Tag Archives: ECR

ECRs at ICaMB – Green transcription: how studying Cyanobacteria could change the world

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In the latest of our series on the Early Career Researchers of ICaMB we asked Dr Yulia Yuzenkova to tell us about her research and the route that took her from a PhD in Moscow to being awarded a Royal Society University Research Fellowship in Newcastle.

Yulia
Dr Yulia Yuzenkova

My early training was in Moscow, firstly as an undergraduate at Moscow State University and then for a PhD from the Russian Academy of Sciences. However, during my PhD I moved to the USA to study at the Waksman Institute (Rutgers University) with Prof Konstantin Severinov, where I was also a postdoc. My work in the laboratory was dedicated to the molecular mechanisms of inhibition of bacterial transcription by antibacterial peptides and small proteins from viruses. Transcription is the first step and critical regulatory checkpoint of gene expression. In all living organisms transcription is performed by multi-subunit RNA polymerases (RNAP). The central role of transcription in cellular metabolism and the presence of domains specific for bacteria make RNAP an obvious target for antibiotics. Yet, for decades, only one inhibitor of RNAP, rifampicin, has been used in the clinic to treat tuberculosis, while only recently, in 2011, lipiarmycin (fidaxomicin) was approved to eradicate Clostridium difficile. Two is a very small number, but this indicates that there is a good probability of finding new drugs that also work by targetting RNAP. Apart from their clinical significance, antibiotics and inhibitors of transcription in general have been proven to be very efficient molecular tools (see “RNAP details” below for more info).

During my second PostDoc in the lab of Prof Nikolay Zenkin in Newcastle, I have focused on the mechanisms controlling the fidelity of transcription. The copying of genetic information by RNAP is far from being absolutely precise, and RNAP whimsically dislikes reading some DNA sequences, resulting in ‘pauses’. RNAP is able to correct its own mistakes using a proofreading mechanism and the help of small proteins called transcript cleavage factors (explained in detail below). It seems to be important to have at least one cleavage factors; otherwise, the RNAP molecules stop, resulting in “traffic jams” as trailing molecules keep moving and bump into it.

Batch culturing of cyanobacteria

Batch culturing of cyanobacteria

It was therefore a big surprise for me to learn that one large group of bacteria, cyanobacteria (details below), do not encode anything even remotely resembling cleavage factors. I started to look for an explanation for this extraordinary fact. Are there any factors that might compensate for the absence of cleavage factors? Is cyanobacterial RNAP so accurate and at the same time processive that it does not need them? In searching for the answer, I realised that almost nothing is known about the molecular details of transcription in cyanobacteria, and so I decided to apply for a Royal Society University Research Fellowship to try to answer these questions.

Microscopy image of membrane-stained cell of Synechocystis sp 6803

Microscopy image of membrane-stained cell
of Synechocystis sp 6803

In performing preliminary experiments for my application, I became fascinated with cyanobacteria, as they are truly amazing organisms to work with. They are the only prokaryotes that exhibit the classic circadian clock, and are the bacteria with the most complex intracellular organisation. Moreover, they are one of the most ecologically important groups on Earth. They live everywhere where sunlight is available and produce 30% of atmospheric oxygen; some can even convert inert atmospheric nitrogen into a digestible form.

 

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

The 2D projection of the membrane structure of cyanobacterium reminds me of a labyrinth

With my Royal Society University Research Fellowship I am planning to investigate the molecular details of the transcription machinery and will look for novel transcription factors that regulate this process. I am also going to test the metal requirements of cyanobacterial transcription, because metal composition of cyanobacterial cells is very different from other bacteria to suit the need of photosynthesis. Another fascinating question is how fast in the cyanobacterial cell, which is tightly packed with photosynthetic organelles, can molecules find their way through the membrane labyrinth.

 

 

The nitty-gritty science:

RNAP in detail:

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Inhibitors of RNAP as molecular tools for understanding its functions. A wide range of targets of known inhibitors is mapped on the structure of bacterial RNAP. We contributed to understanding the modes of action of inhibitors marked in bold.

