An Interview with Manuel Varela and Ann Varela: Masayasu Nomura: The Ribosome Protein Factory is a Super Multiplex Molecular Machine!

Jun 29, 2020 by

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Figure Ribosome subunit – small 30S structure

Michael F. Shaughnessy

1) A fascinating scientist—Masayasu Nomura—where was he born and where did he go to school, and what do we know about his early childhood?

Dr. Masayasu Nomura was a molecular biologist and biochemist whose main pioneering work about the Escherichia coli and yeast ribosomes paved a path towards the molecular mechanisms for ribosome assembly, gene regulation, and protein synthesis. Nomura was born on the twenty-seventh of April, 1927, in Hyogo-ken, Japan. He attended high school, called the general school, in Tokyo, during the Second World War.

In an autobiography, he reported that his educational grounding in the sciences had been hampered by the war and post-war adversity. First, many of his science teaching laboratory facilities had been destroyed by allied bombings. Another consequence was the lack of excellent teachers or mentors who could provide advice on career and scientific matters. Other adverse outcomes encompassed negligible income, food shortages, and the onset of disease.

When the war ended in 1945, he was 18 years old, and he was relieved that he did not have to sacrifice his life for the war effort, an accomplishment that was thought to be the natural order in Japan during that era.

Nomura earned his B.S. in microbiology at the University of Tokyo in 1951. He took his Ph.D. in microbiology at the University of Tokyo in 1957, in the laboratory of his graduate advisor, Professor Kin-Ichiro Sakaguchi.

Upon completing his Ph.D., Nomura spent the next few years in the position of postdoctoral researcher at the University of Illinois, Harvard University, and Purdue University in the United States. From 1960 to 1963, Nomura was employed as an assistant professor at the Osaka University Institute of Protein Research. In 1963, Nomura joined the faculty in the Department of Genetics at the University of Wisconsin in Madison. He was bestowed full professor status in 1966. By 1970, he was selected co-director of the Institute for Enzyme Research at the University of Wisconsin, where he remained until 1984. In 1984, Namura was nominated and selected as the first recipient of the Grace Beekhuis Bell chair of Biological Chemistry at the University of California, Irvine.

2) Nomura had a rely on various investigatory methods that we would consider as “primitive,” but apparently, he was able to disassemble and reassemble the ribosome in a test tube! What did he learn from this experiment or process, and what have we learned from him?

Nomura began investigating the ribosomes when, in 1960, as a postdoctoral fellow, he entered the laboratory of bacteriologist and professor, Dr. Sol Spiegelman, at the University of Illinois. They studied infection of Escherichia coli by bacteriophage T2. At the time, it was understood that a small amount of RNA was made during T2 phage infection of bacteria. But it was unclear whether the RNA was of viral or bacterial origin. Working with Benjamin Hall, Spiegelman and Nomura examined the RNA produced during the T2 phage infection and found that there were two distinctive RNA types: bacterial and viral.

Furthermore, the bacterial RNA was actively associated with the Escherichia coli ribosome when large concentrations of magnesium were present. They could separate the RNA from the ribosome using low amounts of magnesium. Nomura, Hall, and Spiegelman referred to bacterial RNA type as ribosomal RNA (rRNA), and they published the work in the Journal of Molecular Biology in 1960.

During the summer months, Nomura worked at Harvard University in the laboratory of the famous James Watson. At Harvard, Nomura met Dr. Matthew (Matt) Meselson, who told him about a breakthrough that he had when working with Sidney Brenner and François Jacob. They had performed the “primitive” experiment you spoke of above. First, they exploded bacteria, exposed their internal cytoplasmic contents, added highly concentrated solutions of saturated cesium chloride (CsCl), and spun the mixtures in a test tube at extremely high speeds for several days.

The old-fashioned spinning method was known as the density gradient centrifugation, and it is still used to this day. The method has been necessary for analyzing mixtures of cellular components that differed in terms of molecular weights and densities. The groundbreaking result was two peaks, one for each of the two large ribosome subunits, the 30S, and the 50S, also called the B-band and the A-band, respectively. The “S” had to do with the so-called Svedberg rate obtained during the old-fashioned CsCl density gradient centrifugation experiment. Some sources like to denote the “S” as a “sedimentation coefficient” rate. Together, the two portions produced a massive 70S band, representing an intact ribosome with its 30S and 50S subunits attached.

