EFB325 Cell Physiology

The genetic code

Our studies of the DNA replication have shown how DNA is accurately duplicated and transmitted to daughter cells. Now we will learn how DNA is used as a template for producing proteins, the functional units of metabolism.

Initial experiments describing the relationship between DNA and proteins were based on studying mutations

Beadle and Tatum (mid 50's)

• used UV light to generate mutants of the fungus Neurospora that required a particular nutrient supplement in the medium to grow (for example, one mutant could no longer make its own arginine [an amino acid], so it needed arginine in the media)
• biochemical analysis of the mutants revealed that a single mutation inactivated a single enzyme in the arginine biosynthetic pathway=one gene-one polypeptide hypothesis

sickle-cell anemia

• it was discovered (in the late 50's) that one of the polypeptide subunits of hemoglobin is different in humans with sickle-cell anemia (compared to normal hemoglobin) by only a single amino acid difference [they had techniques to obtain the sequence of proteins since 1953, but sequencing of DNA wasn't developed until 1977]
• this is evidence that DNA determines the sequence of amino acids in proteins

genetic maps of mutations in E. coli

• several different mutations in the same gene of E. coli were mapped, putting them in a specific order on the chromosome
• the proteins produced by the normal gene and by the mutant forms of that gene were sequenced
• the order of mutations on the genetic map was the same order as the amino acid differences in the mutant proteins=colinearity between gene and protein

The genetic code is the deciphering key between nucleic acids and protein

• we know that there are 20 different amino acids in proteins and only 4 different bases in DNA, so how does the DNA sequence encode the information to use a particular amino acid?
• if two bases were used for the code=4x4=16 different arrangements [not enough for 20 amino acids]; if three bases are used for the code=4x4x4=64 different arrangements [more than enough for 20 amino acids]

Crick and Brenner (in 1961)

• studied mutants of a bacteriophage in which the mutations resulted from either an addition or a deletion of a base pair
• by genetically combining two mutants, sometimes they would give back a functional virus (when an addition (+) mutation was combined with a deletion (-) mutation)
• combining two (+) mutations=mutant, combining two (-) mutations=mutant
• BUT combining three (+) or three (-) mutations can result in a functional virus again
• each gene has a proper reading frame, which is thrown off by the addition or subtraction of 1 or 2 base pairs in the gene sequence=frameshift mutations
• addition or deletion of three base pairs restores the proper reading frame of the gene
• ALSO, these results show that the code is non-overlapping (each individual base is a part of only one "word") and degenerate (a particular amino acid can be encoded by more than one three-base sequence=codon)

The genetic code was deciphered by using synthetic RNA with a particular repeated sequence

• Nirenberg and Matthaei approched this by synthesizing an RNA with only U=poly(U); the only codon is UUU
• this RNA was added to a cell extract, which produced a protein which had only phenylalanines; therefore UUU is a codon for phenylalanine
• could also synthesize an RNA as a polymer of UA (=UAUAUAUAU...), which had two codons in the repeating order UAU, AUA; this RNA resulted in synthesis of proteins with tyrosine and isoleucine [don't know which codon for which amino acid though]
• finally, synthesized each codon as a three base-long piece of RNA, then analyzed which amino acid bound to the ribosomes for each sequence
• the genetic code was deciphered by 1966 (in only 5 years) by the work of Nirenberg and Khorana

The genetic code is degenerate and (nearly) universal

• some amino acids are encoded by more than one codon
• three of the codons represent signals to stop translation
• the genetic code is the same for nearly all organisms (genetic code is slightly different in mitochondria, some bacteria, some protozoa)

Back to Cell Phys Syllabus