Har Gobind Khorana, the Son of a Punjab Village Tax Clerk, Helped Complete the Genetic Code, Built a 77-Base-Pair Gene in 1970 and Shared the 1968 Nobel Prize

Har Gobind Khorana shared the 1968 Nobel Prize in Physiology or Medicine with Marshall Nirenberg and Robert Holley “for their interpretation of the genetic code and its function in protein synthesis”, the Nobel Foundation’s wording. His part was chemistry. He built DNA and RNA molecules letter by letter, in a sequence he chose, and used them to read the meaning of the code’s three-letter words. He was born in a Punjab village of about 100 people, in a family that was, in his own words, practically the only literate one there.

Two years after the prize his laboratory announced the first chemical synthesis of a gene, according to the National Academy of Sciences memoir written by Phillip Sharp and Uttam RajBhandary: 77 base pairs of double-stranded DNA, put together from short pieces made by chemistry. India’s science portal calls him the Indian scientist who cracked the genetic code, and the Government of India gave him the Padma Vibhushan in 1969.

What the code is, and what was missing in 1961

DNA and RNA are written in four letters. A group of three letters, a codon, tells the cell which amino acid to add next when it builds a protein, and there are 64 possible groups. According to India’s science and technology portal, the four bases fix the order of the 20 amino acids in proteins. Reading the code means filling in a table of 64 entries.

The first entry came in 1961, when Marshall Nirenberg and Heinrich Matthaei fed a cell-free bacterial extract an RNA made only of uracil, and it built a chain made only of the amino acid phenylalanine. The Nobel presentation speech says Nirenberg reported his first results in August 1961. Filling in the other 63 needed RNA with chosen letters in a known order, and in 1961 chemistry could make only a few RNA units at a time.

Khorana had spent years on that problem. The presentation speech says all the details of the code were established less than five years after Nirenberg’s first report, “mainly from the work of Nirenberg and Khorana”. India’s own large-scale reading of DNA is covered in GenomeIndia, the project that sequenced 10,074 people.

A village of about 100 people

Khorana’s Nobel autobiography gives his birthplace as Raipur, a village in Punjab that is now in Pakistan, and says the correct date of his birth is not known, although documents show 9 January 1922. The India Science and Technology portal and a 2022 article in The Wire Science instead give Multan, the town where he went to school. This article follows his own note.

Youngest of five children, four sons and a daughter, Khorana had a father who was a patwari, a village clerk who assessed agricultural tax under British rule. Money was short at home, the Nobel biography says, but his father was set on educating the children. Raipur’s other residents were all Muslim, the memoir adds, and his was the only Hindu family.

School was DAV High School in Multan, where a teacher named Ratan Lal influenced him, and college was Punjab University in Lahore. A scholarship, a chemistry degree in 1943 and a master’s in 1945 follow in the memoir’s account. Mahan Singh, whom the Nobel note calls a great teacher and an accurate experimentalist, supervised his work there.

Partition came in August 1947, after he had left for England. Raipur became part of Pakistan, and the memoir says the family moved to India as refugees, some of them settling near New Delhi. The political background is in the account of the Radcliffe Line.

Liverpool, Zurich, Cambridge and Vancouver

A Government of India fellowship took him to the University of Liverpool in 1945. Sponsored by the Ministry of Agriculture and meant for work on insecticides and fungicides, it arrived when returning war veterans had left few university places, the memoir says. Roger Beer in the chemistry department supervised his research, and Khorana took his PhD in 1948.

A postdoctoral year, 1948 to 1949, followed at the Swiss Federal Institute of Technology in Zurich. Khorana approached Vladimir Prelog with no letters of recommendation or introduction, the memoir says, and Prelog accepted him. Part of the pull was the language, since much chemical literature was in German, and a German paper he read there, on a reagent called a carbodiimide, played a central role in much of his later research. A search for a post in India in 1949 did not succeed, the memoir reports, and a place opened instead in Alexander Todd’s laboratory in Cambridge, where he stayed from December 1949.

In Cambridge he found that dicyclohexylcarbodiimide, one of those reagents, could couple phosphate esters into pyrophosphates, a reaction of central interest in Todd’s laboratory. Carbodiimide methods later let him make fragments of DNA and RNA with a defined sequence, the memoir says. In 1952 Gordon Shrum offered him a job at the British Columbia Research Council in Vancouver, where, in Shrum’s words quoted in the Nobel autobiography, there was “all the freedom in the world”. The same year he married Esther Elizabeth Sibler. Arthur Kornberg and Paul Berg, later Nobel laureates themselves, visited the Vancouver laboratory to learn his methods, the memoir says.

The University of Wisconsin in Madison recruited him in 1960 as one of three co-directors of its Institute for Enzyme Research, and he became a naturalised United States citizen.

Repeating patterns, 64 triplets and a result with no exceptions

The method, as his Nobel lecture describes it, started with short pieces of DNA made by chemistry, each a repeating pattern of two, three or four letters. DNA polymerase, an enzyme that copies DNA, stretched them 50 to 200 times longer. A second enzyme, RNA polymerase, then copied one strand into RNA, still in its repeating pattern. That RNA went into a cell-free extract of the bacterium Escherichia coli, and the protein chains that came out were analysed.

