Manuel Varela: Who Discovered that RNA acts like an enzyme AND was a member of an Ice Hockey Team at M.I.T.?

Oct 31, 2017 by

An Interview with Manuel Varela: Who Discovered that RNA acts like an enzyme AND was a member of an Ice Hockey Team at M.I.T.?

Michael F. Shaughnessy –

1) Professor Varela, first of all, while people may have HEARD about DNA—what exactly IS it, and why is it important?

Thank you for inquiring about one of my favorite molecules. In short, DNA is considered by many to be the principal molecule of life. As such, DNA can be envisaged as a never-ending living molecular blueprint for specifying all life on Earth.

DNA is a storage form containing a set of biological directions, so to speak, to confer life upon molecules.  This famous DNA molecule directs certain biological functions within all living cells. Along these lines, DNA has associated with it certain molecular biological machinery for mediating the transcription process, also known as RNA synthesis. That is, DNA participates in its conversion of the genetic information stored in it to a messenger RNA transcript (mRNA) version of the code.  The mRNA transcript version, in turn, works in conjunction with other forms of RNA, like ribosomal RNA (rRNA) and transfer RNA (tRNA) to perform translation for the purpose of producing gene products, such as proteins.

Hence, the expression of a gene involves the production of RNA and then protein, encoded within DNA. These proteinaceous gene products then perform their biological functions to produce and maintain new progeny cells and tissues contained within intact living organisms.

In addition to specifying molecular blueprints for living organisms, DNA also has molecular mechanisms for propagating its genetic blueprint information into the next generation, and the process occurs in the cells of all living beings on Earth. The genetic information propagation process also involves the synthesis of new DNA, also called DNA replication.

Many scientific investigators have used DNA to determine, compile, and annotate its nucleotide base sequences, placing the acquired genetic information into very large computer-based databases, for the purposes of identifying gene functions and of facilitating the study of protein biochemistry, physiology, and cell biology. DNA is a central molecule in the biomedical sciences discipline.

DNA has additional relevance. For instance, scientific investigators have used DNA as a tool for the basic and applied study of life. In these regards, molecular biologists have cloned the DNA molecules that code for genes contained with genomes of organisms. Genes that are defective in genetic diseases, and which have been cloned, facilitate the acquisition of new cellular biological and biochemical knowledge that may, in turn, be exploited to treat or circumvent genetic disease, to lessen the suffering connected with genetic diseases. The field of molecular medicine is an active area of investigation.

DNA has practical purposes, too. For example, DNA may be exploited to diagnose diseases, using biotechnological toolkits to do so. Additionally, in forensic medicine, DNA may be used to identify victims and perpetrators of crimes. It may also be effectively used to identify parental lineages in child custody disputes.

In the last several decades, DNA has been used to perform gene therapy, for the specific purpose of correcting a defective gene by replacing it with a properly functioning version of the gene, in order to express the correct gene product properly and reverse the aberrant function that may occur during a genetic disease.  Such measures may restore the clinical utility of the normal gene. More recently, a gene-editing technology, called CRISPR, has emerged as the promising new and upcoming invention for correcting a damaged or defective gene in all cells of a living organism.

2) Now, to set the stage for the next scientist, what exactly is RNA and why is it important for scientists?

I think it is intriguing that the traditional DNA structure primarily has one major and very famous 3-D double helical shape, while RNA molecules, on the other hand, have the ability to form a variety of distinctive 3-D shapes. As I briefly alluded to above, RNA serves as an intermediate molecule between the genetic code embedded in DNA and the proteins specified by the code. The three RNA forms, mRNA, rRNA and tRNA, have their individual functions—all of which are geared towards the production of protein chains, which then fold into their respective and functional 3-D shapes.

In terms of the three various types of RNA, the mRNA type, for example, serves as a messenger of the genetic code information. As such, mRNA constitutes a conduit between the code stored in DNA and the biological functions conducted by protein. The tRNA molecules participate in transferring specific amino acids from the cytoplasmic milieu to the translational machinery; and a specific tRNA molecule exists for each of the 20 amino acids that make up protein molecules. Much of the rRNA molecules form a large part of the cellular ribosomes, which are protein-making entities, and they (rRNA) participate directly in attaching amino acids to each other.

With respect to these RNA functions, the investigative works performed in the laboratories of Drs. Sidney Altman and Thomas Cech revealed a new function for RNA. They found that RNA could act like proteins in the sense that they could catalyze biochemical reactions. This was, in fact, a shocking and unheard of discovery. Altman reported that his laboratory had relatively less trouble publishing their findings as Cech’s work had been already known.

3) Sidney Altman in the 80’s shared a Nobel Prize with Thomas Cech for looking at the catalytic properties of RNA. Why is and was this important?

