Editing Nirenberg and Leder experiment (section)

==Experimental work==
[[File:06 multi pu.jpg|thumb|175px|The Multi-plater, developed by Leder, helped speed up the process of deciphering the genetic code.<ref name="Leavitt_Instruments">{{cite web |last= Leavitt |first=Sarah A.|title=Deciphering the Genetic Code: Marshall Nirenberg. Scientific Instruments |url=https://history.nih.gov/exhibits/nirenberg/instruments.htm |publisher=Stetten Museum, Office of NIH History |year=2004 |accessdate=2009-10-05 |url-status=live |archive-date=9 February 2020 |archive-url=https://web.archive.org/web/20200209013324/https://history.nih.gov/exhibits/nirenberg/instruments.htm }}</ref>]]
 
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.<ref name="Judson H. 1996">{{Cite book | title=The Eighth Day of Creation: Makers of the Revolution in Biology|  author=Judson H.| year=1996|location= Cold Spring Harbor| publisher=Cold Spring Harbor Laboratory Press}}</ref>

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. <ref name="Nirenberg_1964">{{cite journal|author1=Philip Leder |author2=Marshall Nirenberg |name-list-style=amp |title=RNA Codewords and Protein Synthesis. The Effect of Trinucleotides upon the Binding of sRNA to Ribosomes |journal=Science |doi=10.1126/science.145.3639.1399 |volume=145 |issue=3639 |pages=1399–1407|year = 1964 |pmid=14172630 |s2cid=7127820 }}</ref>  

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]]:<ref name="Nirenberg_1964"/> he determined that GUU, UGU, and UUG encoded the amino acids valine,<ref name="Leder_1964a">{{cite journal |author1=Leder P. |author2=Nirenberg M.W. |name-list-style=amp |title=RNA Codewords and Protein Synthesis, II. Nucleotide Sequence of a Valine RNA Codeword |journal=PNAS | pmc=300293 |doi=10.1073/pnas.52.2.420 |volume=52 |issue=2 |pages=420–427 |year=1964 |pmid=14206609 |bibcode=1964PNAS...52..420L |doi-access=free }}</ref> cysteine and leucine,<ref name="Leder_1964b">{{Cite journal|author1=Leder P. |author2=Nirenberg M.W. |name-list-style=amp |title=RNA Codewords and Protein Synthesis, III. On the Nucleotide Sequence of a Cysteine and a Leucine RNA Codeword | journal=PNAS | pmc=300480 | doi=10.1073/pnas.52.6.1521 | volume = 52 | pages =1521–1529| year = 1964 | pmid=14243527| issue = 6|bibcode = 1964PNAS...52.1521L |doi-access=free }}</ref> 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.<ref name="Nirenberg_2004">{{cite journal |doi=10.1016/j.tibs.2003.11.009 |author=Nirenberg, Marshall |year=2004 |title=Historical review: Deciphering the genetic code – a personal account |journal=Trends in Biochemical Sciences |volume=29 |issue=1 |pages=46–54|pmid=14729332 }}</ref> 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.<ref name="Bernfield_1964">{{cite journal|author1=Bernfield M.R. |author2=Nirenberg M.W. |name-list-style=amp |title=RNA Codewords and Protein Synthesis. The Nucleotide Sequences of Multiple Codewords for Phenylalanine, Serine, Leucine, and Proline |journal=Science |doi=10.1126/science.147.3657.479 |volume=147 |issue=3657 |pages=479–484 |year=1965 |pmid=14237203 }}</ref> Many others in the Nirenberg lab and at NIH contributed to the full decipherment of the genetic code.<ref name="Nirenberg_2004"/>