Studying the antibiotics and inhibitors modes of action have helped us and other groups to discover previously unknown functions and structural domains of RNA polymerase. For example, work on rifampicin shed light on geometry of the RNA exit path, long before the crystal structure of RNAP was solved. Moreover, streptolydigin led to the discovery of the novel catalytic domain, while microcinJ25 confirmed the proposed entry channel for substrates and tagetitoxin provided insight into the mechanisms of RNAP translocation along the template.

Newly synthesised RNA participates in the proofreading  in a ribozyme-like manner. This method of proofreading, probably a remnant from the distant past, is extremely slow. To accelerate proofreading (and to escape from pauses), all 3 domains of life encode non-homologous, but very similarly folded, small proteins called transcript cleavage factors. In E.coli, GreA is an example of a protein that fulfils this role. Some bacteria have several homologs of GreA (in E.coli there are at least 6). It seems to be important to have at least one, because if these cleavage factors are depleted in the cell, the RNAP molecules on the actively transcribed genes stop, obstructing transcription, but also the chromosomal replication machinery moving along the same DNA.

Cyanobacteria:

Cyanobacteria have been hailed as future photobioreactors.  Indeed, when supplied with little more than tap water and light, engineered cyanobacteria can produce all sorts of compounds from sunscreen to biofuels. Cyanobacteria can also be used for environmental applications such as greenhouse gas fixing and cleaning water from industrial pollutants. These initiatives, however, are compromised by slow growth of cyanobacteria and limited knowledge of their basic biology. By putting more effort into research, the potential abilities of cyanobacteria can eventually be harnessed on the industrial level. With this we could make a giant leap towards a future “greener” economy. With a little bit of imagination, it is not hard to envisage cyanobacteria helping humanity to colonise new worlds, and to permit them to inhabit the first lunar and martial greenhouses in not so distant future.

Dr Yulia Yuzenkova’s ICaMB website: http://www.ncl.ac.uk/camb/staff/profile/yulia.yuzenkova

Royal Society University Research Fellowship: https://royalsociety.org/grants/schemes/university-research/

ECRs at ICaMB: RNA Quality Control

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Claudia SchneiderIn the latest of our series focussing on the ECRs in ICaMB, we feature Dr Claudia Schneider. Claudia obtained her PhD from the Philipps-University in Marburg, Germany. She then moved to the UK to work with Prof David Tollervey at the Wellcome Trust Centre for Cell Biology in Edinburgh. In 2011, she was awarded a Royal Society University Research Fellowship and started her own lab at ICaMB. Here, Claudia describes her research, and how being alarmed during her postgraduate studies triggered her long-term research interest.

By Dr Claudia Schneider

Hi, my name is Claudia Schneider, and my Royal Society University Research Fellowship has allowed me to set up my own group here at ICaMB to study enzymes involved in RNA processing and quality control.

I am originally from Germany, where I did my undergraduate studies and my PhD. Many people might think that Germany is the land of cars or lederhosen – but in truth it is really the land of bread (and beer!). It might therefore not come as a surprise that baker’s yeast has become my favourite model organism.

Click on the image to find out how to make these budding buns!

Click on the image to find out how to make these budding buns!

During my undergraduate studies I was first introduced to RNA and I was (and am still) amazed by its many known and still emerging functions in the cell. We now know that almost the entire eukaryotic genome is transcribed, but only a small fraction of the transcripts are protein-coding messenger RNAs (mRNAs). The others are stable and unstable non-coding RNAs (ncRNAs), which are involved in all aspects of gene expression. RNA molecules are often extensively processed before they’re functional, and each processing step is subject to quality control mechanisms. If you want to know more about the life and death of non-coding RNAs, have a look at this recent review.

