For Want of a Template [Rich Losick and Jan Pero]

We were not alone in the search for phage-induced subunits of RNA polymerase. The laboratories of Peter Geiduschek and Helen Whiteley were also hot on the trail, having discovered that RNA polymerase from phage-infected cells of B. subtilis was associated with novel proteins and transcribed phage middle genes. But, in a particularly gratifying series of experiments, Tom Fox was able to prove that the three RNA polymerase-associated proteins (σ^gp28, σ^gp33, and σ^gp34) were in fact the products of the SP01 regulatory genes that had previously been shown to be required for middle and late transcription. This was no mean feat as cloning and DNA sequencing were yet to be invented. Instead, Tom grew Okubo’s SP01 phage nonsense mutants for each of the three regulatory genes in two different suppressor strains. These suppressor strains inserted differently charged amino acids at the same nonsense mutation. He then showed that for each phage mutant, one of the three RNA polymerase-associated proteins following growth in the two suppressor strains exhibited two distinct isoelectric points during two-dimensional gel electrophoresis. This could only be explained if each alternative sigma factor was in fact the product of the corresponding regulatory gene. Meanwhile, graduate student Robert (Tij) Tjian, who had joined Jan, Jane, and Tom on the SP01 project, showed that the gene 33 and gene 34 proteins functioned together as an alternative sigma factor. He purified the proteins and added them back to core RNA polymerase from uninfected cells and demonstrated that both were necessary and sufficient to confer late-specific transcription. Thus, the program of SP01 transcription could be explained by a simple cascade in which the house-keeping sigma factor (σ⁵⁵, now called σ^A) of the host turned on phage early genes including the gene for σ^gp28, which in turn activated phage middle genes including the genes for σ^gp33 and σ^gp34, which together activated phage late genes.

“The invention of DNA cloning and a method for detecting specific DNA fragments by hybridization with radioactive nucleic acid probes . . . changed our lives.”

Jan started her own lab at Harvard in 1975, a time when women scientists were finally being recognized with faculty appointments. When Jan started graduate school, there were no women faculty members in the Biological Laboratories, but her appointment took the tally to four. Jan’s first graduate students Carol Talkington and Gloria Lee sequenced five middle promoters recognized by σ^gp28-containing RNA polymerase. Middle promoters exhibited their own distinctive and conserved −10 and −35 sequences, leading us to propose in the pages of this journal that sigma factors work by directly contacting the −10 and −35 regions of their cognate promoters. Our hypothesis was met with skepticism but ultimately proved to be correct.

This was an intensely exciting period for us, but juggling two careers and raising a son (and later a daughter) was no mean feat. Once when we separately had lectures scheduled at conferences in Tennessee and at Princeton University over almost the same dates, the only way that we could make it work was by handing off our 2-year-old son at the Newark airport! Somehow our children managed to survive this career intensity to become successful adults.

The search for sporulation sigma factors continued through the 1970s. The invention of DNA cloning and a method for detecting specific DNA fragments by hybridization with radioactive nucleic acid probes (by Stan Cohen and Herb Boyer and by Ed Southern, respectively) changed our lives. Now, finally, we had the means to obtain our long-sought templates containing sporulation-specific genes! Using these powerful new tools, graduate student Jackie Segall cloned a DNA segment that ultimately proved to contain promoters for multiple alternative sigma factors. Using Segall’s DNA as a template, postdoc Bill Haldenwang purified RNA polymerase containing what proved to be the first alternative bacterial sigma factor, σ³⁷ (now called σ^B). In subsequent work. he purified σ³⁷ and added it back to core RNA polymerase, showing that this polypeptide alone was sufficient to direct the transcription of a cloned gene and proving that it was indeed an alternative sigma factor. We originally believed that σ³⁷ was a sporulation sigma factor, but when we cloned the gene for σ³⁷ and knocked it out, the mutant was unimpaired in spore formation. Although we did not develop this work further, Chet Price at the University of California, Davis went on to demonstrate in beautiful work that σ³⁷ is a stress-response sigma factor.

Haldenwang did not give up the search for sporulation sigma factors. He and graduate student Naomi Lang (later Unnasch) purified a form of polymerase from sporulating cells that contained a sigma factor dubbed σ²⁹ (now σ^E) that was indeed unique to sporulation and, as we later demonstrated, required at a specific stage of spore formation.

