Nirenberg and Leder experiment
Encyclopedia
The Nirenberg and Leder experiment was a scientific experiment performed in 1964 by Marshall W. Nirenberg and Philip Leder
. The experiment elucidated the triplet nature of the genetic code
and allowed the remaining ambiguities codons in the genetic code
to be deciphered.
In this experiment, using a ribosome binding assay, various combinations of mRNA were passed through a filter which contained ribosome
s. Unique triplets promoted the binding of specific tRNAs to the ribosome. By associating the tRNA with its specific amino acid
, it was possible to determine the triplet mRNA sequence that coded for each amino acid.
discovered that the substance responsible for producing inheritable change in the disease-causing bacteria was neither a protein nor a lipid, rather deoxyribonucleic acid (DNA
). He and his colleagues Colin MacLeod
and Maclyn McCarty
suggested that DNA was responsible for transferring genetic information. Later, Erwin Chargaff
discovered that the makeup of DNA differs from one species to another. These experiments helped pave the way for the discovery of the structure of DNA. In 1953, with the help of Maurice Wilkins
and Rosalind Franklin
’s X-ray crystallography
, James Watson
and Francis Crick
proposed DNA is structured as a double helix.
In the 1960’s, one main DNA mystery scientists needed to figure out was in translation
how many bases would be in each code word, or codon. Scientists knew there was a total of four bases (guanine
, cytosine
, adenine
, and thymine
). They also knew that were 20 known amino acids. George Gamow
suggested that the genetic code was made of three nucleotides per amino acid. He reasoned that because there are 20 amino acids and only four bases, the coding units could not be single (4 combinations) or pairs (only 16 combinations). Rather, he thought triplets (64 possible combinations) were the coding unit of the genetic code. However, he proposed that the triplets were overlapping and non-degenerate.
Seymour Benzer
in the late 1950s had developed an assay using phage mutations which provided the first detailed linearly structured map of a genetic region.
Crick felt he could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code.
In the Crick, Brenner et al. experiment
,using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutation
s and a process called reversions, to add and delete various numbers of nucleotides. When a nuleotide triplet was added or deleted to the DNA sequence the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame. They correctly concluded that the code is degenerate, that triplets are not overlapping, and that each nucleotide sequence is read from a specific starting point.
The New York Times reported on Leder and Nirenberg's discovery by explaining that "the science of biology has reached a new frontier," leading to "a revolution far greater in its potential significance than the atomic or hydrogen bomb." Most of the Scientific community saw these experiments as highly important and beneficial. However, there were some who were concerned with the new are of Molecular Genetics. For example, Arne Wilhelm Kaurin Tiselius, the 1948 Nobel Laureate in Chemistry, asserted that knowledge of the genetic code could "lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions."
Philip Leder
Philip Leder is an American geneticist. He was born in Washington, D.C. and studied at Harvard University, graduating in 1956. In 1960, he graduated from Harvard Medical School....
. The experiment elucidated the triplet nature of the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
and allowed the remaining ambiguities codons in the genetic code
Genetic code
The genetic code is the set of rules by which information encoded in genetic material is translated into proteins by living cells....
to be deciphered.
In this experiment, using a ribosome binding assay, various combinations of mRNA were passed through a filter which contained ribosome
Ribosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
s. Unique triplets promoted the binding of specific tRNAs to the ribosome. By associating the tRNA with its specific amino acid
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
, it was possible to determine the triplet mRNA sequence that coded for each amino acid.