Nomura was to recall in later years that in the early 1960s, he wondered by 30S and 50S parts added up to 70S and not 80S. Thus, the first experiment he conducted at Harvard involved a closer examination of the two bands. He showed that each could be further broken down into even smaller proteins. Then, Nomura added back the smaller ribosomal parts and reassembled the entirety of the ribosomes.

For instance, he used pure 16S RNA with 30S proteins to assemble the 30S ribosome subunit. He could disassemble and assemble the 30 ribosomal subunits at will. Furthermore, when these reassembled ribosomes were applied to mRNA, the mixtures were functional in that they could make new proteins!

Next, Nomura turned his attention into the natural process for the assembly of the 30S ribosome subunit in Escherichia coli. The work involved determining the order of the steps involved in the ribosome subunit assembly process. He showed that when two or more sub-components were brought together, the complex would favor the binding of the next component to the assembly complex. In the end, Nomura devised a so-called ribosome assembly blueprint, which he called a map.

Then, Nomura focused his interest in identifying and isolating the genes that encoded individual rRNA molecules and proteins associated with the ribosomes. He also studied the regulation of the ribosome synthesis mechanism in bacteria and later, in eukaryotes, like yeast.

3) Now I surely want to focus on this—how did he find that certain things could make bacteria resistant to antibiotics—and why is this important?

Nomura inadvertently entered the field of bacterial resistance to antibiotics during his unrelated attempts to identify and then isolate the genes that encoded the ribosomal proteins and the mechanisms that regulated their gene expressions. During the course of these investigations, he discovered a new mechanism for antibiotic resistance against the famed antibiotic called streptomycin.

Nomura had heard about the works of others, like Ole Maaløe, Jon Beckwith, who were also trying to find and clone genes. At about that time, during the late 1950s and early 1960s, recombinant DNA technology for gene cloning was not yet fully developed. So, gene identification and isolation methods involved classic genetic approaches.

One such classic gene isolation technique involved bacteriophages and bacteria—in a process known as transduction—using viruses to introduce foreign genes into new host cells. One of these transducing phages was called lambda (λ), whose viral genome inserted itself into a region of the bacterial genome near a gene encoding galactose-metabolizing machinery. Following how an infected bacterium used the galactose sugar for food provided a clear indicator of how close the viral and bacterial genes were located.

Another classic genetic cloning approach that pre-dated recombinant DNA technology was referred to as transposition. A transposon was a genetic element of DNA that could migrate from one location in a genome to another location. In this case, transduction was done with the phage called Φ-80, (phi-80), a virus, to introduce the transposons into bacteria. The transposon insertion occurred near the lactose-utilizing genes, called lac, of the bacterial genome. The bacterial sugar metabolizing genes were, thus, isolated using these transduction and transposition methods.

Nomura used these old classical gene cloning methods, transduction, and transposition, effectively. He combined these methods with his biochemical protein reconstitution approaches, as described above, to study the ribosome protein-encoding genes. In so doing, Nomura was the first investigator to isolate a gene that encoded a ribosomal protein, called strA. The strA gene was the first ribosome protein gene to be identified, and it was Nomura who discovered it, publishing the work in the journal Nature in 1969. It was a significant finding.

In his ribosome protein gene identifying experiments, Nomura exploited the fact that ribosome genes were known to be adjacent to an antibiotic-resistant gene for streptomycin resistance along the Escherichia coli genome, a region called “72 minutes.” The units of time, minutes, refers to the 100 minutes that Escherichia coli took to transfer its genome by mating.

If mating was stopped with a blender at 72 minutes during conjugation, the location of the resistance gene could be mapped by later testing for susceptibility to streptomycin. With the first ribosome protein gene, thus, identified, Nomura now had to isolate the DNA encoding the gene. At first, their efforts to do so failed.

Then, Nomura recalled reading a 1966 paper by Jon Beckwith and others in which they had shown that the strA and lac genes were close to each other in a transposition mutant of Escherichia coli, a strain called EC2. Nomura thought that if he could get his hands on the EC2 mutant, he might be able to use the λ phage system to insert its viral genome with its lac gene into the region along the genome near the strA gene. The approach might be used to obtain the DNA that codes for ribosome proteins.