Yeast has not always been my first choice to study RNA metabolism, since the object of my PhD project in Prof Reinhard Lührmann’s lab turned out to be completely missing in baker’s yeast. Back then I worked on nuclear pre-mRNA splicing, the removal of non-coding introns from precursor mRNAs catalysed by the spliceosome. It was an exciting time in the splicing field: A second low abundance “minor” spliceosome had just been discovered in most multicellular eukaryotes (with the strange and still not readily explainable exception of C. elegans), and this complex is not present in yeast. The minor spliceosome recognises a rare class of introns (<0.5%) with different consensus sequences at the splice sites and has since been linked to a number of human diseases. During my PhD, I purified and biochemically characterised the snRNP components of this unusual pre-mRNA splicing machinery in human and Drosophila cells.

Since then, I have been fascinated by biochemical and enzymatic assays involving RNA such as in vitro splicing assays, where in vitro transcribed pre-mRNAs are mixed with purified spliceosomes. The goal of such an experiment is to observe precise and (hopefully) pretty intron removal in the test tube – but, to my great annoyance, success was every so often hampered by a powerful ribonuclease (RNase) contamination in the assay that completely trashed the precious RNA substrates. Generic and aggressive RNases like RNase A are found on our skin, are incredibly stable and can even survive boiling.

Common decoration on lab surfaces during my PhD

Common decoration on lab surfaces during my PhD

It is therefore fair to say that my scientific career was majorly influenced by constant warnings by my mentor, who told me that all RNases are evil and must be destroyed. However, for my postdoc, I decided to face my fears and look these evil RNases in the eye, in the humble model system yeast. During my time with Prof David Tollervey at the University of Edinburgh, I learned that there are many different types of RNases, and only very few of them are promiscuous and chop RNA to bits.

The majority of RNases are very sophisticated and versatile enzymes. Several RNases are capable of degrading only specific RNA molecules, or only function under certain circumstances, and protein co-factors often assist in substrate recognition. RNases are crucial elements in RNA quality control or surveillance systems, which distinguish aberrant from “normal” RNA molecules. One clinically important RNA surveillance pathway is called “nonsense-mediated decay” or NMD, and this system recognises and degrades a specific class of defective mRNAs to limit the synthesis of truncated and potentially toxic proteins. NMD defects are linked to ~30% of all inherited human diseases (e.g. Duchenne muscular dystrophy and forms of b-thalassemia) as well as certain types of cancer. In addition to quality control/surveillance, where RNAs are mostly completely degraded, a growing number of RNases have been shown to be responsible for precise processing or “trimming” of precursor RNA molecules to produce their functional forms.

Exonucleases were long believed to be the main players involved in RNA recognition and processing/turnover. However, this model was recently challenged by the identification of endonucleases containing PIN (PilT N-terminus) domains, which appear to play key roles in RNA metabolism. Eight PIN domain proteins and therefore putative endonucleases are encoded in the genome of budding yeast, and this includes three largely uncharacterised “orphan” nucleases.

Overall it is still puzzling to me how individual RNases “make the decision” to either completely degrade or carefully process a specific RNA. Given the ever-growing number of non-coding transcripts in the cell, I am also keen to know which RNases are responsible for which substrates and what the so-far uncharacterised putative PIN domain endonucleases in yeast are doing!

To this end, our lab is using an RNA-protein cross-linking method called “CRAC” (UV cross-linking and analysis of cDNA) to identify the targets of PIN domain endonucleases on a transcriptome-wide scale. The CRAC method and the machinery to cross-link yeast cultures were developed by Sander Granneman at the University of Edinburgh, when we were both PostDocs in Prof David Tollervey’s lab. Sander now has his own lab too, and he runs a CRAC-blog. Interestingly, the cross-linking device we are using was originally designed to sterilise sewage water, but it is now also commercially available for research. With this setup, yeast cells are cross-linked while they are growing in culture, which is crucial to identify the often very transient interactions between nucleases and their target RNAs. This system provides a huge advantage over more traditional cross-linkers like the “Stratalinker”, which requires pelleting and cooling the cells on ice before cross-linking. It is, however, also much bigger and takes up a whole bench in the lab – but I guess there is a drawback to everything! In any case: if you want to find RNA targets for your favourite yeast protein – get in touch!!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Cultures of Saccharomyces cerevisiae can be “zapped”, while they are growing: It only takes 100 seconds!

