Podcasts about eukaryotic

Eukaryotic cells manage to pull off a number of remarkable feats. One is packing quite a long DNA molecule, with potentially billions of base pairs, into a tiny central nucleus. A key role is played by histones, proteins that provide scaffolding for DNA to wrap around. Histones also appear in archaea (one of the other domains of life), but until recently there wasn't evidence for them in bacteria (the final of the three domains). Todays guest, Tobias Warnecke, is an author on a recent paper that claims to provide such evidence. We discuss this new result, as well as background questions of how cells evolved and what their current structure can teach us about their histories.Support Mindscape on Patreon.Tobias Warnecke received his Ph.D. in biology from the University of Bath. He is currently a Programme Leader and MRC Investigator at the London Institute of Medical Sciences. He is a co-author on A. Hochner et al. (2023), "Histone-Organized Chromatin in Bacteria."Web pageLab web siteGoogle Scholar publicationsTwitterSee Privacy Policy at https://art19.com/privacy and California Privacy Notice at https://art19.com/privacy#do-not-sell-my-info.

Algae. It's one of the greatest things on the planet and it's responsible for all life on Earth, including your life. But how much do you really know about this incredible species? Is it a plant? Why is it green? Can you eat it? Can we make it into fuel? What's up with algae blooms? Learn more in our newest episode where we talk about the benefits of algae and how it is better than human. Follow us on Twitter @betterthanhuma1on Facebook @betterthanhumanpodcaston Instagram @betterthanhumanpodcastOr email us at betterthanhumanpodcast@gmail.comWe look forward to hearing from you, and we look forward to you joining our cult of weirdness. 

Eukaryotic cells reproduce themselves by going through the cell cycle, which divides one cell into two. The cell cycle comprises two main phases, interphase and mitosis, both of which are further broken down into steps, as well as a separate resting phase. When a cell divides appropriately, this allows our bodies to fix damaged tissue and replace old layers of cells. However, when the cell cycle happens either at an inappropriate time or without stopping, cancers can develop. This is why the cell cycle is highly regulated with multiple checkpoints and myriad regulatory proteins. Next, we'll go over the steps of the cell cycle and dive into the complex regulatory mechanisms that prevent cancers from forming. After listening to this Audio Brick, you should be able to: Outline the four main stages of the cell cycle. Describe the role of cyclins and cyclin-dependent kinases in promoting cell cycle progression. Describe the cell cycle checkpoints. Outline the process of mitosis. You can also check out the original brick from our Cellular and Molecular Biology collection, which is available for free. Learn more about Rx Bricks by signing up for a free USMLE-Rx account: www.usmle-rx.com You will get 5 days of full access to our Rx360+ program, including nearly 800 Rx Bricks.  After the 5-day period, you will still be able to access over 150 free bricks, including the entire collections for General Microbiology and Cellular and Molecular Biology. *** If you enjoyed this episode, we'd love for you to leave a review on Apple Podcasts.  It helps with our visibility, and the more med students (or future med students) listen to the podcast, the more we can provide to the future physicians of the world. Follow USMLE-Rx at: Facebook: www.facebook.com/usmlerx Blog: www.firstaidteam.com Twitter: https://twitter.com/firstaidteam Instagram: https://www.instagram.com/firstaidteam/ YouTube: www.youtube.com/USMLERX Learn how you can access over 150 of our bricks for FREE: https://usmlerx.wpengine.com/free-bricks/ from our Musculoskeletal, Skin, and Connective Tissue collection, which is available for free. Learn more about Rx Bricks by signing up for a free USMLE-Rx account: www.usmle-rx.com You will get 5 days of full access to our Rx360+ program, including nearly 800 Rx Bricks.  After the 5-day period, you will still be able to access over 150 free bricks, including the entire collections for General Microbiology and Cellular and Molecular Biology.

My AP Biology Thoughts  Unit 2 Cell Structure and FunctionWelcome to My AP Biology Thoughts podcast, my name is Chloe and I am your host for episode #49 called Unit 2 Cell Structure and Function: Comparing and Contrasting the Prokaryotic and Eukaryotic Cells. Today we will be discussing the comparison between the functions and structures of these two cell types.  Segment 1: Introduction to Prokaryotes and Eukaryotes The main difference between prokaryotic and eukaryotic cells is the presence of the nucleus and other internal membranes. This lack of membrane in prokaryotic cells often causes them to lack crucial organelles which are present in Eukaryotic cells.  In Eukaryotic cells, the genetic information, the DNA, is held within the nucleus.  In a prokaryotic cell, the genetic material is carried on a singular piece of DNA which is attached to the cell membrane, and there is no enclosing membrane which causes the genetic information to come into direct contact with the cytoplasm. (This whole system is called a nucleoid, a concentration of DNA)  Overall, the main difference is the presence of membrane bound organelles in eukaryotic cells, and absolutely no membrane bound organelles or a nucleus at all in prokaryotic cells.  Segment 2: More About Prokaryotes and EukaryotesGoing more in depth, prokaryotes are ultimately unicellular organisms. In contrast, eukaryotic organisms can be unicellular, but eukaryotes are the building blocks of larger organisms  Two examples of prokaryotes include bacteria and archaea. Eukaryotic cells make up everything besides these two organisms including fungi, plants, and animals. Specific similarities between the organelles present in both prokaryotic and eukaryotic cells is that they both contain a plasma membrane, ribosomes, cytoplasm, and DNA. Although they carry genetic information differently, it is important to remember that they both still possess it.  It's important to understand the origin of these two different cells, and how it came about that they have different contents. According to the endosymbiotic theory, it is believed that two or more prokaryotic cells, living in a symbiotic relationship with each other, ultimately evolved into the mitochondria, present in only eukaryotic cells. One prokaryotic may have engulfed another, created an enclosed membrane for the new organelles that were being created by the presence of two prokaryotic cells.  Segment 3: Connection to the CourseThe endosymbiotic theory is very critical to the evolution aspect of all living things. Because two prokaryotic cells were able to work together in their own beneficial way to make a eukaryotic cell, which now make up all living things besides bacteria and archaea, is very significant. Once the eukaryotic cells were created, evolution was able to take its course, and lead us to where we are now. The creation of the membrane bound nucleus in eukaryotic cells made a huge structural difference, and made complex evolution possible. Overall, both prokaryotic and eukaryotic cells play a major role in the biological world, but it is especially important to appreciate how the eukaryotic cells were created, and how evolution took place after this occurrence.  Thank you for listening to this episode of My AP Biology Thoughts. For more student-ran podcasts and digital content, make sure that you visit http://www.hvspn.com/ (www.hvspn.com). See you next time! Music Credits:“Ice Flow” Kevin MacLeod (incompetech.com) Licensed under Creative Commons: By Attribution 4.0 License http://creativecommons.org/licenses/by/4.0/ Subscribe to our Podcasthttps://podcasts.apple.com/us/podcast/my-ap-biology-thoughts/id1549942575 (Apple Podcasts) https://open.spotify.com/show/1nH8Ft9c9f6dmo75V9imCk?si=IvI4iQV-SSaFb0ZmvTabxg (Spotify) https://podcasts.google.com/feed/aHR0cHM6Ly9mZWVkcy5jYXB0aXZhdGUuZm0vbXlhcGJpb2xvZ3l0aG91Z2h0cw (Google...

DNA structure, Eukaryotic chromosome organization, DNA replication, DNA repair, and recombinant DNA and biotechnology. --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app

On the second episode of 71%, we'll dive deep into the science of marine snow. Hosts: Dr Laura Cappelatti and Dr Ben Whittaker References: Lundgreen et al (2019) Eukaryotic and cyanobacterial communities associated with marine snow particles in the oligotrophic Sargasso Sea. Sci Reports Credits: This episode was edited in Audacity and is hosted by Anchor. The main theme is "Big Gay Waterfight" by Plushgoolash and the soundscape is "Stream, Water" by InspectorJ. Contact: Follow us on Twitter and Instagram or contact us by email (71percent.pod at gmail.com).