Prior to each of the Nobel Prize winning discoveries in the research laboratories of Drs. Altman and Cech, RNA had been thought of primarily as a player in the so-called Central Dogma for the flow of genetic information between generations and in the replication of DNA within generations.

As Profs. Altman and Cech independently demonstrated, RNA was found to confer catalytic reactions, similar to those previously known to be performed mainly by enzymatically based proteins. That is to say, Altman and Cech discovered that RNA could act like an enzyme and catalyze a biochemical reaction.

In fact, in order to accommodate the newly discovered function for RNA, the term ‘ribozyme’ was coined to denote the new fact that RNA could harbor catalytically-based characteristics, similar to those seen in conventional biochemical reactions mediated by proteins.  Sidney Altman and colleague Norman Pace studied one of these ribozymes closely in the bacterium called Escherichia coli. Referred to as ribonuclease P, or RNase P, the new ribozyme was determined to be a combination of RNA with a protein component, the latter of which was found to stabilize the RNase P ribozyme. Altman and Pace took an E. coli RNase P substrate, a tRNA molecule, and observed that the RNase P had cleaved a portion of the tRNA substrate. The result was that the tRNA product was now a bit shorter than before. Thus, cleaving the tRNA eventually allowed it to become a mature tRNA molecule and now bind to an amino acid, making it ready for participation in the protein-making translational process.

For the first time in recorded history, scientific investigators began to think in terms of RNA molecules serving in the additional new role of mediating biochemical reactions, a process that, until this work by Altman and others had become known, was thought to be performed solely by proteins. The now arcane notion that ‘proteins do everything’ had been challenged and in a most direct fashion.

4) Apparently, as a youngster two things impressed him—the periodic table of the elements and the dropping of the Atomic Bomb—how did these two things impact his later learnings?

Interestingly, in his brief autobiographical description for the 1989 Nobel, Dr. Altman nostalgically alludes to a gift consisting of a book detailing the periodic table of the elements. Altman conveyed his fascination with the table of elements and the scientific method in general with its elegance and capacity to make effectual new scientific predictions.  A few years later, as a teenager, he was to invoke elegantly the scientific method of discovery himself to make one of the most important discoveries in molecular biology and biochemistry.

Dr. Altman spoke about the time he learned about the atomic bomb, designed and developed in Los Alamos, NM, when he was a young child. He had become captivated by the realization that human being scientists had had a direct role in its invention. It brought to the young Altman the feeling that scientific progress could play very important roles in society.

5) For a while, he worked at the Medical Research Council Laboratory of Molecular Biology in Cambridge, England. Why was this important in his professional growth and development?

In an interview, published in 2002, Dr. Altman conveyed his experiences as a fledging postdoctoral fellow at the MRC Laboratory for Molecular Biology.  Altman attributes his success in discovering his Nobel-Prize worthy RNase P system to his initial work at the MRC.

Prior to his postdoctoral experience at the MRC, Altman had graduated in 1960 with his undergraduate degree in physics from Massachusetts Institute of Technology (M.I.T.), located in Cambridge, MA, where, as you had briefly alluded, he had become an active participant in M.I.T.’s college hockey team.

Soon after his arrival to the MRC in 1969 to begin a new postdoctoral fellowship, Altman had been initially interested in determining the crystal structure of tRNA. Unfortunately, he had heard through the grapevine by prominent molecular biologists Sydney Brenner and Francis Crick that someone else had already crystalized the tRNA structure.

Thus, Altman decided instead to focus on studying RNA biochemistry in mutational variants of tRNA molecules. The new work led ultimately to his Nobel work.

In order to pursue his interest and conduct these new studies, Altman used a special class of chemical mutagenic compounds, called acridines, to induce the production of mutations in tRNA. Next, he labeled his variant tRNA molecules with a radioisotope to detect his new variant molecules. Then Altman exposed his mutated radioactive tRNA molecules to the cytoplasmic contents of broken bacteria, called a cell lysate; to his astonishment, he found that the front ends of his tRNAs had been clipped off.  He reasoned that there must be some sort of bacterial enzyme activity that chewed off the five-prime (5’) ends of his variant tRNA molecules. The mature forms of the tRNA molecules seemed to lack this 5’-end piece.

Now at Yale as an assistant professor, attempts by Altman and his graduate student, Benjamin Stark, to purify the enzyme grew infuriating as the protein failed to make its appearance unless grown in unusually large quantities, and still, an ‘RNA contaminant’ continually stayed with the protein preparation. It was at this point that Stark had suggested that the enzyme was made of RNA. Being obviously very skeptical at first about the notion of an ‘RNA molecule being an enzyme,’ they conducted a control experiment in which they destroyed the RNA contaminant with another enzyme, called micrococcal nuclease, known to digest RNA molecules quite effectively.

