The Genetic Code

General Features of the Genetic Code

The genetic code is the set of rules by which information encoded in mRNA is translated into proteins. It possesses several key features that ensure accurate and efficient translation.

• Triplet Code: Each codon consists of three RNA bases, specifying one amino acid.

• Nonoverlapping: Once the reading frame is set, each base is used in only one codon.

• Continuous/Commaless: Codons are read sequentially without punctuation between them.

• Unambiguous: Each codon specifies only one amino acid or stop signal.

• Degenerate (Redundant): Most amino acids are specified by multiple codons.

• Start and Stop Signals: AUG is typically the start codon; UAA, UAG, and UGA are stop codons.

• Nearly Universal: The code is conserved across most organisms, with rare exceptions (e.g., some mitochondria).

• Directional: mRNA is read 5′→3′; proteins are synthesized from N-terminus to C-terminus.

• Wobble: Flexibility at the third base of the codon allows one tRNA to recognize multiple codons.

Reading Frame

A reading frame is the way ribosomes group mRNA bases into triplets. There are three possible reading frames on a single strand, depending on the starting base. Shifting the frame (frameshift mutation) alters every downstream codon, often resulting in a nonfunctional protein.

• Significance: Correct reading frame is essential for producing the intended protein sequence.

• Example: Inserting or deleting a base can cause a frameshift, leading to premature stop codons.

Deciphering the Genetic Code: Key Experiments

Several landmark experiments established the correspondence between codons and amino acids.

• Nirenberg and Matthaei: Used cell-free translation systems with synthetic RNAs. Poly-U RNA produced only phenylalanine, showing UUU codes for Phe.

• Nirenberg and Leder: Developed the triplet binding assay. Ribosomes, synthetic trinucleotide codons, and labeled aminoacyl-tRNAs were used to assign codons to amino acids.

• Khorana: Synthesized repeating RNAs (e.g., UCUCUC...) to produce predictable repeating peptide patterns, allowing assignment of remaining codons.

Determining Polypeptides from Repeating RNAs

Given a di-, tri-, or tetranucleotide repeat, the polypeptide produced depends on the reading frame. Each frame yields a different repeating codon pattern and peptide sequence.

• Rule: Write the repeating RNA sequence, list possible codons for each reading frame, and deduce the resulting peptide.

• Example: For a repeating dinucleotide (e.g., UC), possible codons are UCU, CUC, UCU, etc., depending on the frame.

Transcription and Translation: Sequence Determination

To determine the complementary DNA strand, mRNA, and polypeptide:

1. Identify if the given DNA strand is template or coding.

2. Complementary DNA is antiparallel; A pairs with T, C with G.

3. mRNA matches the coding strand (except U replaces T) and is written 5′→3′.

4. Find the start codon (AUG) in mRNA, set the reading frame, and translate until a stop codon.

Example: If given a template strand, transcribe to mRNA (complementary, U for T), then translate using the genetic code.

Translation: Structure and Function

tRNA Structure and Function

tRNA acts as an adaptor molecule in translation, linking mRNA codons to their corresponding amino acids.

• Anticodon Loop: Contains three bases that pair with the mRNA codon.

• 3′ CCA Acceptor Stem: Site of amino acid attachment (to the 3′-OH group).

• L-shaped 3D Structure: Positions anticodon and amino acid for ribosome interaction.

• Modified Bases: Enhance stability and recognition accuracy.

Charging tRNAs: Aminoacylation

Uncharged tRNAs are loaded with amino acids by aminoacyl-tRNA synthetases in a two-step process:

1. Amino acid + ATP → aminoacyl-AMP + PPi (activation)

2. Aminoacyl group transferred to tRNA 3′ end → aminoacyl-tRNA (charged tRNA) + AMP

This process is a major accuracy checkpoint, as synthetases recognize both the amino acid and tRNA identity elements.

Wobble Hypothesis

Wobble refers to flexible base-pairing between the third base of the codon and the first base of the tRNA anticodon. Modified bases (e.g., inosine) allow one tRNA to recognize multiple codons, explaining much of the code's degeneracy.

• Example: tRNA with anticodon containing inosine can pair with U, C, or A at the third codon position.

Ribosome Structure and Function

Ribosomes are complexes of rRNA and proteins, consisting of two subunits. rRNA performs much of the catalytic activity, making the ribosome a ribozyme.

• A Site: Entry point for aminoacyl-tRNA.

• P Site: Holds peptidyl-tRNA (growing peptide chain).

• E Site: Exit site for uncharged tRNA.

• Small Subunit: Decodes codon–anticodon pairing.

• Large Subunit: Catalyzes peptide bond formation (peptidyl transferase activity) and provides the exit tunnel.

Translation Mechanism in Prokaryotes

Components Involved in Translation

Translation in prokaryotes involves several molecular components:

• mRNA: Template with Shine-Dalgarno sequence, start codon, and coding region.

• Ribosome: 30S (small) + 50S (large) = 70S; decodes mRNA and catalyzes peptide bonds.

• tRNAs: Bring amino acids; anticodon reads codon.

• Aminoacyl-tRNA Synthetases: Charge tRNAs with correct amino acids.

• Initiation Factors: IF1, IF2 (GTPase), IF3 assemble initiation complex; IF2 brings initiator tRNA.

• Elongation Factors: EF-Tu (GTPase), EF-Ts, EF-G (translocation) deliver tRNAs and move ribosome.

• Release Factors: RF1/RF2 (+ RF3) recognize stop codons and trigger release.

• Energy: ATP for charging tRNA; GTP for initiation, elongation, translocation, and termination.

• Initiator tRNA: Carries fMet (formyl-methionine) in bacteria.

Steps of Translation in Prokaryotes

Translation proceeds through initiation, elongation, termination, and ribosome recycling.

1. Initiation: 30S subunit binds mRNA at Shine-Dalgarno sequence; initiator fMet-tRNA pairs with start codon in P site; 50S joins (GTP hydrolysis).

2. Elongation: Charged tRNA enters A site (EF-Tu + GTP), peptide bond forms (peptidyl transferase), ribosome translocates (EF-G + GTP), empty tRNA exits E site.

3. Termination: Stop codon in A site recruits release factor; hydrolysis releases polypeptide; complex disassembles.

4. Ribosome Recycling: Ribosome components are released and reused.

Comparisons

Translation: Prokaryotes vs. Eukaryotes

Translation differs between prokaryotes and eukaryotes in several ways:

• Initiation: Prokaryotes use Shine-Dalgarno sequence; eukaryotes use 5′ cap-dependent scanning (Kozak sequence).

• Ribosomes: 70S in prokaryotes; 80S in eukaryotes.

• Initiator Amino Acid: fMet in prokaryotes; Met in eukaryotes.

• mRNA Structure: Prokaryotic mRNAs often polycistronic; eukaryotic mRNAs usually monocistronic.

• Compartmentation: Translation is cytosolic in eukaryotes (separate from transcription); coupled in prokaryotes.

Transcription vs. Translation in Prokaryotes

Transcription and translation are distinct processes but can be coupled in prokaryotes.

• Transcription: RNA polymerase synthesizes RNA from DNA.

• Translation: Ribosomes synthesize protein from mRNA.

• Coupling: Translation can begin on mRNA while it is still being transcribed.

• Processing: Prokaryotic mRNA has minimal processing (no nucleus, no splicing).

Key Equations

Aminoacyl-tRNA Synthetase Reaction

The charging of tRNA involves two steps:

• Activation:

• Transfer:

Summary Table: Comparison of Translation in Prokaryotes and Eukaryotes