Dr. Beckwith kindly provided the needed Escherichia coli strain, and Nomura and his colleagues successfully used the EC2 mutant to isolate a series of infected bacterial strains that carried ribosomal protein genes!

They had tried an elegant two-pronged approach.

First, they conducted an in vivo study. Here, the investigators used phages and infected Escherichia coli bacteria that had been exposed to ultra-violet light radiation. Next, they measured the production of radioactive proteins after exposure of the bacteria to radioactively labeled amino acids.

Second, they performed an in vitro study. In this experiment, Nomura and colleagues measured translation using DNA as a starting point to identify which of the proteins had become labeled in the above in vivo study.

After the studies were finished, they found 28 ribosome protein genes, plus a bonus of genes encoding RNA polymerase (involved in transcription), and a couple of translation proteins, called EF-Tu and EF-G! They even found genes that encoded metabolism-conferring machinery, such as those for aromatic amino acids.

Luckily for Nomura and his research team, the technology for sequencing DNA had just been developed. They initially focused on gene promoter and gene coding regions of the candidate DNA fragments that they found. They used these findings to explore specific gene regulation elements on DNA, explained later in this chapter.

In another groundbreaking study, they discovered a new mechanism for streptomycin resistance in Escherichia coli. In the study using biochemical techniques, they purified 30S ribosome proteins from two sets of Escherichia coli cells, one sensitive and one resistant to streptomycin. Then, they reconstituted the ribosome assemblage using the 30S proteins from the two microbial sets, and they measured translation with or without streptomycin.

They found that in mixtures containing Escherichia coli mutant-derived 30S ribosome proteins, the streptomycin did not stop translation. In contrast, 30S ribosome proteins from wild-type bacteria did inhibit protein synthesis with streptomycin—the antibiotic failed to work, providing resistance. Thus, Nomura discovered a new mechanism for streptomycin resistance, and it was called ribosome alteration. The target of streptomycin is the ribosome. Nomura discovered that bacteria become resistant to the antibiotic streptomycin by altering the target, the ribosome.

File:Ecoli 70S ribosome pair vs gentamicin.png

https://commons.wikimedia.org/wiki/File:Ecoli_70S_ribosome_pair_vs_gentamicin.png

Figure Escherichia coli ribosomes and the antibiotic gentamicin

In modern times, we know about several classes of antibiotics that target the bacterial ribosome. Some antibiotics target the 30S subunit of the bacterial ribosome. Examples include streptomycin, gentamicin (see the figure) tetracycline, spectinomycin, and kanamycin. The streptomycin, in particular, disrupts the translational accuracy of the ribosome, making aberrant proteins that do not function, leading the bacteria to die from the lack of proper proteins.

On the other hand, different antibiotic classes target the 50S ribosomal subunit of bacteria. Examples include clindamycin, chloramphenicol, and macrolides (like erythromycin, clarithromycin, or azithromycin). In both cases, whether the 30S or 50S subunits are targeted, these antimicrobial agents are known as protein synthesis inhibitors. These types of antibiotics prefer to affect bacteria. If bacteria cannot make protein, they cannot survive.

Eukaryotes, like human beings, are not significantly affected by protein synthesis-inhibiting antibiotics. The ribosomes of eukaryotes are sufficiently different such that these antibiotics do not bind to them tightly. Thus, these antimicrobial agents may be prescribed for humans with infectious diseases caused by bacteria without succumbing to too many side effects. Incidentally, side effects that are observed with antibiotics have to do with their alteration of the healthy gut microbiome, rather than with a detrimental effect on human ribosomes or other drug targets.

The antibiotics will hopefully inhibit the growth of infectious bacteria by inhibiting their (and not our) protein synthesis machinery, i.e., ribosomes, thus, effectively treating the illness. In some cases, the infecting bacterium may already be mutated such that they harbor an altered target, like a mutated ribosome, resulting in the antibiotic medicine not working to alleviate the infection. Often, it requires a prescription of another antibiotic with effectiveness. Frequently, the physician will order lab tests to identify the infecting microbes and to obtain a list of effective antibiotics to prescribe to maximize the efficacy of the medicine.

