Nirenberg and Leder experiment

Last updated March 18, 2023

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 ambiguous codons in the genetic code to be deciphered.

In this experiment, using a ribosome binding assay called the triplet binding assay, various combinations of mRNA were passed through a filter which contained ribosomes. 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.

Background

Oswald Avery 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.^[1]

In the 1960s, 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 were 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.^[2]

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.^[3] In the Crick, Brenner et al. experiment, using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutations and a process called reversions, to add and delete various numbers of nucleotides.^[4] When a nucleotide triplet was added to or deleted from 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.^[5] 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

The very first amino acid codon (UUU encoding phenylalanine) was deciphered by Nirenberg and his postdoc Heinrich Matthaei (see Nirenberg and Matthaei experiment ) using long synthetic RNA. However, when similar RNAs are made containing more than one RNA base, the order of the bases was random. For example, a long RNA could be made that had a ratio of C to U of 2:1, and so would contain codons CCU, CUC, UCC at high frequency. When translated by ribosomes, this would produce a protein containing the amino acids proline, leucine, and serine; but it was not possible to say which codon matched which amino acid.^[7]

Instead, Nirenberg's group turned to very short synthetic RNAs. They found that the trinucleotide UUU (which is the codon for phenylalanine), was able to cause specific association of phenylalanine-charged tRNA with ribosomes. This association could be detected by passing the mixture through a nitrocellulose filter: the filter captures ribosomes but not free tRNA; however if tRNA was associated with the ribosome, it would also be captured (along with the radioactive phenylalanine attached to the tRNA). They similarly found that trinucleotides AAA or CCC caused ribosome association of lysine-tRNA or proline-tRNA, respectively. ^[8]

So an experimental plan was clear: synthesize all 64 different trinucleotide combinations, and use the filter assay with tRNAs charged with all 20 amino acids, to see which amino acid associated with which trinucleotide. However, obtaining pure trinucleotides with mixed base sequences, for example GUU, was a daunting challenge. Leder's pioneering studies used trinucleotides made by breaking down long random poly-GU RNA with nuclease and purifying specific trinucleotides by paper chromatography:^[8] he determined that GUU, UGU, and UUG encoded the amino acids valine,^[9] cysteine and leucine,^[10] respectively. Subsequently, Nirenberg's group constructed trinucleotides by using DNA polymerases coupled with nucleotides and RNA polymerases to create the long random poly-GU RNA as well as artificially replicate the purified trinucleotides. Once high enough concentrations of mRNA were produced, degradation and reformation of polymerase products was accomplished through enzymatic processes. For example, AGU could be made from AG and U with polynucleotide phosphorylase; UAG could be made from AG and U with ribonuclease A in a high concentration of methanol.^[11] Nirenberg's postdoc Merton Bernfield used these techniques to determine that UUU and UUC encode phenylalanine, UCU and UCC encode serine, and CCC and CCU encode proline, highlighting a pattern in the way the genetic code redundantly encodes amino acids.^[12] Many others in the Nirenberg lab and at NIH contributed to the full decipherment of the genetic code.^[11]

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 said of Nirenberg's work 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 era of molecular genetics. For example, Arne 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."^[13]

Related Research Articles

<span class="mw-page-title-main">Genetic code</span> Rules by which information encoded within genetic material is translated into proteins

The genetic code is the set of rules used by living cells to translate information encoded within genetic material into proteins. Translation is accomplished by the ribosome, which links proteinogenic amino acids in an order specified by messenger RNA (mRNA), using transfer RNA (tRNA) molecules to carry amino acids and to read the mRNA three nucleotides at a time. The genetic code is highly similar among all organisms and can be expressed in a simple table with 64 entries.

Protein biosynthesis is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.

In molecular biology, a stop codon is a codon that signals the termination of the translation process of the current protein. Most codons in messenger RNA correspond to the addition of an amino acid to a growing polypeptide chain, which may ultimately become a protein; stop codons signal the termination of this process by binding release factors, which cause the ribosomal subunits to disassociate, releasing the amino acid chain.

The central dogma of molecular biology is an explanation of the flow of genetic information within a biological system. It is often stated as "DNA makes RNA, and RNA makes protein", although this is not its original meaning. It was first stated by Francis Crick in 1957, then published in 1958:

The Central Dogma. This states that once "information" has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information here means the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein.

In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription of DNA to RNA in the cell's nucleus. The entire process is called gene expression.