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.13.381962v1?rss=1 Authors: Cheng, C. L., Wong, M. K., Li, Y., Hochstrasser, M. Abstract: The proteasome is a large protease complex that degrades both misfolded and regulatory proteins. In eukaryotes, the 26S proteasome contains six different AAA+ ATPase subunits, Rpt1-Rpt6, which form a hexameric ring as part of the base subcomplex that drives unfolding and translocation of substrates into the proteasome core. Archaeal proteasomes contain only a single type of ATPase subunit, the proteasome-activating nucleotidase (PAN), which forms a trimer-of-dimers and is homologous to the eukaryotic Rpt subunits. A key PAN proline residue (P91) forms cis and trans peptide bonds in successive subunits around the ring, allowing efficient dimerization through upstream coiled coils. The importance of the equivalent Rpt prolines in eukaryotic proteasome assembly was unknown. We show an equivalent proline is strictly conserved in Rpt3 (in S. cerevisiae, P93) and Rpt5 (P76), well conserved in Rpt2 (P103), and loosely conserved in Rpt1 (P96) in deeply divergent eukaryotes, but in no case is its mutation strongly deleterious to yeast growth. However, the rpt2-P103A, rpt3-P93A, and rpt5-P76A mutations all cause synthetic defects with specific base assembly chaperone deletions. The Rpt5-P76A mutation decreases the levels of the protein and induces a mild proteasome assembly defect. The yeast rpt2-P103A rpt5-P76A double mutant has strong growth defects attributable to defects in proteasome base formation. Several Rpt subunits in this mutant form aggregates that are cleared, at least in part, by the Hsp42-mediated protein quality control (PQC) machinery. We propose that the conserved Rpt linker prolines promote efficient 26S proteasome base assembly by facilitating specific ATPase heterodimerization. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.06.371484v1?rss=1 Authors: Basu, I., Gorai, B., Chandran, T., Maiti, P. K., Hussain, T. Abstract: During translational initiation in eukaryotes, the small ribosomal subunit forms a 48S preinitiation complex (PIC) with initiation factors. The 48S PIC binds to the 5' end of mRNA and inspects long untranslated region (UTR) for the presence of the start codon (AUG). Accurate and high speed of scanning 5' UTR and subsequent selection of the correct start codon are crucial for protein synthesis. However, the conformational state of 48S PIC required for inspecting every codon is not clearly understood. Whether the scanning or open conformation of 48S PIC can accurately select the cognate start codon over near/non-cognate codons, or this discrimination is carried out only in the scanning-arrested or closed conformation of 48S PIC. Here, using atomistic molecular dynamics (MD) simulations and free energy calculations, we show that the scanning conformation of 48S PIC can reject all but 4 of the 63 non-AUG codons. Among nine near-cognate codons with a single mismatch, only codons with a first position mismatch (GUG, CUG and UUG) or a pyrimidine mismatch at the second position (ACG) are not discriminated by scanning state of 48S PIC. In contrast, any mismatch in the third position is rejected. Simulations runs in absence of one or more eukaryotic initiation factors (eIF1, eIF1+eIF1A, eIF2[a] or eIF2{beta}) from the system show critical role of eIF1 and eIF2[a] in start codon selection. The structural analysis indicates that tRNAi dynamics at the widened P site of 48S open state drives codon selection. Further, a stable codon: anticodon interaction prepares the PIC to transit to the closed state. Overall, we provide insights into the selection of start codon during scanning and how the open conformation of 48S PIC can scan long 5' UTRs with accuracy and high speed without the requirement of sampling the closed state for every codon. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.04.368092v1?rss=1 Authors: Sinegubova, M. V., Orlova, N. A., Kovnir, S. V., Dayanova, L. K., Vorobiev, I. I. Abstract: The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests - the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 - human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and similar applications. Copy rights belong to original authors. Visit the link for more info

All living things are categorized into one of two cell types: prokaryotes and eukaryotes. Melanie begins Episode 50 with a nerdy word dive (1:13) before distinguishing between prokaryotic and eukaryotic cells (2:04). Settle in for endosymbiosis story time!(5:02) The episode wraps up with unit connections and exam expectations (6:42).The Question of the Day asks (7:58) The liver has many functions, one of which is to process alcohol and detoxify the blood. Which organelle would you expect to find in greater quantity within liver cells?Thank you for listening to The APsolute RecAP: Biology Edition!(AP is a registered trademark of the College Board and is not affiliated with The APsolute RecAP. Copyright 2020 - The APsolute RecAP, LLC. All rights reserved.)Website:www.theapsoluterecap.comEMAILTheAPsoluteRecAP@gmail.comFollow Us:INSTAGRAMTWITTERFACEBOOKYOUTUBE

WHAT EVER HUMANS CREATE IN THEIR LABORATORIES NATURE WON'T RECOGNIZE --- This episode is sponsored by · Anchor: The easiest way to make a podcast. https://anchor.fm/app Support this podcast: https://anchor.fm/area-51-nick-jackson-51-a/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.17.301366v1?rss=1 Authors: Hill, J., Eickhoff, P., Drury, L., Costa, A., Diffley, J. Abstract: Origins of eukaryotic DNA replication are licensed during G1 phase of the cell cycle by loading the six related minichromosome maintenance (MCM) proteins into a double hexameric ring around double-stranded DNA. In S phase, some double hexamers (MCM DHs) are converted into active CMG (Cdc45-MCM-GINS) helicases which nucleate assembly of bidirectional replication forks. The remaining unfired MCM DHs act as dormant origins to provide backup replisomes in the event of replication fork stalling. The fate of unfired MCM DHs during replication is unknown. Here we show that active replisomes cannot remove unfired MCM DHs. Instead, they are pushed ahead of the replisome where they prevent fork convergence during replication termination and replisome progression through nucleosomes. Pif1 helicase, together with the replisome, can remove unfired MCM DHs specifically from replicating DNA, allowing efficient replication and termination. Our results provide an explanation for how excess replication license is removed during S phase. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.14.296129v1?rss=1 Authors: Voegele, A., Sadi, M., O'Brien, D. P., Gehan, P., Raoux-Barbot, D., Davi, M., Hoss, S., Brule, S., Raynal, B., Weber, P., Mechaly, A., Haouz, A., Rodriguez, N., Vachette, P., Durand, D., Brier, S., Ladant, D., Chenal, A. Abstract: The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells have thus far remained elusive. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. We have previously identified a translocation region in CyaA that contains a segment, P454 (residues 454-484), exhibiting membrane-active properties related to antimicrobial peptides. Herein, we show that this peptide is able to translocate across membranes and interact with calmodulin. Structural and biophysical analyses have revealed the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrated that these residues play a crucial role in CyaA translocation into target cells. We have also shown that calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. We propose that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of the CyaA polypeptide chain by the CaM:P454 interaction in the cytosol may assist the entry of the N-terminal catalytic domain by converting the stochastic process of protein translocation into an efficient vectorial chain transfer into host cells. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.29.273474v1?rss=1 Authors: Liu, J., Hansen, D., Eck, E., Kim, Y. J., Turner, M. A., Alamos, S., Garcia, H. G. Abstract: The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel computational technique to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters with high precision. These measurements, combined with theoretical modeling, reveal a significant variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.14.251363v1?rss=1 Authors: Shepherd, J., Lecinski, S., Wragg, J., Shashkova, S., MacDonald, C., Leake, M. C. Abstract: The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.10.245134v1?rss=1 Authors: Bruna, T., Hoff, K., Stanke, M., Lomsadze, A., Borodovsky, M. Abstract: Full automation of gene prediction has become an important bioinformatics task since the advent of next generation sequencing. The eukaryotic genome annotation pipeline BRAKER1 had combined self-training GeneMark ET with AUGUSTUS to generate genes coordinates with support of transcriptomic data. Here, we introduce BRAKER2, a pipeline with GeneMark EP+ and AUGUSTUS externally supported by cross-species protein sequences aligned to the genome. Among the challenges addressed in the development of the new pipeline was generation of reliable hints to the locations of protein-coding exon boundaries from likely homologous but evolutionarily distant proteins. Under equal conditions, the gene prediction accuracy of BRAKER2 was shown to be higher than the one of MAKER2, yet another genome annotation pipeline. Also, in comparison with BRAKER1 supported by a large volume of transcript data, BRAKER2 could produce a better gene prediction accuracy if the evolutionary distances to the reference species in the protein database were rather small. All over, our tests demonstrated that fully automatic BRAKER2 is a fast and accurate method for structural annotation of novel eukaryotic genomes. Copy rights belong to original authors. Visit the link for more info

