Allow me to tell you about how shy this young man Nirenberg was at this meeting. When he walked out onto the stage at this meeting in Moscow to give his talk, he was shocked to find that as few as five people were in his audience. He turned his back on the audience and began his slide talk. When he had finished he couldn't wait to get through with the questions he knew must be coming. He couldn't wait, and rushed off behind the curtains.
The five in his audience sat there in wonderment, and finally got up and left telling others in the lobby what a great talk they had just heard and why hadn't others come to hear it. They said, arrogantly, that because Nirenberg wasn't a member of the prestigious research clubs of Cambridge, Paris, Cal Tech or Boston, they felt it would have been a waste of their time. Jim Watson, who had been one of the five, petitioned the meeting organizers that Nirenberg must give his talk again. It was scheduled for the last hour of the convention week.
Understanding that Nirenberg was pathologically shy, he was led out onto the stage again, but this time lots of bright lights were aimed at him so he couldn't see anyone in the audience except for the same five guys in the front row. He gave his talk - again with his back to the audience. When finished, he was allowed to skip the usual questioning period, and was allowed to leave - but not before they turned up the house lights and he saw that he had an audience of hundreds. He nearly fainted in fright.
Two years later he won the Nobel Prize. Let's now tell his story!
His title speaks of translation so let's understand what is meant by that in genetic terms.
The "code" is stored in the DNA, and that code is transferred to RNA in a process called "transcription." Transcription is a secretarial word for processing - say - spoken English words into English writing. In this case transcription is from DNA's nucleic acids language into RNA's nucleic acid language - essentially the same language - thus transcription.
Now when the nucleic acid language is converted to another language such as that of proteins, it is termed "translation." Thus that line of DNA → RNA → Protein is the central dogma of life, and is commonly referred to as "gene expression."
As far as the title's "cell-free" part, let's consider that if we can take something apart and put it back together, we must understand the process fairly well as we know all the parts and haven't lost any. The same with the translation process of making proteins.
Nirenberg took some E. coli and broke them open and collected all their cytoplasmic juices in a test tube. He then added some DNase to eliminate any influence of DNA in the translation process - he eliminated transcription by doing this.
He then added the whole collection of 18 different radioactive amino acids (C-14), allowed the process to go for awhile, and then did isopycnography and found that much of the radioactivity was now found in a few discrete bands that were macromolecular (Svedberg!) and were further shown to be Biuret-positive (i.e.: polypeptides and not merely bunched free amino acids).
This rapidly became known as the Nirenberg System.
He and Matthaei then wondered what would happen if they threw in some synthetic RNAs that chemists down the hall had made for them. They first tried poly-U:
By golly! Look! It produced poly-phenylalanine!
they then tried the other three homopolymers:
...and got homopolymers of different amino acids. They were really on to something now!
A new approach needs to be made.
Of course we still don't know what the "reading frame" is - is it a pair of bases, or three taken at a time, or four or five??? Their chemist friends provided them with a copolymer of UC... that would test this out, as you see on the figure, above, with this result:
This of course is pretty much what statistics would indicate.
Their chemist associates then provided them with these. See that only the first "base" is changed. And they got new amino acids hitched to the front end of the polypeptides. When these synthetic RNAs were mixed into the "system", quickly new amino-acid assignments were identified.
An interesting aspect of this research is that most of it cannot be found in journals because it was such "hot news" that it was leaked to the NY Times, and every week more assignments were published and corroborated out of various labs.
As the synthetic RNAs became more and more complicated and revealing more and more of the assignments, some perplexing problems arose as the copolymers shown here, produced nothing at all!
Only much later, was it discovered that AUG was a "stop" signal, and UAA, UAG and UGA were "start" signals. So, of course, a copolymer of these would not be translated.
In summary then, here is the complete genetic code as known today:
The above codon assignment chart shows what the code is for E. coli, but what about other living things? Here is the power of the Nirenberg System:
It doesn't make a difference which sources for ribosomes, tRNAs, etc., are used. They all give the SAME polypeptides!!!
THE GENETIC CODE IS UNVERSAL!