In molecular biology, a reading frame is a way of dividing the sequence of nucleotides in a nucleic acid molecule into a set of consecutive, non-overlapping triplets. Where these triplets equate to amino acids or stop signals during translation, they are called codons.

<span class="mw-page-title-main">Wobble base pair</span> RNA base pair that does not follow Watson-Crick base pair rules

A wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules. The four main wobble base pairs are guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C). In order to maintain consistency of nucleic acid nomenclature, "I" is used for hypoxanthine because hypoxanthine is the nucleobase of inosine; nomenclature otherwise follows the names of nucleobases and their corresponding nucleosides. The thermodynamic stability of a wobble base pair is comparable to that of a Watson-Crick base pair. Wobble base pairs are fundamental in RNA secondary structure and are critical for the proper translation of the genetic code.

A frameshift mutation is a genetic mutation caused by indels of a number of nucleotides in a DNA sequence that is not divisible by three. Due to the triplet nature of gene expression by codons, the insertion or deletion can change the reading frame, resulting in a completely different translation from the original. The earlier in the sequence the deletion or insertion occurs, the more altered the protein. A frameshift mutation is not the same as a single-nucleotide polymorphism in which a nucleotide is replaced, rather than inserted or deleted. A frameshift mutation will in general cause the reading of the codons after the mutation to code for different amino acids. The frameshift mutation will also alter the first stop codon encountered in the sequence. The polypeptide being created could be abnormally short or abnormally long, and will most likely not be functional.

The Crick, Brenner et al. experiment (1961) was a scientific experiment performed by Francis Crick, Sydney Brenner, Leslie Barnett and R.J. Watts-Tobin. It was a key experiment in the development of what is now known as molecular biology and led to a publication entitled "The General Nature of the Genetic Code for Proteins" and according to the historian of Science Horace Judson is "regarded...as a classic of intellectual clarity, precision and rigour". This study demonstrated that the genetic code is made up of a series of three base pair codons which code for individual amino acids. The experiment also elucidated the nature of gene expression and frame-shift mutations.

The Nirenberg and Matthaei experiment was a scientific experiment performed in May 1961 by Marshall W. Nirenberg and his post-doctoral fellow, J. Heinrich Matthaei, at the National Institutes of Health (NIH). The experiment deciphered the first of the 64 triplet codons in the genetic code by using nucleic acid homopolymers to translate specific amino acids.

Marshall Warren Nirenberg was an American biochemist and geneticist. He shared a Nobel Prize in Physiology or Medicine in 1968 with Har Gobind Khorana and Robert W. Holley for "breaking the genetic code" and describing how it operates in protein synthesis. In the same year, together with Har Gobind Khorana, he was awarded the Louisa Gross Horwitz Prize from Columbia University.

Johannes Heinrich Matthaei is a German biochemist. He is best known for his unique contribution to solving the genetic code on 15 May 1961.

The history of molecular biology begins in the 1930s with the convergence of various, previously distinct biological and physical disciplines: biochemistry, genetics, microbiology, virology and physics. With the hope of understanding life at its most fundamental level, numerous physicists and chemists also took an interest in what would become molecular biology.

The adaptor hypothesis is a theoretical scheme in molecular biology to explain how information encoded in the nucleic acid sequences of messenger RNA (mRNA) is used to specify the amino acids that make up proteins during the process of translation. It was formulated by Francis Crick in 1955 in an informal publication of the RNA Tie Club, and later elaborated in 1957 along with the central dogma of molecular biology and the sequence hypothesis. It was formally published as an article "On protein synthesis" in 1958. The name "adaptor hypothesis" was given by Sydney Brenner.

Ribosomal frameshifting, also known as translational frameshifting or translational recoding, is a biological phenomenon that occurs during translation that results in the production of multiple, unique proteins from a single mRNA. The process can be programmed by the nucleotide sequence of the mRNA and is sometimes affected by the secondary, 3-dimensional mRNA structure. It has been described mainly in viruses, retrotransposons and bacterial insertion elements, and also in some cellular genes.

An expanded genetic code is an artificially modified genetic code in which one or more specific codons have been re-allocated to encode an amino acid that is not among the 22 common naturally-encoded proteinogenic amino acids.

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The RNA Tie Club was an informal scientific club, meant partly to be humorous, of select scientists who were interested in how proteins were synthesised from genes, specifically the genetic code. It was created by George Gamow upon the suggestion by James Watson in 1954, at the time the relationship between nucleic acids and amino acids in genetic information was unknown. The club consisted of 20 full members, each representing an amino acid, and four honorary members, representing the four nucleotides. The functions of the club members were to think up possible solutions and share in writing the other members.