Two letters repeating in turn gave a protein with two amino acids alternating. The lecture’s example is an RNA of alternating U and C, which produced serine and leucine, but the experiment alone could not say whether the codon UCU meant serine and CUC meant leucine or the reverse. Nirenberg and Philip Leder had devised a second test, which asked which amino-acid carrier sticks to the ribosome, the cell’s protein factory, when a specific three-letter piece is present. Khorana’s group synthesised all 64 trinucleotides for it, the lecture says. Khorana also wrote that the test “has not proved to be completely reliable”, so the final table came from combining it with the repeating polymers and with other laboratories’ work.

In the lecture, Khorana reports that the predictions drawn from the repeating polymers were borne out “without a single exception” and calls 1963 to 1967 a period of essentially uninterrupted success. The memoir sums up the result. Every codon is a triplet, 61 of the 64 stand for amino acids, and the other three, UAG, UAA and UGA, tell the cell to stop. Nirenberg’s team and others contributed other parts of the table, and each of the three laureates received a one-third share of the prize. In the presentation speech, Professor P Reichard of the Karolinska Institute said: “Much of the final work was done by Khorana.” He added that Khorana’s synthetic nucleic acids were a prerequisite for the final solution.

One more story from the memoir concerns the morning of the announcement. Khorana had gone to a rented lake cottage without radio or television to work, and Esther drove out to tell him. At the celebration in the lab that day he talked to two colleagues about his next aim, the chemical synthesis of a gene.

A 77-base-pair gene in 1970, and the fully working one

The target was the gene for yeast alanine transfer RNA, a small RNA that carries amino acids to the ribosome. According to the memoir, it was chosen because in 1965 it was the only gene product whose sequence was known, the work of Holley. Two enzymes found in the mid-1960s, polynucleotide kinase and DNA ligase, let the team prepare short pieces five to twenty units long, line them up on their complementary partners and seal the joins.

Nature carried the paper on 4 July 1970, under 13 authors’ names. Its abstract says chemically synthesised segments were combined into a double-stranded DNA corresponding to the gene for the earliest characterised tRNA. Its length was 77 base pairs, the memoir states, and thirteen detailed papers filled an entire issue of the Journal of Molecular Biology in 1972.

Sharp and RajBhandary separate this from a fully active tRNA gene “with all the necessary sequences for its expression”, which they say was completed in 1979. The Science paper of 16 February 1979 describes the work: a 126-nucleotide DNA for a precursor of an E. coli tyrosine suppressor tRNA, completed with control sequences to a total the abstract gives as 207 base pairs and the memoir as 208. According to the abstract, the gene was transcribed in a test tube and showed activity in living cells as a suppressor of a stop signal. MIT’s obituary and the University of Wisconsin date the first fully working gene in a living cell to 1976, and the memoir notes that twelve consecutive papers in a 1976 issue of the Journal of Biological Chemistry preceded the 1979 summary.

MIT recruited him in 1970, and he held the Alfred P. Sloan chair in biology and chemistry until his retirement in 2007. Sharp and RajBhandary argue that the gene work was part of the groundwork for the recombinant DNA methods of the mid-1970s. They also point to a 1970 paper from his Wisconsin group describing a cycle of heating a DNA duplex, adding primers and polymerase, and repeating, which they say seems, in hindsight, very close to the polymerase chain reaction that Kary Mullis and colleagues described in 1985.

Rhodopsin, and genes for the proteins that sense light

From the mid-1970s, India’s science portal says, Khorana turned to membrane proteins, starting with bacteriorhodopsin, which pumps protons using light, and then rhodopsin, the light-sensing protein of the retina. The memoir says he showed that bacteriorhodopsin could be fully unfolded and then refolded to an active form, which let researchers test the effect of single amino-acid changes. It calls rhodopsin a prototype of the G-protein-coupled receptors, a family of about 750 genes in humans.

The gene-building skill carried over. Reports followed: a 1,057-base-pair synthetic gene for bovine rhodopsin, joined from 72 chemical pieces, in PNAS in February 1986, and a 757-base-pair gene for bacterio-opsin, the protein part of bacteriorhodopsin, in the Journal of Biological Chemistry in July 1987. Each was designed with evenly spaced restriction sites, spots where a short stretch can be swapped out to make a chosen mutation.

Honours, and a programme that carries his name

The Nobel Foundation’s nomination archive lists 15 nominations for Khorana between 1963 and 1974, all in chemistry; he won in medicine. MIT’s obituary lists the Louisa Gross Horwitz Prize and the Lasker Award for Basic Medical Research, both in 1968, the Willard Gibbs Medal in 1974, the Gairdner award in 1980 and the Paul Kayser award in 1987. He was elected to the National Academy of Sciences in 1966, according to the memoir. For another Nobel in the sciences that began in an Indian laboratory, see CV Raman and the effect that bears his name.

Khorana died of natural causes in Concord, Massachusetts, on 9 November 2011, aged 89, MIT reported. His colleague RajBhandary told MIT that “as good as he was, he was one of the most modest people I have known”.

The Khorana Program was set up in 2007 by Aseem Ansari and Kenneth Shapiro at the University of Wisconsin-Madison, the university’s agricultural college reports, to exchange students between select Indian institutions and Wisconsin. In summer 2008 it placed 14 Indian undergraduates in Wisconsin laboratories for ten weeks: ten from five IITs, two from Aligarh Muslim University, one from Jawaharlal Nehru University and one from Punjab Agricultural University, according to the programme’s own update. The Indo-U.S. Science and Technology Forum’s page describes it as run with India’s Department of Biotechnology and WINStep Forward, with placements of 10 to 12 weeks that cover airfare, a stipend and health insurance.