Episode 1 of Teach Me Biology we learn about the structure of Eukaryotic Cells.Find us on the internet!Our website - Teachmescience.co.ukEmail - teachmebiologycast@gmail.comTwitter - twitter.com/teachmebiocastInstagram - @teachmebiologycast

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.21.214403v1?rss=1 Authors: Hunyady, J. P. Abstract: The present paper represents a hypothetical structure, the structure for energy transformation (SET), and its proposed evolution, which might be responsible for the proper energy transformation steps leading to the continuous production of H+ and ATP in living cells. We predict that the electron flow is realized through the electron flow device (EFD). We suppose that there are several versions of the SET with three of them being described below. The hypothesis is based on the properties of the atoms of the protonated adenine molecule and docking computations of molecular mechanics involved, suggesting that two ascorbate molecules may occupy the empty NADPH pocket, preferably binding to the adenine binding site. We hypothesize that the adenine originates from uric acid (UA), resulting in an ATP-UA-ADP-ATP cycle. It would also mean that UA is one of the oxygen carriers in aerobic glycolysis. We also suppose that the EFD contains the well-known molecules and clusters of the electron transport chain supplemented with two additional UA originated adenine molecules and two L-ascorbic acid molecules. Based on all this, we surmise that a tetra adenine octo phosphate ring (TAR) exists, in which the UA originated adenine molecules form a ring. The molecules are linked to each other through the N7-C2 and C8-N1 atoms of the adenine molecules by H2PO4e- molecules. The four N10 atoms of the adenine molecules bind one flavin, one nicotinamide, and two L-ascorbic acid molecules. Six D-glucose molecules complete one unit of the structure. Both Fe-S and cytochrome clusters and dehydrogenases ensure the continuous operation of the unit. Eukaryotic cells are equipped with the mechanisms of the SET, using aerobic glycolysis of Warburg and the SET of oxidative phosphorylation; thus, they can live in an anoxic environment as well. It is hoped that the concept of the SET developed here will help to better understand the complex process of energy transformation. Copy rights belong to original authors. Visit the link for more info

(Content heavy!) Transcription, Structural Differences of Nucleic Acids, mRNA Processing, and RNA Interference

Episode 38 is our second listener’s choice recAP from Instagram topic requests. Pedigrees are used to study the pattern of inheritance of a particular trait through several generations in a family (1:20). Signal transduction pathways link signal reception of a ligand with a cellular response through phosphorylation (2:38). Cellular respiration oxidizes glucose to form ATP (3:30). Eukaryotic gene regulation occurs throughout the central dogma (4:50). Successful FRQ writing involves intentional application of your words (5:44).The Question of the Day asks (7:14) “What is a prokaryote cluster of genes called when it has a single promoter? ”Thank you for listening to The APsolute RecAP: Biology Edition!(AP is a registered trademark of the College Board and is not affiliated with The APsolute RecAP. Copyright 2020 - The APsolute RecAP, LLC. All rights reserved.)Website:www.theapsoluterecap.comEMAIL:TheAPsoluteRecAP@gmail.comFollow Us:INSTAGRAMTWITTER

Around 3.5bn years ago the first forms of life emerged: bacteria and archaea. These so-called prokaryotes had the Earth to themselves for a very, very long time. Then, for some mysterious reason, another new microbial kingdom formed. Eukaryotic cells came into being and complex life began. But how and why did this happen? Hannah Devlin dives into the 12-year scientific odyssey that gives us an important piece of the puzzle. Help support our independent journalism at theguardian.com/sciencepod

Algae. It's one of the greatest things on the planet and it's responsible for all life on Earth, including your life. But how much do you really know about this incredible species? Is it a plant? Why is it green? Can you eat it? Can we make it into fuel? What's up with algae blooms? Learn more in our newest episode where we talk about the benefits of algae and how it is better than human. Follow us on Twitter @betterthanhuma1on Facebook @betterthanhumanpodcaston Instagram @betterthanhumanpodcastOr email us at betterthanhumanpodcast@gmail.comWe look forward to hearing from you, and we look forward to you joining our cult of weirdness.

Comparing the Cell to a Factory a breakdown of organelles in a eukaryotic cell

The tetracoccal TWiM team visits Tardigrades on the Moon, and the twelve year quest to isolate an archaeon that provides insights into the emergence of the first eukaryotic cell. Links for this episode: Tardigrades on the moon (Mashable) Meet the Tardigrade (WaPo) Archaeon at prokaryote-eukaryote interface (bioRxiv) Subscribe to TWiM (free) on iTunes, Google Podcasts, Stitcher, Android, RSS, or by email. You can also listen on your mobile device with the Microbeworld app. Become a Patron of TWiM! Music used on TWiM is composed and performed by Ronald Jenkees and used with permission. Send your microbiology questions and comments to twim@microbe.tv

Eukaryotic cells have many different membrane-bound organelles with distinct functions and characteristic shapes. How does this happen? Dr. Tom Rapoport explains the important role of protein sorting in determining organelle shape and function.

Eukaryotic cells have many different membrane-bound organelles with distinct functions and characteristic shapes. How does this happen? Dr. Tom Rapoport explains the important role of protein sorting in determining organelle shape and function.

The Leishmaniacs solve the case of the Child With Band Keratopathy, and reveal a eukaryotic parasite with functional mitochondria but without mitochondrial genomes. Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin Subscribe (free): iTunes, Google Podcasts, RSS, email Become a patron of TWiP. Links for this episode: Elmer Pfefferkorn, 87 Eukaryotic parasite without mitochondrial genomes (Sci Adv) Hero: Charles Wardell Stiles Letters read on TWiP 171 Case Study for TWiP 171 By boat to remote village, young boy (

Liz looks at more eukaryotic organelles for your A Level Biology exam. In this episode, she will look at the Golgi apparatus and vesicles, the rough and smooth endoplasmic reticulum, cell wall and vacuoles. Ideal for preparing for your A Level Biology exam. For more info visit https://www.senecalearning.com/blog/a-level-biology-revision/

Liz looks at organelles for your A Level Biology exam. In this episode, she will look at cell surface membranes, cytoplasm, nucleus, mitochondria and chloroplasts. Ideal for preparing for your A Level Biology exam. For more info visit https://www.senecalearning.com/blog/a-level-biology-revision/

Franklin M. Harold, noted author of The Way of the Cell: Molecules, Organisms and the Order of Life, discusses his research in complex biochemistry and thoughts on the origin of life. Harold's long career has impacted many areas of science. Harold received his PhD in Comparative Biochemistry from U.C. Berkeley in 1955, and has spent a lifetime as a physiologist, specializing as a cell physiologist. It is the machinery of life, not its molecular constituents, that continues to fascinate him. As a young man, Harold was heavily influenced by the work of esteemed microbiologist, Roger Stanier, by whom he was taught that bacteria are not only the smallest creatures but also the simplest, and to truly understand life, one should study bacteria, not lab rats. Harold talks about his long career in science and biology, starting with a passion for chemistry, which he developed at the age of fifteen. Over the years, he became more interested in cellular and evolutionary biology, and embarked on a prolific career in the scientific community. Harold discusses the tenets of Neo-Darwinism, and its need for tweaking, updating. He states that the early study was primarily focused on mutations in genes, but according to Harold, that doesn't tell the full story. He expounds on the properties of eukaryotic cells and their origin. Eukaryotic cells contain a nucleus and organelles, and they are encompassed by a plasma membrane. Some of the many organisms that possess eukaryotic cells are as follows: protozoa, fungi, plants, and animals. The biochemistry expert states that organisms throw off new species because their environment changes. He states that in order to see the evolution of organisms we would have to change their environment in a novel way, and give it time, lots of time. Harold remarks that the major changes that resulted in new animal species have to do with the genes that affect the web of regulation, the regulatory elements that control how much, and to what extent, a particular gene is expressed. Harold goes on to say that he believes in the theory that life must have begun with very simple chemical systems, not necessarily involving a gene, but a system sufficiently complex and interactive to be able to reproduce itself. And while he states there is zero evidence for it, nor good models, it is however a speculation that Harold finds particularly interesting. Harold served on the research staff of the National Jewish Hospital and Research Center in Denver, and was a longtime member of the faculty at the University of Colorado School of Medicine. Harold retired from Colorado State University in 2000 as Professor Emeritus of Biochemistry and Molecular Biology.

