RNA Replicase and Spiegelman's "Little Monster," 1961-1969

In 1961, Spiegelman became intrigued by the "strange biological situation" of a recently discovered bacterial virus (phage) called MS2. This phage had no DNA--its genetic material was RNA. How, then, did it complete its life cycle in a cell dominated by DNA? Spiegelman reasoned that the RNA strand injected into its host (in this case, E. coli) must serve directly as its own "translator" for directing its replication. But how did it do this, when there was no known mechanism in bacterial cells for making RNA copies from RNA? Ironically, in light of later discoveries showing just such a mechanism in tumor viruses, his first idea was that RNA viruses make DNA copies of their genes, using a reverse transcription enzyme. The DNA would then direct the virus replication. But in looking at E. coli infected with MS2, he could find no evidence of a "reverse transcriptase" or of DNA that would hybridize with the viral RNA. He was forced to assume that the RNA virus was not making DNA copies of itself.

Working on this assumption, Spiegelman looked for an enzyme that would replicate RNA directly. The quest, which occupied the better part of two years, was complicated for several reasons. First, bacteria contain a variety of enzymes that can incorporate ribonucleotides into preexisting RNA chains. They also contain DNA transcriptase, which can sometimes use certain types of RNA as substitute templates for RNA synthesis. Distinguishing these from a direct RNA replicating enzyme would be challenging. Second, Spiegelman later noted, the RNA virus had a special dilemma:

Consider an RNA virus approaching a cell some 106 times its size and into which the virus is going to inject its only strand of genetic information. Even if the protein-coated ribosomal RNA molecules are ignored, the cell cytoplasm still contains of the order of 10,000 free RNA molecules of various sorts. The viral RNA contains the information required for the synthesis of the new kind of polymerase designed to make RNA copies from RNA. If this replicase were indifferent and accepted any RNA it happened to meet, what chance would the single original strand injected have of ever being replicated?

The most likely solution would be an enzyme that could recognize one specific RNA and ignore all other RNA molecules encountered--that is, a template-specific enzyme. Working on this assumption, using purified, intact viral RNA, Spiegelman and his team found the RNA-dependent polymerase specific to phage MS2. When RNA bases and strands of RNA from MS2 were combined with this polymerase, the RNA replicated; when the polymerase was added to any other RNA strands, nothing happened. Reception of this discovery was cautious, for all the nucleic acid polymerases known at the time were not specific. A few years later, however, Spiegelman's teams demonstrated that the RNA polymerase coded for by a second RNA phage--Q-beta--was specific to Q-beta RNA and would replicate no other.

The replicase of RNA phage Q-beta turned out to be "a nice, stable, clean enzyme," relatively easy to isolate and purify, and within a year Spiegelman could easily make phage RNA in the test tube that was physically and chemically indistinguishable from the original virus RNA. In 1965, to prove that the test-tube RNA was infective, he first set up a serial transfer experiment, adding phage RNA to RNA base materials and Q-beta replicase, letting the RNA replicate, then taking a little of the resulting RNA and adding it to a second replicating system. This process was repeated fifteen times, so that at the end the original RNA would be diluted out by the transfers almost completely, leaving in the final tube RNA that was virtually all lab-generated. When added to E. coli protoplasts (bacteria lacking a cell wall) this RNA produced virus particles just as well as RNA from a native Q-beta virus. It was the first time that a piece of nucleic acid made in the test tube worked as well as the stuff made in nature. Spiegelman had achieved the first synthesis of a biologically competent, infective viral nucleic acid. This feat (termed the "dream experiment of modern biology" by one writer) created a sensation in the scientific world, and brought a wave of "life in a test tube" newspaper stories. Spiegelman was quick to qualify his accomplishment, however, and explained, "When you create a living object the presumption is that the object didn't exist before. This I did not do. Working with simple chemical compounds, I take a primer of a living object and I generate many living objects from it."

The synthetic Q-beta RNA (which Spiegelman called "the little monster") yielded other scientific dividends during the next few years. Because he could produce unlimited generations of RNA, Spiegelman was able to watch Darwinian selection in action at the molecular level. By varying the selective pressure on the system (such as temperature, nutrient mix, or the reproduction time allowed) he could "create" mutant RNA molecules with a wide range of properties. One of these, for instance, gradually trimmed itself down from the original 3,300 nucleotides to 470--just big enough to grab the replicase enzyme and copy itself. It had apparently jettisoned sections that coded for protein coats and other components it didn't need in the artificial environment. Other mutations were resistant to a variety of inhibitors of RNA synthesis. For Spiegelman, these experiments solved an important puzzle of pre-cellular evolution, for they showed that, "long before cells were invented, there could have been selective forces that pushed nucleic acid molecules to greater and greater length and complexity in order to solve the selective problems." In 1969 Spiegelman was awarded a patent for the synthetic virus RNA, one of the first genetic patents.