Life's Greatest Secret (32 page)

There are so many criticisms which can be brought against this type of experiment that one hardly knows where to begin.

Crick’s critique was rock solid: the composition of the polynucleotides apart from poly(U), poly(A), etc. was completely unknown (it was not even certain that the incorporation of the different bases into synthetic RNA molecules was truly random), the levels of amino acid incorporation in the ‘cell-free’ experiments were often worryingly low, and even the strongest effect – poly(U) coding for phenylalanine – was weakened by the fact that poly(U) sometimes seemed to code for leucine. Having grudgingly accepted that two codons could be reliably identified (only one – the inevitable UUU = phenylalanine – was in fact correct), Crick concluded that the methodological problems he had outlined ‘make the allocation of further triplets very precarious’.
⁴⁰
He continued:

although not one single codon can be said to be known with certainty we do know something: one codon for phenylalanine contains Us, one for proline contains Cs, and so on. The coding problem has moved out of the realm of rather abstract speculation into the rough and tumble of experimentation.

Crick was not even convinced by the evidence that the code possessed redundancy – the evidence, he said, ‘is of two types, direct and indirect, and with one exception, none of it is satisfactory’.
⁴²
Having surveyed the various options, including Roberts’s code, Crick put his finger on what was the true situation: ‘if the code is really a pure triplet code its degeneracy makes it look at times more like a doublet one’.
⁴³

Crick was not scornful of theory – after all, it was theory that had underpinned most studies of the coding problem over the previous nine years, including many of his own contributions – but ultimately, more precise experimentation was needed. No matter how elegant a theoretical solution might be, the data would determine whether it was correct. Crick recognised that his own pursuit of theoretical models, such as the commaless code, had not led to any breakthroughs (he subsequently described the commaless code as ‘one of those nice ideas which is, nevertheless, completely wrong’
⁴⁴
). He gave his readers a clear outline of how he thought research on the topic should proceed:

In the long run we do not want to guess the genetic code, we want to know what it is. … The time is rapidly approaching when the serious problem will not be whether, say, UUC is likely to stand for serine, but what evidence can we accept which establishes this beyond reasonable doubt. What, in short, constitutes proof of a codon? Whether theory can help by suggesting the general structure of the code remains to be seen. If the code does have a logical structure there is little doubt that its discovery would greatly help the experimental work. Failing that, the main use of theory may be to suggest novel forms of evidence and to sharpen critical judgement. In the final analysis it is the quality of the experimental work which will be decisive.

About a week after sending off his article, Crick heard that he, Jim Watson and Maurice Wilkins had won the 1962 Nobel Prize in Physiology or Medicine, for their work on the structure of nucleic acids and its significance for information transfer in living material.
⁴⁶
The debates on the Nobel Prize committee are closed, but it seems probable that the renewed interest in the significance of the sequential structure of DNA produced by the cracking of the code convinced the committee that Watson, Crick and Wilkins’s time had come.

*

In early June 1963, just two years after Nirenberg and Matthaei’s discovery, Cold Spring Harbor Laboratory held its annual meeting under the title ‘Synthesis and structure of macromolecules’. This time, not only was Nirenberg allowed to attend, he had pride of place on the programme. In the ten years since gangly Jim Watson had presented the double helix structure of DNA in a stifling Cold Spring Harbor lecture theatre, the field of molecular biology had been utterly transformed – this was the largest ever meeting held at Cold Spring Harbor, with more than 300 scientists attending, about one-fifth of them from outside the US.

