Experimental Work Investigating the Role of Nucleic Acids (AQA A-Level Biology): Revision Notes

Experimental Work Investigating the Role of Nucleic Acids

Understanding how scientists determined which codons code for specific amino acids represents a landmark achievement in molecular biology. The experimental work by Nirenberg and other researchers in the 1960s provided the foundation for deciphering the entire genetic code.

Nirenberg's groundbreaking approach

Scientists faced the challenge of determining how the sequence of bases in DNA and RNA corresponds to the sequence of amino acids in proteins. Nirenberg developed an innovative experimental strategy using synthetic mRNA molecules to investigate this relationship directly.

The experimental design involved creating artificial mRNA chains composed of repeating nucleotides, then observing which amino acids became incorporated into the resulting polypeptides. This approach bypassed the complexity of natural gene expression and allowed researchers to test specific codon-amino acid relationships.

Experimental methodology

Cell-free system preparation

Researchers obtained cell extracts containing all the necessary molecular machinery for protein synthesis, including ribosomes, tRNAs, and enzymes. The extracts were treated with DNase to remove genomic DNA, ensuring that only the added synthetic mRNA would direct protein synthesis.

Radioactive labelling technique

The key innovation involved using radioactive amino acids to track which amino acids became incorporated into newly synthesised polypeptides. In the crucial experiment investigating phenylalanine incorporation:

• One amino acid (phenylalanine) was labelled with radioactive carbon-14
• The remaining 19 amino acids contained normal carbon-12
• After incubation, researchers measured the radioactivity levels in the extracted polypeptides

Testing different synthetic mRNAs

Four different experimental conditions were tested using synthetic mRNA molecules:

• Poly A - mRNA containing only adenine nucleotides
• Poly U - mRNA containing only uracil nucleotides
• Poly C - mRNA containing only cytosine nucleotides
• Control - no synthetic mRNA added

Results and interpretation

The experimental results provided clear evidence for specific codon-amino acid relationships:

The low background counts in other conditions (under 100) demonstrated the specificity of the codon-amino acid relationship and the effectiveness of the experimental design.

Extending the genetic code analysis

Khorana's contributions

Building upon Nirenberg's initial findings, scientist Khorana developed techniques to create longer synthetic mRNA molecules with repeating sequences of multiple nucleotides. For example, sequences like GUGUGUGUGUG allowed researchers to determine codons for additional amino acids by analysing the alternating amino acid patterns in the resulting polypeptides.

Completing the genetic code

Through systematic variation of synthetic mRNA sequences, researchers eventually deciphered all 64 possible triplet codons. This comprehensive analysis revealed that 61 codons specify amino acids, while 3 serve as stop codons to terminate translation.

Properties of the genetic code

Degeneracy but not ambiguity

The experimental work revealed that the genetic code is degenerate - multiple different codons can specify the same amino acid. For instance, phenylalanine is coded by both UUU and UUC. However, the code is not ambiguous - each codon specifies only one amino acid (or stop signal).

This degeneracy provides protection against mutations, as changes in the third codon position often do not alter the amino acid incorporated, particularly for hydrophobic amino acids.

Universal nature

The codon assignments determined through these experiments proved to be nearly universal across all forms of life, demonstrating the fundamental importance of the genetic code in biological systems.

Links to protein synthesis

These experiments directly connect to the process of translation, where ribosomes read mRNA codons and incorporate the corresponding amino acids carried by tRNA molecules. The codon-amino acid relationships established through this work form the basis for understanding how genetic information becomes expressed as functional proteins.