Marat Yusupov, IGBMC, France, speaks on "Crystal structures of eukaryotic and bacterials ribosomes". This movie has been recorded at ICGEB Trieste and is part of "RNA structure and function" Course 2018.

This episode: Eukaryotic ocean microbes have surprisingly diverse and complex ballistic weapons! Download Episode (8.2 MB, 9 minutes) Show notes: Microbe of the episode: Mycoplasma arginini Cool videos of microbial weapons firing Journal Paper: Gavelis GS, Wakeman KC, Tillmann U, Ripken C, Mitarai S, Herranz M, Özbek S, Holstein T, Keeling PJ, Leander BS. 2017. Microbial arms race: Ballistic “nematocysts” in dinoflagellates represent a new extreme in organelle complexity. Sci Adv 3:e1602552. Other interesting stories: Astronaut Kate Rubin's experience as microbiologist in space (paywall) Engineered nitrogen-fixing bacteria can reduce need for fertilizer (paper) Diversity of bacterial communities on leaves linked with ecosystem productivity (paper) Using parts of phages to do rapid detection of pathogens (paper) Microbiome modification contributes to effect of diabetes medication   Post questions or comments here or email to bacteriofiles at gmail dot com. Thanks for listening! Subscribe: iTunes, RSS, Google Play. Support the show at Patreon, or check out the show at Twitter or Facebook

Shannon just had a baby! We take the week off and celebrate with this classic episode of the geocast... Don't Panic, we'll be back next week! Having just got back from vacationing near a river, Shannon’s mind is wondering (and wandering!) about dams and their impacts, both good and bad. People use dams for electricity, recreation, flood control, and a myriad of other things. Let’s take a look at some huge dams and what they do to both communities and rivers. Oroville (1968) is the tallest in the US, and earthen dam in CA 770’ high Hoover (1935) is next at 726’ high on the border of AZ and NV Tallest dam in the world is Jingping-I dam (2013) in China is 1000’ high Largest reservoir in the US is Lake Mead, which holds 29 million acre-feet of water Glen Canyon dam Lake Powell on the AZ/UT border, and is a close second at 26 million acre-feet capacity. Colorado River WPA Why build dams at all? Water supply Irrigation and flood control Power supply Recreation Hydroelectric Power Hoover dam generates 4.5 billion kw-hr per year, serving 8 million people in AZ, southern CA and NV Itaipu dam, on the border of Brazil and Paraguay, dams the Parana River, the 7th largest in the world. Penstock Fun Paper Friday Borgonie, G., et al. “Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.” Nature communications 6 (2015). Contest Write us a geoscience themed limerick! This is a family show, so remember…nothing that rhymes with “Nantucket” Please email us your limericks by August 12, 2016 and we’ll be judging them along with Dr. Katie Schearer, an english professor. The prize? One of the awesome creations from Chris at Taylor Custom. Thanks for listening everyone! Contact us Show - www.dontpanicgeocast.com - @dontpanicgeo - SWUNG Slack - show@dontpanicgeocast.com John Leeman - www.johnrleeman.com - @geo_leeman Shannon Dulin - @ShannonDulin  

Hosts: Vincent Racaniello, Dickson Despommier, and Daniel Griffin The TWiP troika solve the case of the Female from the Bronx, and reveal how feeding on different plants affects mosquito capacity to transmit malaria. Links for this episode: Plant mediated effects on malaria transmission (PLoS Path) Image credit Letters read on TWiP 114 This episode is sponsored by CuriosityStream, a subscription streaming service that offers over 1,400 documentaries and non­fiction series from the world's best filmmakers. Get unlimited access starting at just $2.99 a month, and for our audience, the first two months are completely free if you sign up at curiositystream.com/microbe and use the promo code MICROBE. This episode is also sponsored by Drobo, a family of safe, expandable, yet simple to use storage arrays. Drobos are designed to protect your important data forever. Visit www.drobo.com to learn more. Become a patron of TWiP. Case Study for TWiP 114 12 year old boy brought to hospital ER by parents with severe headache, stiff neck, fever, decreased alertness. No rashes. Has been healthy with no prior medical problems. No one else in family is ill. In summer, boy has been engaged in usual summertime activities: soccer, swimming in warm freshwater, playing outside. Undergoes lumbar puncture for CSF: start on meningitis treatment. No surgeries, no allergies. Not on any meds. Lives with Mom, Dad, few brothers. No substance abuse. Not a geographically limited illness. Has had bug bites - lots of mosquito bites. Dogs around as well. Symptoms began a day or two before hospital visit. Eats whatever family eats, food is cooked. Exam: 39.4C, bp low, heart rate up, resp up, decreased responsiveness, stiff neck, looks ill. WBC elevated, neutrophil predominant, eosinopenia. CSF glucose low, cells increased, no bacteria, fungi, acid fast bacilli on stain. CT scan, diffuse swelling of brain. Doing poorly, not a good outcome. Send your case diagnosis, questions and comments to twip@microbe.tv

Having just got back from vacationing near a river, Shannon’s mind is wondering (and wandering!) about dams and their impacts, both good and bad. People use dams for electricity, recreation, flood control, and a myriad of other things. Let’s take a look at some huge dams and what they do to both communities and rivers. Oroville (1968) is the tallest in the US, and earthen dam in CA 770’ high Hoover (1935) is next at 726’ high on the border of AZ and NV Tallest dam in the world is Jingping-I dam (2013) in China is 1000’ high Largest reservoir in the US is Lake Mead, which holds 29 million acre-feet of water Glen Canyon dam Lake Powell on the AZ/UT border, and is a close second at 26 million acre-feet capacity. Colorado River WPA Why build dams at all? Water supply Irrigation and flood control Power supply Recreation Hydroelectric Power Hoover dam generates 4.5 billion kw-hr per year, serving 8 million people in AZ, southern CA and NV Itaipu dam, on the border of Brazil and Paraguay, dams the Parana River, the 7th largest in the world. Penstock Fun Paper Friday Borgonie, G., et al. “Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa.” Nature communications 6 (2015). Contest Write us a geoscience themed limerick! This is a family show, so remember…nothing that rhymes with “Nantucket” Please email us your limericks by August 12, 2016 and we’ll be judging them along with Dr. Katie Schearer, an english professor. The prize? One of the awesome creations from Chris at Taylor Custom. Thanks for listening everyone! Contact us Show - www.dontpanicgeocast.com - @dontpanicgeo - SWUNG Slack - show@dontpanicgeocast.com John Leeman - www.johnrleeman.com - @geo_leeman Shannon Dulin - @ShannonDulin  