The seventy-four presentations at the meeting were focused on DNA, on various forms of RNA and on protein synthesis, and the framework was resolutely biochemical. Nirenberg’s talk was entitled ‘On the coding of genetic information’, but after the introductory paragraph he immersed himself in the biochemical details, even reverting back to the old vague language of specificity rather than giving any content to the idea of information. Nirenberg’s talk revealed that, as Crick had pointed out, the race to crack the code had hit an experimental bottleneck. The techniques that were employed – a combination of synthetic RNA of unknown sequence and data from the effects of mutations on viruses – could not crack the code. Worse, they could not even settle the question of whether the code was composed of groups of two, three or more bases. Furthermore, Nirenberg sounded a new note of caution about the technique that had made his name: it was possible, he argued, that natural messenger RNA found in cells might not use all sixty-four potential triplet codons; as a result, the randomly ordered synthetic molecules might ‘test the cell’s potential to recognise code words’, he said.
⁴⁷

Progress was certainly being made – increasingly detailed experiments and more accurately assembled synthetic RNA allowed Nirenberg’s group to suggest that a number of amino acids, including proline and phenylalanine, were coded by more than one codon, but there was still no absolute proof. The actual code remained out of reach, because the sequence of bases on the RNA molecules used in the cell-free system remained unknown. At the end of his talk, Nirenberg described how his group and that of Indian-born biochemist Gorind Khorana, who was based at the University of Wisconsin, were separately using two techniques for synthesising short bits of DNA, or oligodeoxynucleotides (oligo- is a Greek prefix meaning few). When these pieces of DNA were transcribed into RNA, it was shown that molecules composed of only four bases could still produce detectable levels of amino acids in the cell-free system. Nirenberg’s understated conclusion pointed the way forward:

It is possible that defined oligodeoxynucleotides may be useful in the determination of nucleotide sequence and polarity of RNA code words, and also in the study of control mechanisms related to DNA-directed protein synthesis.

The next talk was by Joe Speyer from Ochoa’s laboratory, who summarised their two-year-long attempts to correlate the theoretical frequency of different triplets in a synthetic RNA molecule with the levels of different amino acids.
⁴⁹
There was little new there, beyond a summary of research from a variety of species indicating that the code was universal. The Ochoa group had made a substantial impact in the field, recovering the initiative from Nirenberg, and showing what focused, large-scale molecular research could achieve, but the limits of their techniques were now apparent. Two weeks later, Ochoa gave a talk in Switzerland in which he inadvertently outlined the impasse his group was in; unlike Nirenberg, he had no solution to the problem.
⁵⁰

The summer of 1963 represented a double shift in the race to crack the code. New techniques had been developed for creating small RNA molecules of known sequence, while the competing laboratories had changed. Ochoa’s group effectively bowed out of trying to determine which triplets coded for which amino acids; Gorind Khorana, the expert in RNA synthesis, took their place.

Over the next two years, Khorana’s group refined its technique for creating small RNA molecules of a known sequence, and in 1964 Nirenberg’s laboratory solved the problem from the other direction – they worked out how to identify the nucleotide sequence on a piece of RNA that had just led to the incorporation of a particular amino acid into a protein chain. This bit of heavy-duty biochemistry involved trapping a complex of molecules – radioactive transfer RNA (tRNA; this was Crick and Brenner’s adaptor molecule), nucleotides and ribosomes – on a Millipore filter. Using this technique, Nirenberg and his colleague Phil Leder were able to show that a UUU triplet led to the binding of phenylalanine tRNA, whereas a UU doublet did not.
⁵¹
There was no evidence for any of the fancy doublet-based codes that had been suggested in the previous couple of years – a codon was composed of three bases.

With the help of NIH colleagues and highly skilled visitors such as Marianne Grunberg-Manago, Nirenberg’s lab was soon able to use a variety of techniques to synthesise triplets with known composition, and then put them through the Millipore ‘plater’ device to demonstrate which amino acid they coded for. One of Nirenberg’s trusted technicians, Norma Heaton, recalled that during this period the atmosphere in the laboratory was ‘intense … busy … crowded … competitive’. She described how they would suck up radioactive reagents with their mouths (‘that would never be allowed today’, she said) and that members of the lab would crowd round the data coming out of the radioactivity counter, eager to know what new codon had been discovered, ‘Then you would hear this shout, like “Oh, we discovered a new one.”’
⁵²

Heaton also gave some insight into the prosaic work that takes place in a laboratory – exactly what has to be done to obtain the data that is interpreted to make scientific breakthroughs. The routine she described resembles the precise, repetitive gestures of a worker on a production line, which, in a way, is what she was:

Initially we used single platers. It was a little round, stainless steel tube, just big enough to hold the Millipore filter, and about so high, and it screwed onto a base. You had a glass Erlenmeyer flask connected to a vacuum, and then you had a rubber gasket at the top, and you plunked this thing down.