A codon table can be used to translate a genetic code into a sequence of amino acids. The standard genetic code is traditionally represented as an RNA codon table, because when proteins are made in a cell by ribosomes, it is messenger RNA (mRNA) that directs protein synthesis. The mRNA sequence is determined by the sequence of genomic DNA. In this context, the standard genetic code is referred to as translation table 1. It can also be represented in a DNA codon table. The DNA codons in such tables occur on the sense DNA strand and are arranged in a 5′-to-3′ direction. Different tables with alternate codons are used depending on the source of the genetic code, such as from a cell nucleus, mitochondrion, plastid, or hydrogenosome.

Degeneracy or redundancy of codons is the redundancy of the genetic code, exhibited as the multiplicity of three-base pair codon combinations that specify an amino acid. The degeneracy of the genetic code is what accounts for the existence of synonymous mutations.

References

↑ Russell P. (2010). iGenetics: A Molecular Approach, 3rd edition. Pearson/Benjamin Cummings.
↑ Leavitt, Sarah A. (2004). "Deciphering the Genetic Code: Marshall Nirenberg. The Coding Craze". Stetten Museum, Office of NIH History. Archived from the original on 9 February 2020. Retrieved 2009-10-05.
↑ Yanofsky C. (2007). "Establishing the Triplet Nature of the Genetic Code" (PDF). Cell. 128 (5): 815–818. doi: 10.1016/j.cell.2007.02.029 . PMID 17350564 . Retrieved 2018-01-24.
↑ Crick F.H.C.; Brenner S.; Barnett L. & Watts-Tobin R.J. (1961). "General Nature of the Genetic code for Proteins" (PDF). Nature. 192 (4809): 1227–1232. Bibcode:1961Natur.192.1227C. doi:10.1038/1921227a0. PMID 13882203. S2CID 4276146 . Retrieved 2009-10-10.
↑ Matthaei, H.J., Jones, O.W., Martin, R.G., and Nirenberg, M.W. (1962). "Characteristics and Composition of RNA Coding Units". Proceedings of the National Academy of Sciences of the United States of America. 48 (4): 666–677. Bibcode:1962PNAS...48..666M. doi: 10.1073/pnas.48.4.666 . PMC 220831 . PMID 14471390.{{cite journal}}: CS1 maint: multiple names: authors list (link)
↑ Leavitt, Sarah A. (2004). "Deciphering the Genetic Code: Marshall Nirenberg. Scientific Instruments". Stetten Museum, Office of NIH History. Archived from the original on 9 February 2020. Retrieved 2009-10-05.
↑ Judson H. (1996). The Eighth Day of Creation: Makers of the Revolution in Biology. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
1 2 Philip Leder & Marshall Nirenberg (1964). "RNA Codewords and Protein Synthesis. The Effect of Trinucleotides upon the Binding of sRNA to Ribosomes". Science. 145 (3639): 1399–1407. doi:10.1126/science.145.3639.1399. PMID 14172630. S2CID 7127820.
↑ Leder P. & Nirenberg M.W. (1964). "RNA Codewords and Protein Synthesis, II. Nucleotide Sequence of a Valine RNA Codeword". PNAS. 52 (2): 420–427. Bibcode:1964PNAS...52..420L. doi: 10.1073/pnas.52.2.420 . PMC 300293 . PMID 14206609.
↑ Leder P. & Nirenberg M.W. (1964). "RNA Codewords and Protein Synthesis, III. On the Nucleotide Sequence of a Cysteine and a Leucine RNA Codeword". PNAS. 52 (6): 1521–1529. Bibcode:1964PNAS...52.1521L. doi: 10.1073/pnas.52.6.1521 . PMC 300480 . PMID 14243527.
1 2 Nirenberg, Marshall (2004). "Historical review: Deciphering the genetic code – a personal account". Trends in Biochemical Sciences. 29 (1): 46–54. doi:10.1016/j.tibs.2003.11.009. PMID 14729332.
↑ Bernfield M.R. & Nirenberg M.W. (1965). "RNA Codewords and Protein Synthesis. The Nucleotide Sequences of Multiple Codewords for Phenylalanine, Serine, Leucine, and Proline". Science. 147 (3657): 479–484. doi:10.1126/science.147.3657.479. PMID 14237203.
↑ Fee, E. (2000). "Profiles in Science: The Marshall W. Nirenberg Papers. Public Reaction". National Library of Medicine. Archived from the original on 9 April 2020. Retrieved 9 April 2020.

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.