Lecture 09. RNA.

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Lecture 09. RNA.
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Lec 09. Introduction to Chemical Biology -- RNA -- Part 2
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UCI Chem 128 Introduction to Chemical Biology (Winter 2013) Description: Introduction to the basic principles of chemical biology: structures and reactivity; chemical mechanisms of enzyme catalysis; chemistry of signaling, biosynthesis, and metabolic pathways. Index of Topics: 0:21:06 RNA and Transcription Factors 0:26:04 Comparing Bacterial and Eukaryotic mRNA Processing 0:29:08 CTP Cap Methylation 0:30:30 Using PolyA Tails to Isolate mRNA 0:34:29 Eukaryotic Splicing of mRNAs 0:37:38 RNA Degredation Plays a Major Role 0:40:40 Therapeutic Anti-Sense 0:42:32 Modifying the Oligo Backbone 0:44:20 RNA Interference Used Extensively in the Lab 0:47:57 Where Peptide Synthesis Starts 0:58:26 The Genetic Code: The Language of the Codons 1:00:33 Decoding the DNA to Protein Sequence 1:01:43 How to Load the Amino Acyl tRNA 1:06:38 Post-Translational Modification of the N-Terminus 1:07:25 Inhibiting Methionine Aminopeptidase 1:09:59 Binding to mRNA Provides Further Regulation of Translation 1:10:59 Incorporating Unnatrual Amino Acids 1:12:38 Expanding the Protein Palette 1:13:48 mRNA Aptamer Libraries 1:18:18 Puromycin Allows Covalent Linkage to the Growing Peptide During Translation
Biosynthesis RNA Chemist Chemische Biologie Translation <Genetik> Functional group Lactitol Protein Amino acid Amino acid Library (computing) Chemical structure Translation <Genetik> Messenger RNA
Sense District Dietary supplement Beer Explosion Walking Chemist Topicity Bock Wursthülle Chemistry Acid Portable Document Format Wine tasting descriptors Chemische Biologie Hardness Vancomycin Cell (biology) Polymorphism (biology) Materials science Cell (biology) Pharmaceutics Locus (genetics) Explosion Stuffing
Sense District Enzyme inhibitor Weinfehler Activity (UML) Cell disruption Gold Korngrenze Wursthülle Cancer Man page Molecule Human subject research Cell (biology) Lactitol Protein Explosion Library (computing) Process (computing) Setzen <Verfahrenstechnik> Area Grading (tumors) Enzyme inhibitor Öl Explosion Proline White blood cell Starvation response Asset River source Cancer Nobelium Sausage Toxicity Chemische Biologie Coordination number Chemical compound Cell (biology) Bile acid ISO-Komplex-Heilweise Stuffing
Meat Binding energy Protein domain Gene Chemistry Molecule Enzyme Transkriptionsfaktor Polymorphism (biology) Protein Propylthiouracil <6-Propyl-2-thiouracil> Crack cocaine Biopolymer Nucleobase Chemical property Multiprotein complex Electronic cigarette Branch (computer science) Chemische Biologie Acid Protein Optische Analyse RNA Tool steel Polymer Thermoforming Biomolecular structure Activity (UML) Sunscreen RNA Wursthülle Nucleic acid sequence Hybridisierung <Chemie> DNS-Synthese Lead Sand Cell (biology) Transcription (genetics) Tool steel Genregulation Translation <Genetik> Sea level Motion (physics) Process (computing) Setzen <Verfahrenstechnik> Area DNS-Synthese Molekulardynamik Water Biochemistry Molecular biology Korken Amine Functional group Chemical structure Ductility Screening (medicine) Basenpaarung Messenger RNA Biopolymer
DNS-Synthese Intron RNA Wursthülle Electronic cigarette Korken Water Protein Protein Transcription (genetics) Gap junction Posttranslational modification Polymer Genregulation Translation <Genetik> Medical history Messenger RNA Process (computing) Stuffing Biomolecular structure