Transcriptome-wide RNA-protein interaction analyses generate huge datasets and we use RNA binding and nuclease assays, as well as co-precipitation studies, to validate the in vivo cross-linking results for individual PIN domain endonucleases.

With the help of an ERASMUS exchange student, Franziska Weichmann, who spent 6 months in my lab last year, we have made good progress with two putative PIN domain endonucleases that are linked to ribosome biogenesis. We were able to identify their binding sites on the pre-ribosomal RNAs, as well as co-factors that are important to recruit them into the pre-ribosome. We have also set up an in vitro system with recombinant proteins, and we are currently trying to convince one of them to specifically cleave its proposed rRNA substrate in the test tube – and we are slowly getting there…..

Like the other ICaMB ECRs, who posted on this Blog before, I would like to finish by saying that having my own lab has been an exciting and (on most days) enjoyable adventure so far – and I am looking forward to the next set of challenges…

ECRs at ICaMB: Copying the blueprint of life – Understanding DNA replication

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Heath MurrayIn the latest of our series focussing on the Early Career Researchers (ECRs) in ICaMB, we feature Dr Heath Murray.  After completing undergraduate studies at the University of California, Los Angeles and then obtaining his Ph.D. from the University of Wisconsin-Madison, Heath came to the UK to join the lab of Prof Jeff Errington in Oxford. From there he re-located to ICaMB, and in 2009 was awarded a Royal Society University Research Fellowship. Here, Heath describes his research into the mechanisms of DNA replication, and explains why he became interested in this field.

By Dr Heath Murray

Hello, my name is Heath Murray and I’m a Royal Society University Research Fellow in ICaMB’s Centre for Bacterial Cell Biology (CBCB) studying DNA replication. DNA is one of the most important molecules required for life because it encodes the information, or the blueprint, used to build a cell (i.e. the most basic unit of an organism). In order for a cell to create new cells it must synthesize an exact copy of its DNA, an extraordinary process when you consider that the genomes of most cells contain millions of individual DNA subunits!

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacillus subtilis is a useful model system as it proliferates rapidly and is amenable to genetic, cell biological, biochemical, and structural analyses

Bacteria are ideal model systems to study this fundamental process because they are much less complex than human cells (e.g. all of their DNA is encoded by a single chromosome, whereas humans have 23), and this allows us to understand how they work at the greatest possible level of detail.

I was introduced to bacteria when I was an undergraduate student and the effect was transformative. My mentors taught me how to add a specific gene (a DNA sequence) to a bacterial cell, and if it worked properly then the bacteria would turn blue!

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

Bacterial colonies turn blue if they contain a gene that degrades specific sugars.

That basic genetic experiment was one of the coolest things I had ever done, and from that point on I worked hard to learn the trade of “bacterial genetics”.

Today my research group focuses on understanding how DNA replication is controlled so that each new cell will end up containing an exact copy of the genetic material from its predecessor. We employ a wide range of complementary experimental techniques: genetic engineering of bacterial strains, biochemical analysis of purified proteins, and fluorescence microscopy.

In the hot room to check my plates.... I haven't even stopped to take my jacket or backpack off yet!

In the hot room to check my plates…. I haven’t even stopped to take my jacket or backpack off yet!

Fluorescence microscopy is a particular strength of the CBCB because there are several bespoke systems specifically designed for bacterial cells (bacteria are 10-100 times smaller than most human cells). One of the core approaches we use is to genetically engineer a protein we want to study so that it will be fused to a special reporter protein called GFP (Green Fluorescent Protein, originally isolated from jellyfish!) within the cell. Using this approach we can then visualize where our test protein is because it fluoresces when exposed to a specific wavelength of light. Some of our microscopes are so sophisticated that we can observe the location of single proteins and track their movements within living cells.