In modern microbiology laboratory courses, students may conduct exercises to hunt for streptomycin-resistant bacteria. Such resistant mutants of bacteria can be readily obtained—one merely needs to incubate the sensitive bacteria in the presence of the antibiotic for one to two weeks! Soon, rare mutants appear and grow, and they can be isolated for closer study. Frequently, the students find that the bacteria have altered their ribosomes.

More recently, it was discovered that bacteria employ other mechanisms for streptomycin resistance. For example, the bacteria can use streptomycin-degrading enzymes to destroy the antibiotic, or bacteria can actively export the antibiotic, or even prevent antibiotic entry into the bacterial cell. Additionally, bacteria can make new proteins that bind to the ribosome to block the antibiotic from binding the target site on the ribosome.

4) How do cells seem to control growth via the forming of proteins and RNA in ribosomes?

Living cells can control their growth by regulating the activities associated with the ribosomes, the factories of protein synthesis. The process protein synthesis, i.e., translation, involves several molecular players.

One prime player is, of course, the ribosome itself. On the molecular level, it is a massive multiplex machine. It consists of many proteins (about 55) and RNA molecules. These are usually referred to as ribosomal proteins and ribosomal RNA (rRNA), respectively. There are three main types of rRNAs, called 5S rRNA, 23S rRNA, and 16S rRNA.

Another translation player is the transfer RNA (tRNA). These tRNA molecules serve to deliver specific amino acids to the translational machinery. There is quite frequently one specialized tRNA molecule, which harbors the anti-codon, for every different amino acid. Thus, at least 20 individual tRNA molecules are known.

Another key player in translation is the messenger RNA (mRNA), which harbors the codons and carries the genetic code of DNA in the form of RNA. The mRNA contains the genetic message, i.e., codons, which in turn specify each of the various 20 amino acids in their correct order within the protein.

The ribosome itself has two portions, called the small (30S) and large (50S) subunits. When bound together, they form an intact 70S macromolecular complex. The 30S subunit harbors 16S rRNA plus 21 proteins, whereas the 50S subunit contains the 5S and 23S rRNAs plus 34 proteins. The large subunit harbors two active sites. The A-site is called as such because it is the aminoacyl-binding site—the location where the transfer RNA brings an amino acid to the machine. The P-site is called the peptidyl-site, and it is a location where the growing protein (i.e., a polypeptide) emerges from the translational machine.

https://upload.wikimedia.org/wikipedia/commons/thumb/1/10/TRNA_ribosomes_diagram_en.svg/373px-TRNA_ribosomes_diagram_en.svg.png

https://upload.wikimedia.org/wikipedia/commons/thumb/1/10/TRNA_ribosomes_diagram_en.svg/373px-TRNA_ribosomes_diagram_en.svg.png

Figure Ribosome and RNA

The translation mechanism involves three distinct phases. The first phase is called initiation, where the individual players come together to assemble into the protein-making machinery. The second phase is called elongation, where the growing polypeptide molecule form by attaching amino acids to form chains. Interestingly, the enzyme that performs the amino acid attachments is called peptidyl transferase. A fascinating property of the ribosome protein-making function is that the enzyme called peptidyl transferase is RNA! The enzyme is RNA! Therefore, the protein-making enzyme is called a “ribozyme.” One of the first ribozymes, RNase P, was discovered by Sidney Altman, who was featured in one of our earlier books.

The third phase of translation is called termination, where the ribosome reaches a termination codon on the mRNA molecule, and the release factor then frees up the nascent polypeptide so that it can fold into a three-dimensional structure to become active. Typically, when complete, the translation machine players come apart from the assemblage and float away. The newly folded active protein will then proceed with its cellular function to keep a cell alive.

5) What would you say are his most relevant and salient accomplishments?

Dr. Masayasu Nomura made many scientific discoveries, any of which is worthy of mention in modern scientific textbooks dealing with biochemistry, and molecular and cell biology. Below is a summary of some of his scientific accomplishments.

One of his earliest scientific contributions as an independent investigator, first at Osaka in the early 1960s and later in the decade at the University of Wisconsin, dealt with the mode of antibacterial action of the colicins. In modern times, the colicins are referred to as the bacteriocins. These antibiotics are produced by bacteria to inhibit the growth of neighboring competitor microbes.

