C L A S S I C E X P E R I M E N T 4 . 1

CRACKING THE GENETIC CODEM. W. Nirenberg and P. Leder, 1964, Science 145:1399

By the early 1960s molecular biologistshad adopted the so-called “centraldogma,” which states that DNA directssynthesis of RNA (transcription), whichthen directs assembly of proteins(translation). However, researchers stilldid not completely understand howthe “code” embodied in DNA andsubsequently in RNA directs proteinsynthesis. To elucidate this process,Marshall Nirenberg embarked upon aseries of studies that would lead to thesolution of the genetic code.

BackgroundProteins are made from combinationsof 20 different amino acids. The genesthat encode proteins—that is, specifythe type and linear order of their com-ponent amino acids—are located inDNA, a polymer made up of only fourdifferent nucleotides. The DNA code istranscribed into RNA, which is alsocomposed of four nucleotides. Niren-berg’s studies were premised on the hy-pothesis that the nucleotides in RNAform “codewords,” each of which cor-responds to one of the amino acidsfound in protein. During protein syn-thesis, these codewords are translatedinto a functional protein. Thus, to un-derstand how DNA directs proteinsynthesis, Nirenberg set out to under-stand the relationship between RNAcodewords and protein synthesis.

At the outset of his studies, muchwas already known about the processof protein synthesis, which occurs onribosomes. These large ribonuleopro-tein complexes can bind two differenttypes of RNA: messenger RNA (mRNA),which carries the exact protein-specifyingcode from DNA to ribosomes, andsmaller RNA molecules now known astransfer RNA (tRNA), which deliveramino acids to ribosomes. tRNAs existin two forms: those that are covalentlyattached to a single amino acid, known

as amino-acylated or “charged” tRNAs,and those that have no amino acidattached, called “uncharged” tRNAs.After binding of the mRNA and theamino-acylated tRNA to the ribosome,a peptide bond forms between theamino acids, beginning protein synthe-sis. The nascent protein chain is elon-gated by the subsequent binding ofadditional tRNAs and formation of apeptide bond between the incomingamino acid and the end of the growingchain. Although this general processwas understood, the question re-mained: How does the mRNA directprotein synthesis?

When attempting to address com-plex processes such as protein synthe-sis, scientists divide large questionsinto a series of smaller, more easily ad-dressed questions. Prior to Nirenberg’sstudy, it had been shown that whenphenylalanine-charged tRNA was incu-bated with ribosomes and polyuridylicacid (polyU), peptides consisting ofonly phenylalanine were produced.This finding suggested that the mRNAcodeword, or codon, for phenylalanineis made up of the nucleosides contain-ing the base uracil. Similar studieswith polycytadylic acid (polyrC) andpolyadenylic acid (polyrA) showedthese nucleosides containing the basescytadine and adenine made up thecodons for proline and lysine, respec-tively. With this knowledge in hand,Nirenberg asked the question: What isthe minimum chain length required fortRNA binding to ribosomes? The sys-tem he developed to answer this ques-tion would give him the means to de-termine which amino-acylated tRNAwould bind which m-RNA codon,effectively cracking the genetic code.

The ExperimentThe first step in determining the mini-mum length of mRNA required for

tRNA recognition was to develop anassay that would detect this interac-tion. Since previous studies had shownthat ribosomes bind mRNA and tRNAsimultaneously, Nirenberg reasonedthat ribosomes could be used as a bridgebetween a known mRNA codon and aknown tRNA. When the three compo-nents of protein synthesis are incu-bated together in vitro, they shouldform a complex. After devising amethod to detect this complex,Nirenberg could then alter the size ofthe mRNA to determine the mini-mum chain length required for tRNArecognition.

Before he could begin his experi-ment, Nirenberg needed both a meansto separate the complex from unboundcomponents and a method to detecttRNA binding to the ribosome. To iso-late the complex he exploited the abil-ity of nylon filters to bind large RNAmolecules, such as ribosomes, but notthe smaller tRNA molecules. He used anylon filter to separate ribosomes (andanything bound to the ribosomes)from unbound tRNA. To detect thetRNA bound to the ribosomes,Nirenberg used tRNA charged withamino acids that contained a radioac-tive label, 14C. All other componentsof the reaction were not radioactive.Since only ribosome-bound tRNA isretained by the nylon membrane, allradioactivity found on the nylon mem-brane corresponds to tRNA bound toribosomes. Now a system was in placeto detect the recognition between amRNA molecule and the properamino-acylated tRNA.