Eukaryotic genomes are organized inside the cell nucleus in a structured macromolecular DNA-protein polymer named chromatin, formed by single discrete unites called Nucleosomes. The packing of the genetic information into chromatin allows the efficient regulation of several nuclear processes, such as gene expression and transcription, DNA replication, cell cycle progression, chromosome segregation and DNA damage repair. Chromatin comes in two flavors: a transcriptionally active, more loosened state, called euchromatin and a transcriptionally silent or low expressed, more compact state, called heterochromatin. The assembly of silent chromatin or heterochromatin is fundamental for the regulation of every nuclear process and it is driven in most Eukaryotes by the deposition and the read-out of the histone H3 lysine 9 methylation (H3K9me) post-translational modification (PTM). H3K9me on the nucleosome is specifically bound by chromatin readers called chromodomains (CD) and this recognition is fundamental for the downstream processes that lead to the formation of heterochromatin and shut down the expression of single genes or entire gene clusters. Despite several studies have been done on different chromodomains binding to H3K9me histone tail peptides, to date there was no structural information on how chromodomains interact with their natural binding partners, the H3K9me3 Nucleosomes. In a preliminary structural study carried out in our laboratory we solved the cryo-electron microscopy (Cryo-EM) structure of the chromodomain of the fission yeast Chp1 protein (Chp1CD) in complex with an H3K9me nucleosome. The structure showed that the Chp1CD interacts not only with the histone H3 tail but also with the histone globular domains in the Nucleosome core, primarily with histone H3. Mutations in the residues of Chp1CD that form the binding interface with the Nucleosome core (two loops in the β-sheet of the domain) caused a drop of the affinity in vitro for the H3K9me Nucleosome, which was independent from the histone H3K9me tail interaction. Cells harboring the same Chp1CD loop mutations were defective in silencing centromeric transcripts and maintain the deposition of the H3K9me mark for heterochromatin formation. This indicated that Chp1CD-nucleosome core interaction is fundamental for heterochromatin formation in fission yeast and opened up to the possibility that chromodomains could read multiple histone PTMs, on both the recruiting histone tail and on the nucleosome core. This study substantially contributes to understand how chromodomains interact with chromatin, how much the nucleosome core interaction is conserved among different CDs and how different chromodomain proteins are regulated at the same loci. Understanding how chromodomain readers recognize nucleosomes is fundamental to uncover the basics of gene silencing and heterochromatin formation.

Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin to deep sequencing and the occupancies were analyzed using advanced analog signal processing methods. We found discontinuous and discrete patches of regularly positioned nucleosomes in wild-type tissue, referred to as ‘regularity regions’. These regions span actively transcribing and silent chromatin domains and show associated variation in the linker lengths across them. A subset of these regions located at sides remote from the transcriptional start sites loses regularity upon ACF1 deletion and show presence of a novel DNA sequence motif. Analyzing nucleosome periodicity by autocorrelation function revealed that nucleosome linker length is longer in ACF1-deficient embryos. Despite profound quantifiable changes in the chromatin organization the RNA expression analyses did not show any major changes. Genomewide localization of ACF1 was studied using by chromatin immunoprecipitation. We observed a strong enrichment of ACF1 along active promoter regions, coinciding strikingly well with another remodeling factor, RSF-1. However, careful analyses using mutant tissues for both proteins demonstrated that the observed enrichments were in fact false positive. We define 3100 genomic sites as false positive ‘Phantom Peaks’ that tend to enrich in the ChIP-seq experiments. By comparing publicly accessible profiles and the Phantom regions, we showed that several ChIP-seq profiles of the epigenetic regulators show strong enrichment along the Phantom Peaks. In conclusion, we identify regions of regularly organized nucleosomes across the genome and show that a subset localized in silent chromatin regions is affected by ACF1 deletion. Moreover, we identified a class of false positive ChIP-seq peaks at active promoters. This list of Phantom Peaks can be used to assess potential false positive signal in a ChIP-seq profile, especially when mutant tissue is not available as a control.

Gene transcription is a fundamental process of the living cell. Eukaryotic transcription of messenger RNA requires the regulated recruitment of the conserved transcribing enzyme RNA polymerase (Pol) II to the gene promoter. The most heavily regulated step is transcription initiation that involves the ordered assembly of Pol II, the general transcription factors (TF) -IIA, -IIB, -IID, -IIF, -IIE, -IIH and the co-activator Mediator complex. Mediator communicates between transcription regulators and Pol II, and is associated with human disease. Mediator from the yeast Saccharomyces cerevisiae (Sc) has a molecular mass of 1.4 megadaltons and contains 25-subunits that constitute a head, middle, tail and kinase module. The core of Mediator contains the head and middle modules that are essential for viability in Sc, and directly contact Pol II. Mediator co-operates with TFIIH, to assist assembly and stabilization of the transcription initiation complex and stimulate TFIIH kinase activity. Because of the large size and complexity of Mediator and the initiation machinery, the underlying mechanism remains poorly understood. In this work we studied the structure and function of Mediator head and middle modules, the structure of the reconstituted Pol II-core Mediator transcription initiation complex, and reveal mechanisms of transcription regulation. We report the crystal structure of the 6-subunit Schizosaccharomyces pombe Mediator head module at 3.4 Å resolution. The structure resembles the head of a crocodile and reveals eight elements that are part of three domains named neck, fixed jaw and movable jaw. The neck contains a spine, shoulder, arm and finger. The arm and essential shoulder elements contact the remainder of Mediator and Pol II. The head module jaws and central joint, important for transcription, also interact with Mediator and Pol II. The Sp head module structure is conserved and revises a 4.3 Å model of the Sc head module, explains known mutations, and provides an atomic model for one half of core Mediator. We further propose a model of the Mediator middle module based on protein crosslinking and mass spectrometry. To determine how Mediator regulates initiation, we prepared recombinant Sc core Mediator by co-expression of its 15 subunits in bacteria. Core Mediator is active in transcription assays and bound an in vitro reconstituted core initially transcribing complex (cITC) that contains Pol II, the general factors TFIIB, TBP, TFIIF, and promoter DNA. We determined the cryo-electron microscopy structure of the initially transcribing core initiation complex at 7.8 Å resolution. The structure reveals the arrangement of DNA, TBP, TFIIB, and TFIIF on the Pol II surface, the path of the complete DNA template strand and three TFIIF elements. The ‘charged helix’ and ‘arm’ of TFIIF subunit Tfg1, reach into the Pol II cleft and may stabilize open DNA. The linker region of TFIIF subunit Tfg2 extends between Pol II protrusion and TFIIB, and may stabilize TFIIB. The structure agrees with its human counterpart, and suggests a conserved architecture of the core initiation complex. Finally, we determined the cryo-electron microscopy architecture of the cITC-core Mediator complex at 9.7 Å resolution. Core Mediator binds Pol II at the Rpb4/Rbp7 stalk close to the carboxy-terminal domain (CTD). The Mediator head module contacts the Pol II dock and TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module ‘plank’ domain touches the Pol II foot and may control polymerase conformation allosterically. The Med14 subunit bridges head and middle modules with a ‘beam’, and connects to the tail module that binds transcription activators located on upstream DNA. The ‘arm’ and ‘hook’ domains of core Mediator form part of a ‘cradle’ that may position CTD and the TFIIH kinase to stimulate Pol II phosphorylation. Taken together, our results provide a structural framework to unravel the role of Mediator in transcription initiation and determine mechanisms of gene regulation.