Then you had the vacuum on, and you took one of the test tubes that had your experiment in it, and you would precipitate the complex with TCA. Then you would pour it through the plater and the precipitated complex would be collected on the filter.

Then you would unscrew it, take out the Millipore filter with forceps, and put it in order onto a piece of aluminium foil. Initially, we used what was called a Nuclear-Chicago planchet counter. You placed the dried filter onto little copper or aluminium planchets, about so big around, they had a little, tiny lip, and you would put the filter on that, and then you would stack them up and you would put them into the Nuclear-Chicago, and they would drop down and as they went across, the level of radioactivity would be counted. …

It all had to be timed. When you got good at this, you knew how many seconds it took you to unscrew this single plater, take it out, put it down, set it up with a new Millipore. I think I got so I could do it every thirty seconds, or maybe every twenty-five seconds.

As the data came tumbling out of the Nirenberg laboratory, Crick, like so many before him, tried to find the reason why some amino acids were coded by more than one codon – the logic behind the degenerate nature of the code – simply by thinking about it. He wondered how the codon on the messenger RNA molecule bound with a complementary set of bases – what he called an anticodon – on the small transfer RNA (tRNA) molecule.* It was not clear whether there was one tRNA molecule per RNA codon (so sixty-four different versions), or one molecule per amino acid (in which case there would there would be twenty), or some intermediate situation. There was some experimental evidence that the tRNA that attaches to phenylalanine could recognise both the UUU and the UUC codons; to explain this curious phenomenon, Crick resorted to the precise molecular modelling that had preoccupied him during the race to discover the structure of the DNA molecule at the beginning of 1953. He came up with the idea that there was a degree of what he called molecular wobble in the binding of the third base in the RNA codon with its equivalent in the tRNA anticodon. Crick provided a masterly survey of the situation at the time, and then concluded with a smile: ‘In conclusion it seems to me that the preliminary evidence seems rather favourable to the theory. I shall not be surprised if it proves correct.’
⁵⁴

It was correct – we now know that most organisms have more than twenty but less than sixty-four tRNAs (for example, there are forty-eight tRNA ‘anticodons’ in humans, but only thirty-one in bacteria), which is explained by the wobble in the anticodon’s ability to recognise more than one base in the third position of the codon.

By the middle of 1965, Nirenberg’s group had identified the function of fifty-four out of the sixty-four RNA codons; at around the same time, Khorana confirmed these data by using synthetic codons of known sequence.
⁵⁵
None of the theoretical schemes that had been so carefully developed over the previous decade proved to be correct. The genetic code is highly redundant, so that in many cases a base in a codon can alter without changing the amino acid that is being coded for. Most of these silent changes in DNA occur in the third base – this was the reason why theoreticians had wondered whether in fact the code was basically a doublet code. In some cases the third base in the codon provides no additional information because all four alternatives code for the same amino acid, as a result of the wobble in codon–anticodon binding.

Three interlinked issues remained: understanding which way the genetic message is read, and finding out how the cell knows where the genetic message begins and ends. A sequence of bases can be read in either direction, with completely different meanings: a DNA codon reading AGG codes for serine, whereas GGA codes for proline. Furthermore, because of the complementary nature of the two DNA strands a given stretch of DNA contains four possible alternatives – in this example, AGG and GGA on one strand, and TCC and CCT on the complementary strand. The genetic code seemed to be becoming even more complicated, but this mystery was soon solved.