Nirenberg and Matthaei experiment
Encyclopedia
The 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
. The experiment cracked the genetic code
by using nucleic acid
homopolymers to translate specific amino acid
s.
In the experiment, an extract from bacterial cells that could make protein
even when no intact living cells
were present was prepared. Adding an artificial form of RNA
, poly-U, to this extract caused it to make a protein composed entirely of the amino acid phenylalanine
. This experiment cracked the first codon of the genetic code
showed that RNA controlled the production of specific types of protein.
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 1960s, one main DNA mystery scientists needed to figure out was in transcription
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 (triplets are not overlapping) and that each nucleotide sequence is read from a specific starting point.
Marshall Nirenberg and Johann Matthaei both longed to understand how information gets transmitted from DNA to protein. At this time there was a race to crack the code of the DNA language. At the same time, Severo Ochoa
was busy working on the coding problem with the help of Leon Heppel, a skillful biochemist
capable of making artificial RNA
s of defined compositions. Ochoa had a big staff, and Nirenberg was worried he would not be able to keep up. Many NIH scientists helped Nirenberg in deciphering the mRNA codons for amino acids.Nirenberg and his post doctoral fellow Matthaei started their experiments in a lab in Germany and completed them in a National Institutes of Health
(NIH) laboratory campus in Maryland.
or Tyrosine
.
At 3 am on May 27 Matthaei used phenylalanine for the "hot" test tube. After an hour, the control tubes showed a background level of 70 counts, whereas the hot tube showed 38,000 counts per milligram of protein. The experiment showed that a chain of the repeated uracil bases produced a protein chain made of one repeating amino acid, phenylalanine. Therefore, polyU coded for polyphenylalanine, consistent with UUU coding for phenylalanine. At the time the number of bases per codon could not be determined. The two kept their breakthrough a secret from the larger scientific community until they could complete further experiments with other strands of synthetic RNA (such as Poly-A) and prepare papers for publication. Using the three-letter poly-U experiment as a model, the research team discovered that AAA (three adenosines) was the code word or "codon" for the amino acid lysine, and CCC (three cytosines) was the code word for proline. They also discovered that by replacing one or two units of a triplet with other nucleotides, they could direct the production of other amino acids. They found, for example, that a synthetic RNA GUU codes for a valine be added to a developing amino acid chain.
In 1964 and 1965, Nirenberg's postdoctoral researcher, Philip Leder, developed a filtration machine that allowed the NIH research team determine the order of the nucleotides in the codons. This development sped up the process of assigning code words to amino acids. By 1966, Nirenberg announced that he had deciphered the sixty-four RNA codons for all twenty amino acids.
For his ground-breaking work on the genetic code, Nirenberg was awarded the 1968 Nobel Prize
in Physiology or Medicine. He shared the award with Har Gobind Khorana of the and Robert W. Holley
. 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 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 area 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."
Heinrich J. Matthaei
J. Heinrich Matthaei is a German biochemist. He is best known for his unique contribution to solving the genetic code on May 15, 1961...
. The experiment cracked 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....
by using nucleic acid
Nucleic acid
Nucleic acids are biological molecules essential for life, and include DNA and RNA . Together with proteins, nucleic acids make up the most important macromolecules; each is found in abundance in all living things, where they function in encoding, transmitting and expressing genetic information...
homopolymers to translate 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...
s.
In the experiment, an extract from bacterial cells that could make protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
even when no intact living cells
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
were present was prepared. Adding an artificial form of RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
, poly-U, to this extract caused it to make a protein composed entirely of the amino acid phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
. This experiment cracked the first codon 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....
showed that RNA controlled the production of specific types of protein.
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 1960s, one main DNA mystery scientists needed to figure out was in transcription
Transcription (genetics)
Transcription is the process of creating a complementary RNA copy of a sequence of DNA. Both RNA and DNA are nucleic acids, which use base pairs of nucleotides as a complementary language that can be converted back and forth from DNA to RNA by the action of the correct enzymes...
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 (triplets are not overlapping) and that each nucleotide sequence is read from a specific starting point.