At the bench.

At the bench.

One of the approaches we often use is to visualize specific regions of the genome within living cells. First, a specific DNA binding protein ( called “LacI”) is genetically fused to GFP. Second, the DNA sequence recognized by LacI (called “lacO”) can be genetically integrated into any location of the genome. Since I study DNA replication, I am particularly interested in the site of the bacterial chromosome where DNA synthesis is initiated (called the “replication origin”). Third, fluorescent dyes are added to cells that bind to the cell membrane and the DNA. Finally, we utilize our fluorescent microscopes to visualize the location of replication origins within individual cells. In the image shown, the live bacterial cells contain chromosomes that are in the process of being replicated, and therefore they have duplicated and separated their replication origins!! This image also emphasizes the fact that although bacteria lack the organelles found in eukaryotic cells, they are nonetheless highly organized (notice how the replication origins are characteristically located at the outer edge of each chromosome).

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

The GFP protein from jellyfish can be used to fluorescently tag proteins in vivo. Fluorescence microscopy can then be used to localise the tagged protein within the bacterial cell.

Well that’s it for my first ICaMB blog! I hope you enjoyed hearing about how I became interested in bacterial genetics and about my work on bacterial DNA replication. Please feel welcome to contact me if you have any questions or if you would like further information regarding my research.

 

Links

Royal Society URF http://royalsociety.org/grants/schemes/university-research/

Centre for Bacterial Cell Biology (CBCB) http://www.ncl.ac.uk/cbcb/

 

ECRs at ICaMB: Solving 3D puzzles

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by Dr Paula Salgado

After nearly one year editing the ICaMBlog, the time has come for me to tell you about my science and work since I joined ICaMB almost 18 months ago.

The fact that it has been 18 months since I moved up North to establish my own research group seems to have snuck up on me… Don’t get me wrong, so much has happened that, if anything, it’s surprising it all took place in 1 and a half years. At the same time, the feeling of a new adventure is still there.

Science is a constant adventure to seek new knowledge, to understand new mechanism, to see new things. In my case, to see into the very core of life’s machines: proteins. I use X-ray protein crystallography to probe the structure of proteins. It’s a bit like solving a puzzle: fitting the pieces of information together until we have a 3D view of the protein.

It is actually fitting that my blog post is the first ICaMB publishes in 2014 as this is the International Year of Crystallography. I could write a lot about it, but for now, I’ll leave you with an amazing video made by the Royal Institution that explains it all – in cartoons! If you want to know more about Crystallography, the Ri has a great collection of videos there, including Prof Stephen Curry’s Friday Evening Discourse, which I strongly recommend.

Freezing protein crystals for data collection at Diamond Light Source. H&S warning: liquid nitrogen is a hazard and we do handle it safely. At this point, I was just dipping the crystals into a small volume, all other procedures handling larger volumes involve wearing appropriate protection.

As a protein crystallographer, I’ve always been interested in proteins that have a relevance to human disease and used this technique to understand their structure and function. In the last few years, I’ve worked on proteins from human pathogens associated with hospital acquired infections, particularly Clostridium difficile and Candida albicans. However, protein structures don’t necessarily give us all the answers and they must be complemented with biochemical studies, as well as in vivo experiments. So my long term goal has become to establish a Structural Microbiology group, where we focus on structure determination of key proteins and complexes involved in pathogenicity as well as on their functional in vivo characterisation.

This is a challenge as it means stepping out of my structural biology comfort zone into the world of microbiology and cell biology. Not that I haven’t stepped out of my comfort zone before – if anything, those are areas that featured strongly during my undergraduate training as a Biochemistry student at the University of Porto in Portugal. In those days, choosing to do protein crystallography as my undergraduate project was the big step into the unknown. A trend that continued as a post-doc, when I joined Dr Cota and Prof Mathews group, a Nuclear Magnetic Resonance (NMR) lab at Imperial College, learning a completely different approach to protein structure determination. And just before coming to ICaMB, I worked in Prof Fairweather’s microbiology lab and always tried to learn a bit about the techniques others were using. So the current idea of bringing structural biology and microbiology expertise together in my lab is the natural evolution of these experiences.