In eukaryotes, mRNA turnover starts with the truncation of 3′ poly(A) tail and proceeds with either 3′-to-5′ degradation by the exosome complex or with decapping followed by 5′-to-3′ degradation by Xrn1. mRNA decapping is catalyzed by the decapping enzyme complex Dcp1-Dcp2 and is regulated by a highly conserved set of decapping activator proteins, including Pat1, Dhh1, Edc3 and the heptameric Lsm1-7 complex. The mechanisms regarding the interplay of mRNA decapping activators remains elusive owing to limited structural and biochemical understanding. My doctoral research was focused on elucidating the structural and functional roles of mRNA decapping activators involved in mRNA decay. Pat1 has a modular domain architecture that allows it to interact with multiple decapping activators simultaneously. Pat1 acts as a bridging factor between the 3′-end and the 5′-end of the mRNA by interacting with multiple proteins involved in decapping. The interaction of S. cerevisiae Pat1 N-terminus with the DEAD-box protein Dhh1 was characterized by biochemical pull-down assays and binding affinities were determined quantitatively by isothermal titration calorimetery. Based on these experiments, the crystal structure of Dhh1 bound to Pat1 was determined at 2.8 Å resolution. The structure reveals that Pat1 wraps around RecA2 domain of Dhh1 via evolutionary conserved interactions. This conserved surface of Dhh1 is also implicated in interaction with another decapping activator, Edc3, rationalizing why Pat1 and Edc3 binding to Dhh1 is mutually exclusive. These interactions were supported by testing mutations in in vitro assays with the yeast proteins and in co-immunoprecipitation assays with the corresponding human orthologs. Furthermore, structural analysis combined with RNA pull-down assays and a crosslinking mass spectrometry based approach gave definitive evidence that Dhh1 engages with Pat1, Edc3 and RNA in a mutually exclusive manner. The Lsm1-7 complex is another important activator of mRNA decapping. It protects the mRNA transcripts from 3′-end degradation and enhances the mRNA decapping. I determined the crystal structure of the Lsm1-7 complex at 2.3 Å resolution showing a hetero-heptameric complex of Lsm1-7 proteins that make a ring-like overall topology. Furthermore, an unusual helical structure of Lsm1 C-terminal extension and protrudes into the central channel of the heptameric ring, explaining how it is modulates the RNA binding properties of the complex. The Lsm1-7 complex interacts with the C-terminal domain of Pat1. Structure determination of this octameric Lsm1-7-Pat1 complex at 3.7 Å gave insights into the interaction of Pat1 with Lsm1-7 complex. Unexpectedly, Pat1 binds to Lsm2 and Lsm3 but not with the cytoplasmic specific subunit Lsm1. The Pat1 C-terminus makes a super-helical structure consisting of HEAT-like repeats of anti-parallel helices similar to the structure of its human ortholog. Structure based mutagenesis analysis by in vitro pull-downs showed that these interactions are conserved. This doctoral thesis gives structural and mechanistic insight into the role of multi-domain protein Pat1 and how it engages at two distinct ends of mRNA by interacting with Dhh1 at 5′-end and with Lsm1-7 complex that at 3′-end. Combining these results present a model of dynamic interplay of these activators and gives a better understanding of protein-protein and protein-RNA interaction network in the decapping machinery.

Summary: Structure of the FACT chaperone domain in complex with histones H2A-H2B, and a model for FACT-mediated nucleosome reorganization Nucleosomes are the smalles unit of chromatin: two coils of DNA are wrapped around a histone octamer core, which neutralizes its charge and `packs' the lengthy molecule. Nucleosomes confer a barrier to processes that require access to the eukaryotic genome such as transcription, DNA replication and repair. A variety of nucleosome remodeling machines and histone chaperones facilitate nucleosome dynamics by depositing or evicting histones and unwrapping the DNA. The eukaryotic FACT complex (composed of the subunits Spt16 and Pob3) is an essential and highly conserved chaperone. It assists the progression of DNA and RNA polymerases, for example by facilitating transcriptional initiation and elongation. Further, it promotes the genome-wide integrity of chromatin structure, including the suppression of cryptic transcription. Genetic and biochemical assays have shown that FACT's chaperone activity is crucially mediated by a direct interaction with histones H2A-H2B. However, the structural basis for how H2A-H2B are recognized and how this integrates with FACT’s other functions, including the recognition of histones H3-H4 and of other nuclear factors, is unknown. In my PhD research project, I was able to reveal the structure of the yeast chaperone domain in complex with the H2A-H2B heterodimer and show that the Spt16M module in FACT’s Spt16 subunit establishes the evolutionarily conserved H2A-H2B binding and chaperoning function. The structure shows how an alpha-helical `U-turn' motif in Spt16M interacts with the alpha-1-helix of H2B. The U-turn motif scaffolds onto a tandem pleckstrin-homology-like (PHL) module, which is structurally and functionally related to the H3-H4 chaperone Rtt106 and the Pob3M domain of FACT. Biochemical and in vivo assays validate the crystal structure and dissect the contribution of histone tails and H3-H4 toward FACT binding. My results show that Spt16M makes multiple interactions with histones, which I suggest allow the module to gradually invade the nucleosome and ultimately block the strongest interaction surface of H2B with nucleosomal DNA by binding the H2B alpha-1-helix. Together, these multiple contact points establish an extended surface that could reorganize the first 30 base-pairs of nucleosomal histone–DNA contacts. Further, I report a brief biochemical analysis of FACT’s heterodimerization domain. Its PHL fold indicates shared evolutionary origin with the H3-H4-binding Spt16M, Pob3M and Rtt106 tandem PHL modules. However, the Spt16D–Pob3N heterodimer does not bind histones, rather it connects FACT to replicative DNA polymerases. The snapshots of FACT’s engagement with H2A-H2B and structure-function analysis of all its domains lay the foundation for the systematic analysis of FACT’s vital chaperoning functions and how the complex promotes the activity of enzymes that require nucleosome reorganization.

Eukaryotic genomes are organized into highly condensed chromatin. This packaging obviously impedes essential DNA mediated processes. ATP-dependent chromatin remodelers are therefore required to establish a dynamic chromatin environment. The chromatin remodeler INO80 is involved in various fundamental nuclear processes such as DNA repair, DNA replication and transcription. INO80 is thought to contribute to these processes by controlling genome wide levels of the histone variant H2A.Z. The INO80 chromatin remodeler is a macro-molecular complex composed of >15 subunits and a molecular mass of ~1.3 MDa. INO80 is found in human, fly and yeast. INO80 contains core subunits, which are conserved across species, as well as species-specific proteins. Not much was known about the organization of the INO80 subunits and their contribution to chromatin remodeling. Therefore, a hybrid approach was applied on yeast INO80 combining chemical cross-linking and mass spectrometry (XL-MS) (in collaboration with Franz Herzog, Ruedi Aebersold’s group, ETH, Zurich), electron microscopy (EM) (in collaboration with Caroline Haas, Roland Beckmann’s group, Gene Center, Munich) and biochemical analysis. For this, firstly the purification of INO80 was established. In order to yield sufficient amounts of highly purified and monodisperse complex, INO80 was purified endogenously from yeast by a combination of affinity and chromatography methods. In addition, nanobodies targeting the INO80 complex were generated that could yield even larger amounts of INO80 in the future. EM analysis revealed that INO80 is an embryo-shaped particle with a dynamic head-neck-body-foot architecture that can undergo large conformational changes. XL-MS unraveled the interaction map of the INO80 complex. The analysis of INO80 deletion mutants verified the observed interactions in vivo and proved the modular architecture of INO80. Additionally, the gained knowledge allowed the design and purification of stable and novel sub-complexes that could improve crystallization behavior. An integration of the results from different techniques deepened our understanding of the molecular architecture of INO80. The enigmatic subunits Rvb1 and 2 assemble as a dodecamer composed of two hetero-hexameric rings within the head of the INO80 complex. Rvb1/2 is flanked by the Swi2/Snf2 ATPase of Ino80 and the actin related protein (Arp) 5 in the neck. The Nhp10-module localizes to the body and the Arp8-module to the foot. Biochemical analysis showed that the Nhp10-module is a high affinity DNA/nucleosome binder. The Nhp10-module might together with the Arp8-module target INO80 to chromatin. The Arp5-module is catalytically crucial for nucleosome remodeling and senses the histone entity in chromatin. In order to map interaction sites to the substrate, INO80-nucleosome complexes were analyzed by XL-MS and were visualized by EM. Two-dimensional class averages showed that the nucleosome bound to the central groove of INO80 and was flanked by the head and foot module. The nucleosome was oriented in respect to INO80 as the H2A/H2B dimer- the moiety to be exchanged- was in contact with subunits situated in the neck. All INO80 modules contribute to nucleosome binding and the observed flexibility proposes a mechanism of how INO80 may remodel its substrate. This study established a structural and functional framework of these large remodelers. The investigation of the interaction with the checkpoint kinase Mec1 will contribute to the understanding of the obscure signaling of INO80.