Marshall Nirenberg and Johann Matthaei both longed to understand how information gets transmitted from DNA to protein. At this time there was a race to crack the code of the DNA language. At the same time, Severo Ochoa
Severo Ochoa
Severo Ochoa de Albornoz was a Spanish-American doctor and biochemist, and joint winner of the 1959 Nobel Prize in Physiology or Medicine with Arthur Kornberg.-Early life:...
was busy working on the coding problem with the help of Leon Heppel, a skillful biochemist
Biochemist
Biochemists are scientists who are trained in biochemistry. Typical biochemists study chemical processes and chemical transformations in living organisms. The prefix of "bio" in "biochemist" can be understood as a fusion of "biological chemist."-Role:...
capable of making artificial RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
s of defined compositions. Ochoa had a big staff, and Nirenberg was worried he would not be able to keep up. Many NIH scientists helped Nirenberg in deciphering the mRNA codons for amino acids.Nirenberg and his post doctoral fellow Matthaei started their experiments in a lab in Germany and completed them in a National Institutes of Health
National Institutes of Health
The National Institutes of Health are an agency of the United States Department of Health and Human Services and are the primary agency of the United States government responsible for biomedical and health-related research. Its science and engineering counterpart is the National Science Foundation...
(NIH) laboratory campus in Maryland.
Experimental Work
In order to decipher this biological mystery, Nirenberg and Matthaei needed a cell-free system that would build amino acids into proteins. Following the work of Alfred Tissieres and after a few failed attempts, they created a stable system by rupturing E. coli bacteria cells and releasing the contents of the cytoplasm. This allowed them to synthesize protein, but only when the correct kind of RNA was added, allowing Nirenberg and Matthaei to control the experiment. They created synthetic RNA molecules outside the bacterium and introduced this RNA to the E. coli system. The experiment used 20 test tubes, each filled with a different amino acid. For each individual experiment, 19 test tubes were "cold", and one was radioactively tagged with 14C so they could detect the tagged amino acid later. They varied the "hot" amino acid in each round of the experiment, seeking to determine which amino acid would be incorporated into a protein following the addition of a particular type of synthetic RNA. In their experiments in late May 1961 they had narrowed down the amino acids encoded by Poly-U to PhenylalaninePhenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
or Tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
.
At 3 am on May 27 Matthaei used phenylalanine for the "hot" test tube. After an hour, the control tubes showed a background level of 70 counts, whereas the hot tube showed 38,000 counts per milligram of protein. The experiment showed that a chain of the repeated uracil bases produced a protein chain made of one repeating amino acid, phenylalanine. Therefore, polyU coded for polyphenylalanine, consistent with UUU coding for phenylalanine. At the time the number of bases per codon could not be determined. The two kept their breakthrough a secret from the larger scientific community until they could complete further experiments with other strands of synthetic RNA (such as Poly-A) and prepare papers for publication. Using the three-letter poly-U experiment as a model, the research team discovered that AAA (three adenosines) was the code word or "codon" for the amino acid lysine, and CCC (three cytosines) was the code word for proline. They also discovered that by replacing one or two units of a triplet with other nucleotides, they could direct the production of other amino acids. They found, for example, that a synthetic RNA GUU codes for a valine be added to a developing amino acid chain.
Reception and Legacy
In August, at the International Congress of Biochemistry in Moscow, Nirenberg presented his paper. The experimentation with synthetic RNA in a cell-free system was a key technical innovation. In 1961, when they announced their methods for decoding the relationship of mRNA to amino acids, there was still a lot of experimentation required before the entire code was deciphered. The scientists had to determine which bases made up each codon, then determine the sequence of bases in the codons. This proved to be a tremendous amount of work.In 1964 and 1965, Nirenberg's postdoctoral researcher, Philip Leder, developed a filtration machine that allowed the NIH research team determine the order of the nucleotides in the codons. This development sped up the process of assigning code words to amino acids. By 1966, Nirenberg announced that he had deciphered the sixty-four RNA codons for all twenty amino acids.
For his ground-breaking work on the genetic code, Nirenberg was awarded the 1968 Nobel Prize
Nobel Prize
The Nobel Prizes are annual international awards bestowed by Scandinavian committees in recognition of cultural and scientific advances. The will of the Swedish chemist Alfred Nobel, the inventor of dynamite, established the prizes in 1895...
in Physiology or Medicine. He shared the award with Har Gobind Khorana of the and Robert W. Holley
Robert W. Holley
Robert William Holley was an American biochemist. He shared the Nobel Prize in Physiology or Medicine in 1968 for describing the structure of alanine transfer RNA, linking DNA and protein synthesis.Holley was born in Urbana, Illinois, and graduated from Urbana High School in 1938...
. 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 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 area of Molecular Genetics
Molecular genetics
Molecular genetics is the field of biology and genetics that studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology...
. 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."