MicrobiologyBytes

“In my day” i.e. when I started my PhD back in 197<cough>, the first few weeks were spent joining the Enzyme Club. This encompassed all the biomedical researchers at the University of Leicester. Each new student would prepare a batch enzyme for recombinant DNA work. In my case, I made Hsu I (an isoschizomer of Hae III but allegedly easier to prepare). Since it was years ago, I can’t remember how many litres of the organism I grew up, but I remember very clearly doing the first assay on two litres of crude extract, and figuring out I was holding £40 million pounds worth of enzyme at the then current market prices. The first affinity column cut it down to £15 million, and a quick gel filtration to couple of millions pounds worth – still pretty good for two weeks work, especially when you remember that two million pounds was enough to buy you a house back in the 1970s!

Why did the Enzyme Club exist? Because these reagents were scarce in the 1970s, and rationed both by price and availability. Only a few years before, the only way to get hold of any of these enzymes was to make your own. This type of open science made sense. Why did the Enzyme Club cease to exist? Gradually, it became clear that the batch of enzyme I made wasn’t very good. It had a persistent exonuclease activity which meant it was fine for restriction analysis but rubbish for cloning, and it went off very quickly in storage, so that after three months there wasn’t much activity left. And although I’ve always been a rubbish protein chemist, that was a pretty common experience. Gradually, the companies dropped their prices and improved both the quality and availability of commercial enzymes. The day came when the Enzyme Club didn’t make sense any more, and it quietly died. It’s probably still moldering in the back of a coldroom over in the MSB.

So boys and girls, this is a story of the economics of open science, which made sense in response to scarce resources. When the availability of enzymes was limiting, this open approach made sense. When time became limiting, we all retreated back into our laboratories and got on with whatever we needed to do to get a PhD. The moral of this story is that open science pops up it’s head when times are hard and resources are scarce, but retreats quickly as the balance changes.

Transcription, the process of converting DNA into RNA (which in turn is translated into proteins by ribosomes) is carried out by the multisubunit RNA polymerase (RNAP) enzyme. Transcription is fundamental to all organisms across the three kingdoms of life – Eukarya, Bacteria, and Archaea – and can be divided into three major steps: initiation, transcription/elongation, and termination. Eukaryotes have three different nuclear RNAPs, whereas Archaea and Bacteria have one. Archaeal transcription is similar to that of eukaryotes, but initiation requires only two accessory proteins bound to DNA: transcription factor B (TFB) and TATA-box binding protein (TBP). It is believed that studies of the archaeal enzyme may shed light on the more complex eukaryotic RNAP. New research has shown that organisms which live in boiling acid read their DNA using enzymes surprisingly similar to our own, providing insight into the way in which the information stored in DNA is unlocked. This new work has shown that the enzyme that converts DNA into RNA is conserved between simple single-celled microorganism Sulfolobus and more complicated “higher” organisms, including human beings, despite a staggering 2 billion year evolutionary gulf. A new paper explores how evolution has shaped our own RNAP enzyme to accomplish more complex functions.

Many Archaea are extremophiles; living in high salt, acid or temperature environments. Transcription, the process of reading DNA to make RNA (which is in turn translated into proteins by ribosomes) is a fundamental process common to all organisms, and is carried out by the enzyme multisubunit RNA polymerase (RNAP). Eukaryotes have three different RNAPs, whereas Archaea and Bacteria have one. Archaea can serve as a wonderful model system because their simpler RNA polymerase machinery is related to the more complex eukaryotic RNAP. To start transcription, the archaeal enzyme requires two accessory proteins whilst the eukaryotic counterpart needs at least two more. This increased complexity prompts two important questions: how did our polymerase evolve from the ancestral enzyme; and how does Archaea bypass the requirement of further co-factor proteins?

New work investigates the polymerase from the Archaeon Sulfolobus shibatae using X-ray crystallography. This reveals the enzyme’s architecture which confirms its close evolutionary relationship with the eukaryotic RNAP. The research also identified a subunit novel to Sulfolobus which has no equivalent in the eukaryotic enzyme. The striking structural similarities suggest that the ancestral eukaryote used the same enzyme as the Archaeon, and that modern eukaryotic RNAP evolved by the addition of bolt-on proteins that regulate eukaryotic-specific processes. From the location and topology of the newly identified, Archaeon-only subunit, the scientists have suggested a mechanism by which Archaea do without the additional cofactors required by eukaryotes for initiating transcription. The scientists also noted that the complete structure of the archaeal polymerase illustrates how the ancestral core enzyme was modulated by addition of novel subunits, an evolutionary process that has facilitated the complexity that we see today in Eukarya.

Evolution of complex RNA polymerases: The complete archaeal RNA polymerase structure. 2009 PLoS Biol 7(5): e1000102
The archaeal RNA polymerase (RNAP) shares structural similarities with eukaryotic RNAP II but requires a reduced subset of general transcription factors for promoter-dependent initiation. To deepen our knowledge of cellular transcription, we have determined the structure of the 13-subunit DNA-directed RNAP from Sulfolobus shibatae at 3.35 A° resolution. The structure contains the full complement of subunits, including RpoG/Rpb8 and the equivalent of the clamp-head and jaw domains of the eukaryotic Rpb1. Furthermore, we have identified subunit Rpo13, an RNAP component in the order Sulfolobales, which contains a helix-turn-helix motif that interacts with the RpoH/Rpb5 and RpoA9/Rpb1 subunits. Its location and topology suggest a role in the formation of the transcription bubble.

Related:

• Unique cell division machinery in the Archaea