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Biophysicist Jens Michaelis takes a look into the molecular machine room

Jens Michaelis is extremely interested in the molecular machines that control the gene expression process. He has developed a method that enables basic researchers to localise biomolecules in real time as well as gaining insights into their spatial order. In fact what the method does is actually allow researchers to watch proteins at work.

Basic researchers like Jens Michaelis are still a long way off their goal of monitoring proteins in their physiological cellular environment. However, complex physical methods are providing increasing insights into gene expression.

Using one molecule to investigate other structures

Prof. Jens Michaelis studies the molecular details of gene expression. © Univ. Ulm

Prof. Michaelis has been the director of the Institute of Biophysics at the University of Ulm since 2011. He studied physics at the University of Ulm, did his master’s degree at the University of Oregon, USA, and his doctorate at the University of Konstanz in the laboratory of Jürgen Mylnek. In Konstanz, Michaelis dealt with the quantum optics of dye molecules. His doctoral thesis also involved the development of a microscope that used a single molecule as light source – a kind of molecular torch – that illuminated other structures. 

As with the work of other researchers, Michaelis’ research efforts need to be understood as an attempt to overcome the limits of traditional light microscopy resulting from the physical phenomenon of refraction limit. Traditional light microscopy can only be used for looking at a large number of proteins, but not for analysing individual proteins, which is of course of particular interest for biological research.

Michaelis had to carry out complicated experiments before he finally succeeded in obtaining microscope images that resulted from the light of a single molecule. The physicist had to ensure that the dye molecule did not fade (i.e. stop illuminating); dye molecules are very unstable and tend to change their spectral properties. Michaelis managed to stabilise the dye molecule by “shock freezing” it at a temperature of minus 271˚C (only around 2˚C from “absolute zero”, the temperature which is equal to 0 kelvins).

After obtaining his doctoral degree, Michaelis was not quite sure whether he should focus on quantum optics or biophysics. He therefore applied for and got a position as post-doc in the laboratory of David Wineland, who was awarded the 2012 Nobel Prize in Physics, and later moved on to the field of biophysics. Michaelis continued to focus on individual molecules, in this case biomolecules rather than dye molecules, and was working on elucidating the function of individual protein molecules.

NPS – Satellite system on the nanometre scale

Transcription complex of RNA polymerase II. Single molecule fluorescence microscopy and NPS provide Michaelis with dynamic information about the structure and conformational changes of flexible and temporary complexes. © Michaelis/Uni Ulm

Working with colleagues at the University of Munich, his last stop before accepting a position at the University of Ulm, Michaelis used single-molecule spectroscopy to develop a kind of satellite system known as Nano-Positioning System, NPS. This propelled him into the premier league of biophysicists; the European Research Council is funding his research on chromatin for five years, which will enable him to crack some harder nuts than would have been possible with short-term funding.

NPS allows the localisation of flexible domains in macromolecules. It is based on the energy transfer of fluorescent dyes. Dye molecules are attached to the mobile region and to the regions within the macromolecule. Three different distances are required for determining the desired relative position of a mobile region with respect to known positions within a structure. This works in exactly the same way as satellite navigation does. The ability to observe molecules in this way is an important requisite for achieving a detailed understanding of how genes are regulated. 

The method is based on single-molecule FRET – fluorescence resonance energy transfer – measurements with a fluorescence microscope: a donor fluorescent dye molecule in its excited electronic state transfers energy to an acceptor fluorescent dye moledule. The transferred energy, and hence the intensity of the FRET signal measured, depends on the distance between the two molecules. FRET is very sensitive to distances in the nanometre range. Michaelis and his co-workers combined single-molecule measurements with a method that is also applied in global positioning systems (GPS). Fluorescent dyes are fixed at known and unknown positions and their distance determined using FRET. In analogy to satellite navigation, the distances calculated can be used to determine unknown positions. The NPS can now be used as a quantitative tool (probability analysis) for investigating the position and dynamics of flexible domains within macromolecular complexes (e.g. proteins) (Muschielok, 2008).

How do proteins change?

NPS can be used to determine the relative position of individual proteins. This enables Michaelis to find answers to one of the issues that interests him the most: how do proteins change their conformation, what effects do the altered conformations of a macromolecule have? In analogy to structural biology, which is able to recognise rigid structures without time resolution, Michaelis can determine dynamic structures and observe how these structures change in real time. He can also glean information on how a protein segment moves during a biochemical process. The NPS method works well with proteins that can be isolated.