Wed, 20 Nov 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16307/ https://edoc.ub.uni-muenchen.de/16307/1/Anger_Andreas.pdf Anger, Andreas ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Eukaryotic gene transcription is highly complex and regulation occurs at multiple stages. RNA Polymerase II (Pol II) is recruited to promoter regions of the DNA to initiate transcription. Shortly after initiation, Pol II exchanges initiation factors for elongation factors. After Pol II passes termination signals, the RNA is cleaved and Pol II eventually released from the DNA template. pre-mRNAs are polyadenylated and exported to the cytosol for translation and ultimately degradation. Mechanisms regulating transcription have been studied extensively, but mechanisms of mRNA degradation are less well understood. To monitor mRNA synthesis and degradation, we developed the comparative dynamic transcriptome analysis (cDTA). cDTA provides absolute rates of mRNA synthesis and decay in Saccharomyces cerevisiae Sc cells with the use of Schizosaccharomyces pombe Sp cells as internal standard. We show that Sc mutants can buffer mRNA levels and that impaired transcription causes decreased mRNA synthesis rates compensated by decreased decay rates. Conversely, impairing mRNA degradation causes decreased decay rates, but also decreased synthesis rates. Thus, although separated by the nuclear membrane, transcription and mRNA degradation are coupled. In addition to regulated mRNA synthesis, pervasive transcription can be found throughout the genome, governed by an intrinsic affinity of Pol II for DNA. These divergent noncoding RNAs (ncRNAs) stem to a large extent from bidirectional promoters. However, global mechanisms for the termination of ncRNA synthesis that could act as a transcriptome surveillance mechanism are not known. It is also unclear if such a surveillance system protects the transcriptome from deregulation. Here we show that ncRNA transcription in Sc is globally restricted by early termination which relies on the essential RNA-binding factor Nrd1. Depletion from the nucleus results in Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) throughout the genome and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at essentially all promoters and antisense NUTs in most 3’-regions of genes. Nrd1 preferentially binds RNA motifs which are enriched in ncRNAs and depleted in mRNAs except in some mRNAs whose synthesis is controlled by transcription attenuation. These results describe a mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and prevent transcriptome deregulation

Mon, 8 Apr 2013 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15724/ https://edoc.ub.uni-muenchen.de/15724/1/Ruessmann_Florian.pdf Rüßmann, Florian ddc:540, ddc:500, Fakultät für Chemie und

Wed, 25 Jul 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15499/ https://edoc.ub.uni-muenchen.de/15499/1/Treutlein_Barbara.pdf Treutlein, Barbara ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 8 May 2012 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15076/ https://edoc.ub.uni-muenchen.de/15076/1/Hartmann_Holger.pdf Hartmann, Holger ddc:540, ddc:500, Fakultät für Chem

Eukaryotic nuclear transcription is carried out by three different Polymerases (Pol), Pol I, Pol II and Pol III. Among these, Pol I is dedicated to transcription of the rRNA, which is the first step of ribosome biogenesis, and cell growth is regulated during Pol I transcription initiation by the conserved factor Rrn3/TIF-IA in yeast/human. A wealth of structural information is available on Pol II and its general transcription factors (GTFs). Recently, also the architectures of Pol I and Pol III have been described by electron microscopy and the additional subunits that are specific to Pol I and Pol III have been identified as orthologs of the Pol II transcription factors TFIIF and TFIIE. Nevertheless, we still lack information about the architecture of the Pol I initiation complex and structural data is missing explaining the regulation of Pol I initiation mediated by its central transcription initiation factor Rrn3. The Rrn3 structure solved in this study reveals a unique HEAT repeat fold and indicates dimerization of Rrn3 in solution. However, the Rrn3-dimer is disrupted upon Pol I binding. The Rrn3 structure further displays a surface serine patch. Phosphorylation of this patch represses human Pol I transcription (Mayer et al, 2005; Mayer et al, 2004), and a phospho-mimetic patch mutation prevents Rrn3 binding to Pol I in vitro, and reduces S. cerevisiae cell growth and Pol I gene occupancy in vivo. This demonstrates a conserved regulation mechanism of the Pol I-Rrn3 interaction. Crosslinking indicates that Rrn3 does not only interact with Pol I subunits A43/14, but the interface further extends past the RNA exit tunnel and dock domain to AC40/19. The corresponding region of Pol II binds the Mediator head (Soutourina et al., 2011) that co-operates with TFIIB (Baek et al, 2006). Consistent with this, the Rrn3 binding partner, core factor subunit Rrn7, is predicted to be a TFIIB homologue. Taken together, our results provide the molecular basis of Rrn3-regulated Pol I initiation and cell growth and indicate a universally conserved architecture of eukaryotic transcription initiation complexes.

Thu, 21 Jul 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13400/ https://edoc.ub.uni-muenchen.de/13400/1/Schiller_Christian_Bernd.pdf Schiller, Christian Bernd ddc:540, ddc:500, Fakultät für Chem

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. We show here that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP-dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. We have identified several dMi-2 regions which bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. We suggest that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response.

Fri, 10 Jun 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16606/ https://edoc.ub.uni-muenchen.de/16606/1/Zielinska_Dorota.pdf Zielinska, Dorota ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Ribosomes are macromolecular protein-RNA complexes translating mRNA into protein. To date, crystal structures are available for the bacterial 30S and archaeal 50S subunits, as well as the complete bacterial 70S ribosomes. Eukaryotic ribosomes are much more complex in terms of ribosomal RNA and proteins. However, to date high-resolution crystal structures of eukaryotic ribosomes or ribosomal subunits are lacking. In order to build reliable models for the eukaryotic rRNA, we developed an approach for large scale homology and de novo modeling of RNA and subsequent exible tting into high-resolution cryo-EM density maps. Using this approach we built a model of the T. aestivum and the S. cerevisiae ribosome based on available cryo-EM maps at 5.5 Å and 6.1 Å resolution, respectively. The model comprises of 98% of the eukaryotic rRNA including all 21 RNA expansion segments (ES) and structurally six variable regions. Further, we were able to localize 74/80 (92.5%) of the ribosomal proteins. The model reveals unique ES-ES and r-protein-ES interactions, providing new insight into the structure and evolution of the eukaryotic ribosome. Moreover, the model was used for analyzing functional ribosomal complexes, i.e. the characterization of dierent nascent polypeptide chains within the ribosomal tunnel, intermediates of protein translocation as well as mRNA quality control.

Protein biosynthesis, the translation of the genetic code into polypeptides, occurs on ribonucleoprotein particles called ribosomes. Although X-ray structures of bacterial ribosomes are available, high-resolution structures of eukaryotic 80S ribosomes are lacking. Using cryo-electron microscopy and single-particle reconstruction we have determined the structure of a translating plant (Triticum aestivum) 80S ribosome at 5.5 Å resolution. This map, together with a 6.1 Å map of a Saccharomyces cerevisiae 80S ribosome, has enabled us to model ~98 % of the rRNA and localize 74/80 (92.5 %) of the ribosomal proteins, encompassing 11 archaeal/eukaryote-specific small subunit proteins as well as the complete complement of the ribosomal proteins of the eukaryotic large subunit. Near-complete atomic models of the 80S ribosome provide insights into the structure, function and evolution of the eukaryotic translational apparatus.

Nick Proudfoot, Sir William Dunn School of Pathology, Molecular Biology, University of Oxford, Oxford, UK speaks on "Gene punctuation: multiple roles of transcriptional termination in regulating eukaryotic gene expression". This seminar has been recorded by ICGEB Trieste

In this show, I report on four exciting stories: a probiotic bacteria that can fight cancer, bacteria in dust that affect asthma, microbes living in a lake of asphalt, and a census of marine microorganisms.Download Sections:IntroStory 1Story 2Story 3Story 4Ending CommentsShow notes:Story 1: News item/Journal paperStory 2: News item/Journal PaperStory 3: News item 1/News item 2/Submitted PaperStory 4: News item 1/News item 2/SlideshowOther interesting stories:**Plant fungal disease that uses different tactics depending on which part of the plant it’s infecting**Using microbes to clean up groundwater pollution**The Navy investigating ways to use microbes to power small devices **A species of bacterium that produces lots of hydrogen gas **Using a microbe to protect grapes from grey mold**Study about how probiotics may not help Salmonella food poisoning**Paper about recently-discovered salt-loving archaea found in the human intestine**Eukaryotic plankton in the ocean actually incorporate a lot of carbon dioxide into their cells**Article describing the job of planetary protection officer for NASAPost questions or comments here or email to bacteriofiles at gmail dot com. Thanks for listening!Subscribe at iTunesThis show features music from Mevio's podsafe Music Alley.