C. difficile cells (green rods) lining the microvilli of the human gut. © Wellcome Trust (CC-BY)

Since joining ICaMB, I’ve focused on 2 main projects, both involving proteins from C. difficile. This spore forming strict anaerobe is resistant to most antibiotics and colonises the gut of individuals whose microbiome has been disturbed by these drugs. It is the most prevalent cause of gastrointestinal infections in hospitals and is a major cause of morbidity and mortality in the hospital environment. Despite recent decreases in the number of deaths and infections as hygiene procedures have improved in the UK, over 1600 people died in England and Wales in 2012 due to C. difficile infections (CDI). It also causes a huge burden to health systems, with an estimated €3,000 million per annum costs in the EU.

C. difficile disease symptoms are caused by the toxins it releases in a process that has been extensively studied over the years. However, the mechanisms of colonisation of the gut and spore formation are poorly understood. So we have been focusing on proteins involved in these two mechanisms.

Firstly, I’ve been trying to determine the structure of SlpA, the main protein constituent in C. difficile S-layer. S-layer is a paracrystalline coat that covers the cell and is presumed to act like a defense mechanism, as well as being involved in gut colonisation. This work, initiated a few years ago in Prof Fairweather’s lab is now a joint collaboration between our two labs and Dr Fagan, at Sheffield University.

SlpA crystals viewed under polarised light (protein crystals are birefringent, unlike salt crystals)

 

 

As this protein has tendency to form 2D paracrystalline layers, getting well ordered 3D crystals required for X-ray crystallography has been a challenge, but I have now succeeded in obtaining good crystals. However, other hurdles still need to be overcome to get a structure – but we are getting there!

 

 

Our lab: Adam Crawshaw and Paula Salgado

Last year, Adam Crawshaw joined my group as a BBSRC Doctoral Training Programme (DTP) student and we started a new project, looking at a complex between two membrane proteins that are essential for spore formation. As spores are the infectious agents, revealing the molecular details sporulation is important to understand the pathogenicity and infection cycle of C. difficile.

SpoIIQ and SpoIIIAH localise at the membranes of the forming forespore. Green: Membrane; Red: SNAP-tagged proteins.

When spores are first formed, a small cell (forespore) is engulfed by the larger mother cell, physically isolating it from the environment and nutrients in the medium. So, for the forespore to fully mature, it needs a nurturing channel to the mother cell. The two proteins we are studying  – SpoIIQ from the forespore membrane and SpoIIIAH from the mother cell membrane – create this channel. We have already successfully produced recombinant versions of the proteins and shown their interaction in vitro. In collaboration with Prof Henriques at ITQB, Lisbon, we also established their localisation during C. difficile spore formation. Next: crystals! But we are also investigating potential enzymatic activity both in vitro and in vivo, to bring the structure and biology together.

It has been an exciting year and a half – a steep learning curve with many new tasks, from supervising students to managing a lab and teaching undergraduates and postgraduates. The adventure continues, with new challenges and exciting discoveries ahead.

 

Links

International Year of Crystallography http://www.iycr2014.org/

Royal Institution Crystallography gallery http://richannel.org/celebrating-crystallography

Office for National Statistics (Clostridium difficile data) http://www.ons.gov.uk/ons/rel/subnational-health2/deaths-involving-clostridium-difficile/2012/index.html

European Centre for Disease Intervention and Control on Clostridium difficile  http://www.ecdc.europa.eu/en/healthtopics/healthcare-associated_infections/clostridium_difficile_infection/pages/index.aspx

BBSRC Doctoral Training Programme http://www.ncl.ac.uk/fms/dtp/