To their great surprise, the micrococcal nuclease treatment resulted in a loss of the tRNA cleavage of its 5’-end—the RNase P activity disappeared!  Next, they repeated their experiment, except this time, they removed a requisite cation, called calcium ion, which was needed for the RNA-destroying micrococcal nuclease enzyme to function properly. The calcium removal restored the detection of the putative RNase P enzyme function—they had shown that RNA was an enzyme, and its substrate was tRNA!

In the end, it was shown that RNase P was actually a combination of partly protein and mainly RNA—a two-component ribozyme system. Nevertheless, their experiments definitively showed that an enzyme was RNA.

6) Now, where do ribozymes fit into this big picture? Can you first define them for us and then tell us how they fit in?

Ribozymes are RNA molecules that have the ability to mediate cellular chemistry by catalyzing biochemical reactions.  Before the onset of the ribozyme concept, the biochemical catalytic process was primarily the domain of the proteins. The concept that RNA can mediate biochemical catalysis is important for several reasons. Chief among them is the implication that during the first signs of life in primordial seas, RNA-based life may have been the first, rather than DNA. This ‘RNA-first’ world has important ramifications in investigations of the origin of life on Earth.

Another important implication is that during translation, i.e., protein synthesis, in all living organisms involves a ribozyme type process.  In ribosomes, which are known to function as protein synthesizing biological machinery, RNA plays an integral role in translation. In fact, the actual component in rRNA that acutely participates in attaching amino acids to each other, a ribozyme of sorts called peptidyl transferase, has an active site composed of RNA.

One last example considered here is the idea that ribozymes may be used therapeutically to modulate the infective activities of RNA viruses, which may be pathogenic to humans and other animals. This area of scientific investigation is an active one to this day.

7) At the University of Colorado Medical Center, Altman apparently studied the effects of acridines on the replication of bacteriophage T4 DNA.  Now what exactly are acridines and why are they relevant to the replication of bacteriophage T4 DNA?

Altman obtained his Ph.D. degree in the field of biophysics at the University of Colorado, housed in their medical center, located in Boulder, CO, in 1967.

It is here in Colorado where Altman had studied effects of the acridines upon special viruses with an affinity for bacteria.  Hence, these viruses are known as bacteriophages. The T4 phages that Altman was interested in were already famously known as those that were used in the famous Hershey-Chase experiment to convey the idea that DNA was the heredity material, rather than protein.

The acridine molecules are chemicals with interesting structural and chemical properties. First, these molecules are flat (planar), which permit them to insert themselves in between the nucleotide bases, a process known as intercalation, within a double-stranded DNA molecule. The acridine intercalation distorts the double helical nature of DNA just slightly enough such that when the DNA replication machinery attempts to synthesize new DNA from acridine-associated DNA templates, the replication machinery will invariably make new mistakes when incorporating new nucleotides. Thus, the acridines play a key role in producing DNA mutations.

Furthermore, as a newly minted Ph.D., Altman had taken a postdoctoral position at Harvard University, in Cambridge, MA where he had worked under a prominent molecular biologist, Mathew Meselson, studying DNA endonuclease enzymes involved in performing DNA synthesis and recombination in the T4 bacteriophage viruses.

8) What have I neglected to ask?

I once had an unprecedented opportunity to meet Dr. Altman. As part of an organizing committee in the early 2000s for a small symposium to be held in Taos, New Mexico for biomedical scientists, I was given the charge of picking up Dr. Altman from the Sunport in Albuquerque and driving him to the conference in Taos, NM, some 2.5 hours away through somewhat rugged, but very pretty, mountainous areas.

Since I had not been accustomed to spending extended periods of time alone with such prominent Nobel Laureates, I was a bit apprehensive about the 2.5 hour drive alone with Dr. Altman. As a young assistant professor still thinking about tenure in the faraway future, I desperately wanted to avoid saying anything that might give him pause about my scientific acumen or me.  Any trepidations I had imagined, however, were immediately dispelled from the very moment we drove out of the Albuquerque International Sunport and began our two and a half hour conversation.

We talked freely about science, books, music, and history. It turned out we had been reading similar books; one in particular still stands out in my memory—we had read Frederick Holmes’ thick two-volume biography (more like a tome) about the early life and science of Prof. Hans Krebs—Altman was good friends with the author, who was also at Yale.

During the drive to Taos, I took advantage of the unique opportunity and asked Altman many questions.  He provided practical research advice that I still follow to this day—he encouraged me to continue my then preliminary studies of bacterial resistance and to be open to new research avenues, should they come up. It was sound advice.

It had been clear to me that I had been in the presence of a true genius. Furthermore, Dr. Altman turned out to be an extremely pleasant and friendly person with whom to hold an extended conversation, and I feel tremendously fortunate to have had the opportunity to pick his brain on that lovely drive through the northern New Mexico mountains.

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