Background
Oswald AveryOswald Avery
Oswald Theodore Avery ForMemRS was a Canadian-born American physician and medical researcher. The major part of his career was spent at the Rockefeller University Hospital in New York City...
discovered that the substance responsible for producing inheritable change in the disease-causing bacteria was neither a protein nor a lipid, rather deoxyribonucleic acid (DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
). He and his colleagues Colin MacLeod
Colin MacLeod
Colin Munro MacLeod was a Canadian-American geneticist.- Biography :Born in Port Hastings, Nova Scotia, Canada MacLeod entered McGill University at the age of 16 , and completed his medical studies by age 23.In his early years as a research scientist, MacLeod, together with Oswald Avery and...
and Maclyn McCarty
Maclyn McCarty
Maclyn McCarty was an American geneticist.Maclyn McCarty, who devoted his life as a physician-scientist to studying infectious disease organisms, was best known for his part in the monumental discovery that DNA, rather than protein, constituted the chemical nature of a gene...
suggested that DNA was responsible for transferring genetic information. Later, Erwin Chargaff
Erwin Chargaff
Erwin Chargaff was an American biochemist who emigrated to the United States during the Nazi era. Through careful experimentation, Chargaff discovered two rules that helped lead to the discovery of the double helix structure of DNA...
discovered that the makeup of DNA differs from one species to another. These experiments helped pave the way for the discovery of the structure of DNA. In 1953, with the help of Maurice Wilkins
Maurice Wilkins
Maurice Hugh Frederick Wilkins CBE FRS was a New Zealand-born English physicist and molecular biologist, and Nobel Laureate whose research contributed to the scientific understanding of phosphorescence, isotope separation, optical microscopy and X-ray diffraction, and to the development of radar...
and Rosalind Franklin
Rosalind Franklin
Rosalind Elsie Franklin was a British biophysicist and X-ray crystallographer who made critical contributions to the understanding of the fine molecular structures of DNA, RNA, viruses, coal and graphite...
’s X-ray crystallography
X-ray crystallography
X-ray crystallography is a method of determining the arrangement of atoms within a crystal, in which a beam of X-rays strikes a crystal and causes the beam of light to spread into many specific directions. From the angles and intensities of these diffracted beams, a crystallographer can produce a...
, James Watson
James D. Watson
James Dewey Watson is an American molecular biologist, geneticist, and zoologist, best known as one of the co-discoverers of the structure of DNA in 1953 with Francis Crick...
and Francis Crick
Francis Crick
Francis Harry Compton Crick OM FRS was an English molecular biologist, biophysicist, and neuroscientist, and most noted for being one of two co-discoverers of the structure of the DNA molecule in 1953, together with James D. Watson...
proposed DNA is structured as a double helix.
In the 1960’s, one main DNA mystery scientists needed to figure out was in translation
Translation (genetics)
In molecular biology and genetics, translation is the third stage of protein biosynthesis . In translation, messenger RNA produced by transcription is decoded by the ribosome to produce a specific amino acid chain, or polypeptide, that will later fold into an active protein...
how many bases would be in each code word, or codon. Scientists knew there was a total of four bases (guanine
Guanine
Guanine is one of the four main nucleobases found in the nucleic acids DNA and RNA, the others being adenine, cytosine, and thymine . In DNA, guanine is paired with cytosine. With the formula C5H5N5O, guanine is a derivative of purine, consisting of a fused pyrimidine-imidazole ring system with...
, cytosine
Cytosine
Cytosine is one of the four main bases found in DNA and RNA, along with adenine, guanine, and thymine . It is a pyrimidine derivative, with a heterocyclic aromatic ring and two substituents attached . The nucleoside of cytosine is cytidine...
, adenine
Adenine
Adenine is a nucleobase with a variety of roles in biochemistry including cellular respiration, in the form of both the energy-rich adenosine triphosphate and the cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide , and protein synthesis, as a chemical component of DNA...
, and thymine
Thymine
Thymine is one of the four nucleobases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine nucleobase. As the name suggests, thymine may be derived by methylation of uracil at...
). They also knew that were 20 known amino acids. George Gamow
George Gamow
George Gamow , born Georgiy Antonovich Gamov , was a Russian-born theoretical physicist and cosmologist. He discovered alpha decay via quantum tunneling and worked on radioactive decay of the atomic nucleus, star formation, stellar nucleosynthesis, Big Bang nucleosynthesis, cosmic microwave...
suggested that the genetic code was made of three nucleotides per amino acid. He reasoned that because there are 20 amino acids and only four bases, the coding units could not be single (4 combinations) or pairs (only 16 combinations). Rather, he thought triplets (64 possible combinations) were the coding unit of the genetic code. However, he proposed that the triplets were overlapping and non-degenerate.