Michaelis and some of his co-workers (Treutlein, 2012) carried out numerous experiments and found out how DNA is loaded into the RNA polymerase of baker’s yeast and subsequently transcribed into RNA. These findings contribute to clarifying why conformational changes affect protein function.

The enzyme RNA polymerase II (Pol II) transcribes DNA into RNA. In Michaelis’ words, Pol II functions like a machine as it requires chemical energy for the cyclical process of incorporating bases (30 per second). Pol II is supported in its work and regulated by numerous other proteins (transcription factors). Before Pol II is able to start transcribing DNA into RNA, it needs to attach to a specific site on the gene – the promoter – and unwind several base pairs of DNA. These two steps lead to the formation of an open promoter complex. The open promoter complex accompanies the transition of transcription initiation to the start of RNA synthesis.

Comprehensive structural alterations during transcription

Michaelis and his former colleague from Munich, Patrick Cramer, recently succeeded in characterising for the first time ever the three-dimensional structure of the open promoter complex, which consists of Pol II, open promoter DNA and the initiation factors TBP, TFIIB and TFIIF, using single-molecule FRET and NPS. They found that comprehensive structural changes occur during the transition from initiation to elongation, which is the phase during which the largest part of the DNA is copied into mRNA.

Jens Michaelis describes the reseachers’ interest in solving this puzzle as follows: “We wanted to see movement in order to find out more about the speed of the entire process, at the same time as wanting to find out what happened with the protein itself, how it changed its conformation, etc.” Michaelis and his colleagues were mainly interested in the initiation of transcription; they wanted to find out how the enzyme Pol II found the site on the DNA where the copying process starts and which other proteins (transcription factors) are involved.

Michaelis’ second major topic of interest, namely the structure of DNA in cells, is closely related to his investigations on gene expression. In order to fit into cells, DNA, which is several metres long, needs to fold tightly. In addition, it needs to be packaged in such a way that enables polymerases to bind at any site of the DNA. Michaelis refers to this miracle of nature as a kind of transparent compression. The DNA is packaged by molecular machines which Michaelis likes to refer to as snowploughs. The basic unit of DNA packaging is referred to as a nucleosome, in which the DNA is wrapped twice around a histone octamer consisting of two copies each of the four core histones.

Solving the puzzle of DNA packaging: what is the function of the 100 remodellers?

The chromatin structure has to be flexible and dynamic in order to enable or prevent regulatory proteins having access to the DNA. Several mechanisms ensure controlled chromatin changes, one of which is molecular machines known as remodellers that change the positions of the histones within nucleosomes. But little is yet known about how the complex process of moving the histones around works. The fact that human cells contain around 100 different remodellers is surprising in many ways at the same time as explaining the complexity of the gene expression process. Many questions still require answers: why are there so many protein complexes? Why is one complex not enough? What does each complex specialise in?

Several hypotheses have been put forward to explain these basic mechanisms, which are believed to involve the formation of loops: the DNA wrapped around the histones unwinds at a specific position and forms a loop with the assistance of a remodeller. This loop moves around the nucleosome. However, these models are still highly speculative. Michaelis is convinced that there will still be a lot of work for future generations of researchers to do.

Some of Michaelis’ colleagues have recently focused on chromatin research (Bönisch, 2012) and discovered an alternatively spliced protein in humans that plays a novel role in chromatin structure regulation. This protein is a histone 2A.Z variant that destabilises the nucleosome by attaching to a specific site on the chromatin. 2A.Z causes major structural changes and significantly destablises nucleosomes.

In the near future, Michaelis and his team of 15 physicists, biologists and chemists are going to publish more papers on their EU-funded chromatin research. “We’ve accumulated quite a lot of information, and would like to publish it soon,” said Michaelis who hopes that they will soon have obtained detailed insights into the molecular mechanism underlying the rearrangement of nucleosomes. In the medium term, the researchers hope to carry out similar experiments with living cells.

Literature:
Bönisch, C., et al.: H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization, Nucleic Acid Research, 2012, 1-14, doi: 10.1093/nar/gks267

Treutlein et al., Dynamic Architecture of a Minimal RNA Polymerase II Open Promoter Complex, Molecular Cell (2012), doi: 10.1016/j.molcel.2012.02.008

Muschielok, A. et al.: A nano-positioning system for macromolecular structural analysis, in : Nature methods  5/11, Nov. 2008, S. 965-971, doi:10.1038/NMETH.1259

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