Nomura studied three of these bacteriocins, which he called K, E2, and E3. He found that bacteriocin K killed bacteria by inhibiting the production of DNA, RNA, and proteins, making the antibiotic versatile. Nomura discovered that E2 caused the degradation of DNA. For E3, Nomura discovered that the bacteriocin inhibited translation by causing the cleavage of 16S ribosomal RNA by an RNase action. In the late 1960s and early 1970s, Nomura’s group discovered proteins that conferred immunity to E2 and E3 colicins.

As discussed above, many of Nomura’s studies focused on reconstituting protein subunits of ribosomes. In so doing, he elucidated not only the assembly process of ribosome components, but he also delineated the natural order of the ribosome construction in bacteria. Nomura was a central figure in the investigations of the in vitro ribosome assembly, as well, such as producing a blueprint for the 30S ribosome subunit. His pioneering work contributed to our knowledge of the structure-function relationships with ribosomes. Thus, Nomura provided key mechanistic details fundamental to protein synthesis.

Nomura was much interested in the regulation of these essential biochemical systems in molecular terms. In particular, he was fascinated about how microbes such as Escherichia coli regulated the synthesis of ribosomes and of the components that make up ribosomes. In so doing, he identified and isolated many of the genes that specify ribosome-specific proteins. He then studied the regulatory elements of these genes.

One significant discovery dealt with the mechanism of translation feedback regulation loops for the synthesis of Escherichia coli ribosome proteins. Several of these regulatory systems involved a variety of genetic operons, each of which specified promoter elements, and which were often regulated by RNA products of distinctive operons. Likewise, Nomura developed schemes for regulatory proteins that acted by modulating the translation of other translation products, each tied to each other by a vast regulatory network in bacteria. He identified several repressor proteins that were used in this translational regulation scheme.

After evaluating translation-based gene regulation in bacteria, Nomura shifted to studies involving the eukaryote Saccharomyces cerevisiae, a yeast microbe. The change in Nomura’s research field provided new insights into the regulation of eukaryotic rRNA transcription. He found that the synthesis of these rRNA molecules involved an RNA polymerase I enzyme produced in the yeast organism. Having before studied rRNA genes from bacterial microbes, he moved forward to discover genes uniquely concerned with rRNA synthesis in the yeast microbes. His feedback loop mechanism played essential roles in prokaryotes as well as in eukaryotes.

Lastly, Nomura elucidated central molecular mechanisms for the initiation of rRNA transcription. He discovered three transcription factors, called upstream activation factor (UAF), core factor (CF), and a Pol I transcription initiation factor, called Rrn3p. Regarding UAF, he elucidated its sub-components, which consisted of proteins like Uaf39p and histones H3 and H4. Using these and other molecular players, Nomura constructed a novel mechanism for the initiation of transcription in yeast microorganisms. Such molecular mechanisms of transcription initiation are still featured in molecular and cellular biology textbooks in modern times.

6) Is he still alive, and what is he doing?

Sadly, Professor Masayasu Nomura died in November on the 19th, in 2011, at the age of 84, in California, the U.S. He left behind a legacy of fundamental knowledge in molecular biology and biochemistry. The discoveries made by Dr. Nomura form a basis for our understanding of life at a molecular level. They inform our knowledge base of transcription, translation, antibiotic action, antimicrobial resistance, and regulation of gene expression. The scientific work of Nomura is still covered in popular books, as well as in textbooks relating to biochemistry, molecular biology, and cell biology. His scientific influence will last many centuries.

7) What have I neglected to ask about this famous scientist?

Masayasu Nomura wed Junko Hamashima on the tenth of February 1957. The couple had a daughter named Keiko and a son named Toshiyasu. Nomura’s hobbies included hiking and reading.

Strangely, Masayasu Nomura did not garner a Nobel. He was, however, immensely highly regarded by his scientific colleagues. Nomura was the recipient of numerous prestigious awards, including the 1971 National Academy of Sciences Award in Molecular Biology. He received the UC Irvine Distinguished Faculty Lectureship Award for Research (2000 -2001). Namura was awarded the 2002 Abbott-American Society for Microbiology Lifetime Achievement Award. Nomura was also a Fellow of the American Academy of Microbiology and the American Association for the Advancement of Science. Namura had become a member of the National Academy of Sciences in 1978. He was also a member of the Royal Netherlands Academy of Arts and Sciences and the Danish Academy of Science.

For additional information regarding the distinguished Prof. Masayasu Nomura, visit these sites:

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