Subsequent work (carried out by talented students Barbara Kunkel and Valerie Oke; postdocs and former postdocs, among them Simon Cutting, Bill Haldenwang, Lee Kroos, and Charles Moran; our collaborator Patrick Stragier; and others in the field including Jeff Errington, Patrick Piggot, Pete Setlow, Issar Smith, and Michael Yudkin) led to the discovery of four developmental sigma factors (σ^E plus: σ^F, σ^G, and σ^K) in the small (forespore) and large (mother cell) cellular compartments of the sporangium. The production of these factors unfolds in a cascade, such that σ^F in the small compartment directs the appearance of σ^E in the large compartment. The σ^E factor, in turn, activates σ^G in the small compartment. Then, in a final step, σ^G triggers the appearance of σ^K in the large compartment. Thus, the four factors in the cascade are linked by intercellular signaling pathways and, as we came to learn, two that operated, remarkably, at the level of the processing of pro-protein precursors to σ^E and σ^K. (Activation of transcription factors by pro-protein processing was a major surprise, and indeed, the processing enzyme for pro-σ^K proved to be the founding member of an iconic family of intramembrane proteases that includes the human Site-2 protease for regulatory proteins that control sterol biosynthesis.) We dubbed this cascade crisscross regulation. Using the same templates as Haldenwang, former postdoc Charles Moran, who was now running his own lab at Emory and collaborating with Issar Smith, discovered a fifth sigma factor required for sporulation, σ^H, this time a factor present in growing cells and required for entry into sporulation.

A remarkable feature of the gene for σ^K deserves special mention. As a postdoc, Lee Kroos had identified the gene for σ^K from the N-terminal amino acid sequence of the protein, but the gene was too small to encode the entire sigma factor! Meanwhile, Jeff Errington’s lab had identified a gene whose inferred product resembled the C-terminal region of a sigma factor. The two half-genes were relatively close (∼48 kb apart) on the chromosome. Patrick Stragier, Barbara Kunkel, Lee Kroos, and Rich discovered that the intervening sequence, a prophage-like element, is removed from the chromosome in the mother cell by site-specific recombination. Sonenshein later found that a prophage-like element similarly interrupting the coding sequence for σ^K in certain Clostridia is also removed during sporulation by site-specific recombination. Unexpectedly, phage-like elements were hitchhiking on the sporulation process in a manner that required their removal from the somatic chromosome of the mother cell, which is destroyed after spore formation, but not from the germline chromosome of the forespore, which becomes packaged into the spore.

Over time, the increasing availability of DNA templates and the power of bacterial genetics made it possible for us and others to discover additional sigma factors. And indeed, alternative sigma factors of multiple families emerged as a widespread feature of bacterial taxa. Among these are sigma factors governing the heat-shock response, gene activity in stationary phase, motility, the response to nitrogen limitation, and a large and diverse family of “extra-cytoplasmic function” sigma factors. Each sigma factor became the star of its own intricate and engaging story as unraveled by many outstanding researchers, including Mike Chamberlin, Mark Buttner, Carol Gross, and Sydney Kustu. Over time it became apparent that house-keeping sigma factors and alternative sigma factors are a distinctive feature of bacteria, being absent in the other domains of life. While lacking sigma factors, archaea and eukaryotes typically have a DNA-binding protein called the TATA-binding protein that helps anchor their RNA polymerases, which otherwise share a common evolutionary history with bacterial RNA polymerases, to promoters. One is therefore left wondering about the nature of the transcription machinery in the last common universal ancestor (LUCA) that pre-dated the division into the three domains of life—did it have sigma factors, TATA-binding proteins, both, or something else?

How priviledged we were to participate in such an exciting chapter in the history of molecular biology both as scientific collaborators and as a married couple. And from this vantage point, we got to witness the birth of the field of eukaryotic transcription and the discovery of the myriad transcription factors that govern gene expression in higher cells. Meanwhile, Rich went on to tackle questions of cell biology, multicellularity, and stochasticity in B. subtilis, while Jan helped launch two biotech companies that harnessed the power of B. subtilis genetics and genetic engineering for the production of vitamins and useful enzymes.