To test his system, Nirenberg usedpolyU as the mRNA, and [14C]-phenylalanine-charged tRNA, whichbinds to ribosomes in the presence ofpolyU. Ribosomes were incubated withboth polyU and [14C]-phenylalaninetRNA for sufficient time to allow bothmolecules to bind to the ribosomes; the

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reaction mixtures were then passedthrough a nylon membrane. When themembranes were analyzed using ascintillation counter, they containedradioactivity, demonstrating that inthis system polyU could recognizephenylalanine-charged tRNA. But wasthis recognition specific for the properamino-acylated tRNA? As a control,[14C]-lysine- and [14C]-proline-chargedtRNAs also were incubated withpolyU and ribosomes. After the reac-tion mixtures were passed through anylon filter, no radioactivity was de-tected on the filter. Therefore, the as-say measured only specific binding be-tween a mRNA and its correspondingamino-acylated tRNA.

Now the minimum chain lengthof RNA necessary for proper amino-acylated tRNA recognition could be de-termined. Short oligonucleotides weretested for their ability to bind ribosomesand recognize the appropriate tRNA.When UUU, a trinucleotide, was used,tRNA binding to ribosomes could be

detected. However, when the UU dinu-cleotides were used, no binding wasdetected. This result suggested that thecodon required for proper recognitionof tRNA is a trinucleotide. Nirenbergrepeated this experiment on two otherhomogeneous trinucleotides, CCC andAAA. When these trinucleotides wereindependently bound to ribosomes,CCC specifically recognized prolinecharged tRNA and AAA recognizedlysine-charged tRNAs. Since none ofthe three homogeneous trinucleotidesrecognized other charged tRNAs,Nirenberg concluded that trinu-cleotides could effectively direct theproper recognition of amino-acylatedtRNAs.

This study accomplished muchmore than determining the length ofthe codon required for proper tRNArecognition. Nirenberg realized thathis assay could be used to test all 64possible combinations of trinucleotides(see Figure). A method for cracking thecode was available!

Discussion

Combined with the technology to gen-erate trinucleotides of known se-quence, Nirenberg’s assay provided away to assign each specific amino acidto one or more specific trinucleotides.Within a few years, the genetic codewas cracked, all 20 amino acids wereassigned at least one trinucleotide, and61 of the 64 trinucleotides were foundto correspond to an amino acid. Thefinal three trinucleotides, now knownas “stop” codons, signal terminationof protein synthesis.

With the genetic code cracked, bi-ologists could read the gene in thesame manner that the cell did. Simplyby knowing the DNA sequence of agene, scientists can now predict theamino acid sequence of the protein itencodes. For his innovative work,Nirenberg was awarded the NobelPrize in Physiology or Medicine in1968.

Assay developed by Marshall Nirenberg and his collaborators fordeciphering the genetic code. They prepared 20 E. coli extractscontaining all the aminoacyl-tRNAs (tRNAs with amino acid attached).In each extract sample, a different amino acid was radioactively labeled(green); the other 19 amino acids were present on tRNAs but remainedunlabeled. Aminoacyl-tRNAs and trinucleotides passed through a nylonfilter without binding (left panel); ribosomes, however, bind to the filter(center panel). Each of the 64 possible trinucleotides was testedseparately for its ability to attract a specific tRNA by adding it with

Phe

Trinucleotide and all tRNAspass through filter

Trinucleotide

Aminoacyl-tRNAs

Ribosomes stick to filter

Ribosomes

Complex of ribosome, UUU,and Phe-tRNA sticks to filter

UUU

UUU

UUU

UUU

Phe

Phe

Leu

Leu

Leu

Arg Arg

Arg

Arg

Phe

Leu

ribosomes to different extract samples. Each sample was then filtered.If the added trinucleotide causes the radiolabeled aminoacyl-tRNA tobind to the ribosome, then radioactivity is detected on the filter;otherwise, the label passes through the filter (right panel). Bysynthesizing and testing all possible trinucleotides, the researchers wereable to match all 20 amino acids with one or more codons (e.g.,phenyalanine with UUU, as shown here). [From H. Lodish et al., 1995,

Molecular Cell Biology, 3rd ed. W. H. Freeman and Company. See M. W.

Nirenberg and P. Leder, 1964, Science 145:1399].

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