNA?
What is the synthetase editing site and how does it work collaboratively with the active site?
Understand why co-transcriptional translation can occur in bacteria
Be able to use the genetic code to translate mRNA sequences