Seymour Benzer
Seymour Benzer
Seymour Benzer was an American physicist, molecular biologist and behavioral geneticist. His career began during the molecular biology revolution of the 1950s, and he eventually rose to prominence in the fields of molecular and behavioral genetics. He led a productive genetics research lab both at...
in the late 1950s had developed an assay using phage mutations which provided the first detailed linearly structured map of a genetic region.
Crick felt he could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code.
In the Crick, Brenner et al. experiment
Crick, Brenner et al. experiment
The Crick, Brenner, Barnett, Watts-Tobin experiment of 1961 was a scientific experiment performed in 1961 by Francis Crick, Sydney Brenner, Leslie Barnett and R.J. Watts-Tobin. They demonstrated that three bases of DNA code for one amino acid in the genetic code...
,using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutation
Frameshift mutation
A frameshift mutation is a genetic mutation caused by indels of a number of nucleotides that is not evenly divisible by three from a DNA sequence...
s and a process called reversions, to add and delete various numbers of nucleotides. When a nuleotide triplet was added or deleted to the DNA sequence the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame. They correctly concluded that the code is degenerate, that triplets are not overlapping, and that each nucleotide sequence is read from a specific starting point.
Experimental work
Nirenberg and Leder could not decode the remaining codons in the same manner as Nirenberg did with Mattaei. Because the mRNA bases were taken up at random by the ribosome, it is hard to determine which specific codon correlates with the amino acid. For example, to pick out the correct codon among non repeating codons (UCU, CUU, UUC) is difficult because they couldn't determine the specific sequence. Instead, Leder and Nirenberg used very short artificial RNA sequences (three nucleotides) in the cell-free systems. These shorter length fragments were long enough to allow the ribosome to bind with the type of tRNA molecule that is complementary to the one codon and still be detectable. The key step of the experiment was that they labeled one type of amino acid at a time and then put the mixture through a Millipore filter. This special filter allowed unbound tRNAs to pass through but did not allow the ribosomes with the bound triplet to pass through. The sample was then tested for radioactivity. If there was radioactivity found in the sample that did not pass through the filter the corresponding Amino acid was added.Reception and legacy
By the Cold Spring Harbor Symposium of 1966, between Nirenberg and Khorana the genetic code was almost completely decoded. Nirenberg was awarded the 1968 Nobel Prize in Physiology or Medicine. He shared the award with Har Gobind Khorana of the University of Wisconsin and Robert W. Holley of the Salk Institute. Working independently, Khorana had mastered the synthesis of nucleic acids, and Holley had discovered the exact chemical structure of transfer-RNA.The New York Times reported on Leder and Nirenberg's discovery by explaining that "the science of biology has reached a new frontier," leading to "a revolution far greater in its potential significance than the atomic or hydrogen bomb." Most of the Scientific community saw these experiments as highly important and beneficial. However, there were some who were concerned with the new are of Molecular Genetics. For example, Arne Wilhelm Kaurin Tiselius, the 1948 Nobel Laureate in Chemistry, asserted that knowledge of the genetic code could "lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions."
External links
- Philip Leder's laboratory notebooks, December 1963 to August 1965, can be found at The Center for the History of Medicine at the Countway Library, Harvard Medical School. Digital surrogates are also available through Harvard's HOLLIS catalog.
See also
- Crick, Brenner et al. experimentCrick, Brenner et al. experimentThe Crick, Brenner, Barnett, Watts-Tobin experiment of 1961 was a scientific experiment performed in 1961 by Francis Crick, Sydney Brenner, Leslie Barnett and R.J. Watts-Tobin. They demonstrated that three bases of DNA code for one amino acid in the genetic code...
- Nirenberg and Matthaei experimentNirenberg and Matthaei experimentThe Nirenberg and Matthaei experiment was a scientific experiment performed on May 15, 1961, by Marshall W. Nirenberg and his post doctoral fellow, Heinrich J. Matthaei...
- List of famous experiments