Vincent and Dick classify parasites according to whether or not they are transmitted by a vector, then consider the implications of long-lived parasites. Host links: Vincent Racaniello and Dickson Despommier

Vincent and Dick provide an overview of parasites and parasitism. Host links: Vincent Racaniello and Dickson Despommier Weekly Science Picks Dick The Black Cloud by Fred HoyleVincent Parasitic Diseases by Despommier et al.

Eukaryotic genomes are condensed into a multilevel structure called chromatin which serves to organize and package the DNA, but at the same time needs to be flexible to permit regulated access to the stored information. ATP-dependent chromatin remodelling factors largely contribute to this dynamic nature of chromatin by catalysing processes such as the disruption of histone-DNA contacts, nucleosome repositioning and histone exchange. ATP-dependent remodelling has been well documented on a mononucleosomal level, but little is known about its regulation in a more physiological chromatin environment, where neighbouring nucleosomes and linker histones might interfere with the remodelling reaction. If and to what extent remodelling can work on chromatin bound by linker histones remains controversial, in spite of their high abundance and their strong influence on chromatin folding. We therefore investigated chromatin remodelling in the presence of linker histones H1 or H5 using regularly spaced, oligonucleosomal substrates reconstituted from purified components. Surprisingly, we found that both the remodelling complex ACF – consisting of the ATPase ISWI and the regulatory subunit ACF1 – and ISWI alone were able to catalyse the repositioning of entire chromatosomes (nucleosomes + H1). Linker histones inhibited their remodelling activity by only about 50%. In contrast, the related ATPase CHD1 remodelled chromatin only in the absence of linker histones, suggesting that linker histones allow remodelling by selected factors only. In addition, our data indicate that repositioning in the presence of H1 might be unidirectional. ACF1 is abundant during early Drosophila development, when H1 gradually replaces its early placeholder HMG-D. HMG-D binds to chromatin less tightly than H1 and unlike the latter, did not affect the remodelling activity of ACF in our assay. H1 was able to displace HMG-D from and bind to our reconstituted arrays without the help of cofactors. Strikingly, both H1 and HMG-D are more abundant in embryonic nuclei of acf1 null flies compared to the wild-type, raising the possibility that an ACF1-containing complex controls linker histone incorporation.

Intr Bio Fall lec : Eukaryotic genetics II con't

Intr Bio Fall lec : Eukaryotic genetics II con't

Intr Bio Fall lec : Eukaryotic genetics II

Intr Bio Fall lec : Eukaryotic genetics II

Intr Bio Fall lec : Eukaryotic genetics I

Intr Bio Fall lec : Eukaryotic genetics I

Intr Bio Fall lec : Eukaryotic cell division II

Intr Bio Fall lec : Eukaryotic cell division II

Intr Bio Fall lec : Eukaryotic cell division I

Intr Bio Fall lec : Eukaryotic cell division I

Enhanced Video PodcastAired date: 10/29/2008 3:00:00 PM Eastern Time

Enhanced Audio PodcastAired date: 10/29/2008 3:00:00 PM Eastern Time

Preface This study is focussed on the structural investigation of large molecular assemblies such as the 26S proteasome and the translocation machinery of the outer mitochondrial membrane. It is divided in two chapters and in both parts the structural and further functional analysis is based on X-ray crystallography. Chapter 1: Structural investigation of Rpn13, the multifunctional adaptor protein of the 26S proteasome The results in chapter 1 reveal that the multifunctional adaptor protein Rpn13 acts as a novel ubiquitin receptor of the 26S proteasome and deliver structural and biophysical details of its interaction with ubiquitin and with other proteasomal subunits. The crystal structure of the ubiquitin binding domain of Rpn13 reveals the molecular architecture of a Pleckstrin Homology (PH) domain and the NMR structure of the complex with ubiquitin shows a novel ubiquitin-binding mode. Additional NMR studies and domain mapping by truncation analysis provide further insights in the domain architecture of Rpn13 and the interaction with its partners Rpn2 and Uch37. Chapter 2: Crystallographic studies of the TOM core complex Chapter 2 presents the purification and crystallization of the mitochondrial protein translocase, the TOM core complex, from Neurospora crassa. Preliminary crystallographic data lead to the determination of space group and cell dimensions. This chapter also describes various experiments to improve the diffraction quality of the crystals and the co-crystallization of TOM core complex with specific monoclonal antibody fragments. Furthermore, expression and refolding of the main component Tom40 is raised as an alternative approach in structural investigation of the TOM complex.

Since its first description in 1994 by Yuan Chang and Patrick Moore, Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human Herpesvirus 8 (HHV-8) has emerged as a pathogen of international public health importance. It has been detected in biopsies of all forms of Kaposi’s sarcoma (KS), irrespective of geographic origin, age, or gender of the patient. Moreover, KSHV has been shown to be associated with two other diseases, multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL). In comparison to alpha and beta-herpesvirinae, the understanding of KSHV-related pathogenesis has been hampered by inefficient virus replication in vitro, poor cell culture systems and the lack of an animal model. Thus, many basic questions concerning the biology of KSHV infection remain open. For example, the primary target cell of KSHV and the function of more than 50% of the viral proteins are still unknown. Since the investigation of viral gene functions by virus mutants did not prove to be very efficient for KSHV, a system for a genome-wide screening of viral gene functions by cloning the complete KSHV ORFeome (all open reading frames) and by generating KSHV arrays in a variety of different expression vectors was established in this project. Very often viruses regulate cellular signalling pathways, which favour viral infection and replication in the host cells. The SRE is a transcription factor binding site present in promoters of many genes involved in cell growth and transformation. In this study, a genome-wide screen for KSHV genes inducing the SRE element and AP-1 was performed. A strong induction of SRE by the latency-associated nuclear antigen 1 (LANA-1) was observed. LANA-1 is a multifunctional protein which interacts with the p53 and RB tumor suppressor proteins. This study reveals several novel functions of LANA-1. LANA-1 led to an activation of the ERK-1/2 MAP kinase, but also bound to the Mediator, a multi-subunit transcriptional coactivator complex for RNA polymerase II, via the ARC92/ACID1 subunit. Since LANA-1 interacted with SRF, one of the two transcription factors binding to the bipartite SRE element, a model for LANA-1 as an adaptor between specific transcription factors and the basal transcriptional machinery was hypothesized.

A family of proteins known as 14-3-3 is currently receiving increased attention by investigators studying a broad range of biological systems, including plants and invertebrates. The outstanding feature of this family is the extraordinarily high sequence conservation observed. Current thinking indicates that these proteins may function as regulators in signal tranduction/phosphorylation mechanisms.

Wed, 1 Jan 1992 12:00:00 +0100 http://epub.ub.uni-muenchen.de/4045/ http://epub.ub.uni-muenchen.de/4045/1/023.pdf Zatloukal, Kurt; Wagner, Ernst; Cotten, Matt; Phillips, Stephen; Plank, Christian; Steinlein, Peter; Curiel, David T.; Birnstiel, Max L. Zatloukal, Kurt; Wagner, Ernst; Cotten, Matt; Phillips, Stephen; Plank, Christian; Steinlein, Peter; Curiel, David T. und Birnstiel, Max L. (1992): Transferrinfection. A Highly Efficient Way to Express Gene Constructs in Eukaryotic Cells. In: Annals of the New York Academy of Sciences, Vol. 660, Nr. 1: pp. 136-153. Ch

Thu, 1 Jan 1987 12:00:00 +0100 https://epub.ub.uni-muenchen.de/7649/1/Luckow_Bruno_7649.pdf Schütz, Günther; Luckow, Bruno

H NMR resonance assignments in the spectra of horse, tuna, Neurmpora crassa and Candida krusei cyto-chromes c are described. Assignments have been made using NMR double-resonance techniques in conjunction with electron-exchange experiments, spectral comparison of related proteins, and consideration of the X-ray structure of tuna cytochrome c. Resonances arising from 11 residues of horse cytochrome c have been assigned.