Metal Ionenbindung Polyphosphate Stickstoffatom Aldol RNA Carbon (fiber) Chemical reaction Methionine Methylgruppe Korken Lone pair Functional group Lactitol Ribosome Freezing Sea level Medical history Messenger RNA S-Adenosyl methionine Biomolecular structure
Glucose Sense District Ionenbindung Reaction mechanism RNA Hydroxyl Ribonucleoprotein Organische Verbindungen Wursthülle Man page Cheese Cellulose Cell (biology) Protein Elektronentransfer Periodate Angiotensin-converting enzyme Process (computing) Area Setzen <Verfahrenstechnik> Nucleotide Walking Intron Chemical reaction Electronic cigarette Water Korken Phosphate Soft matter Protein Azo coupling Ribosome Medroxyprogesterone Polymer Chemical structure Repeated sequence (DNA) Base (chemistry) Messenger RNA Extract Thermoforming Stuffing Biomolecular structure
Starvation response Sense District Blood vessel Recreational drug use Reaction mechanism Phosphorus Chloramphenicol RNA Hydroxyl Spaltfläche Wursthülle Man page Ribonuclease Magnesium Molecule Enzyme Cupcake Cell (biology) Orange juice Organic food Translation <Genetik> Golgi apparatus Enzyme inhibitor Sense District DNS-Synthese Chemical reaction Cancer Wine tasting descriptors Acid Phosphate Functional group Azo coupling RNA Pharmaceutics Chemical structure Polymer Base (chemistry) ISO-Komplex-Heilweise Messenger RNA Biomolecular structure Stuffing
Sense District Ionenbindung Recreational drug use Phosphorus RNA Wursthülle Acid Signal peptide Man page Hyperpolarisierung Cell (biology) Posttranslational modification Periodate Cell membrane Nucleic acid Setzen <Verfahrenstechnik> Zentrum für Molekulare Biomedizin Sense District Peptide Nucleotide Proline Omega-3 fatty acid DNS-Synthese Gezeitenküste Sulfur CHARGE syndrome Lipid bilayer Chemische Biologie RNA Guar Cobaltoxide Wasserbeständigkeit Peptide nucleic acid Thermoforming Basenpaarung Biomolecular structure Biopolymer
Chain (unit) Sense District Reaction mechanism Octane rating Mashing Fluorescence RNA interference RNA Antibodies (film) Electronegativity Wursthülle Wine tasting descriptors Man page Ribonuclease Cell (biology) RNA Protein Clay Controller (control theory) Kernproteine Chemical structure Dye penetrant inspection Nucleolus
Sense District Reaction mechanism Activity (UML) Bohrium Plasmid Binding energy Gene Wursthülle Stem-loop Man page Ribosome Cell (biology) Protein Active site Medroxyprogesterone Periodate Process (computing) Area Peptide Biosynthesis DNS-Synthese Topicity Potato chip RNA Ribosome Kernproteine Dye penetrant inspection Messenger RNA Biomolecular structure
Sense District Ionenbindung Ester Polyurethane Activity (UML) Reaction mechanism Peptide RNA Wursthülle Organische Verbindungen Acid Nucleic acid sequence Man page Enzyme Machinability Magnetometer Amine Cell (biology) Boiling N-terminus Protein Data conversion Controller (control theory) Amino acid Elektronentransfer Active site Setzen <Verfahrenstechnik> University of Porto Peptide Arginine Transfer RNA Threonine Chemical reaction Ester Hydroxide Vancomycin Gesundheitsstörung Acid Amine Protein Functional group Ribosome Herzog August Library Carboxylierung Base (chemistry) Thermoforming
Antibiotic resistance Peptide Erdölraffination RNA Binding energy Antibacterial Man page Tetracycline Block (periodic table) Hardness Medicalization Attachment theory Protein Ribosome Cell (biology) Protein Ribosome Active site Translation <Genetik>
Species Reaction mechanism Drainage divide Wursthülle Man page Molecule Magnetometer Cell (biology) Protein Streptomycin Amino acid Translation <Genetik> Angiotensin-converting enzyme Area Setzen <Verfahrenstechnik> DNS-Synthese Formaldehyde Man page Methionine Stockfish Antibacterial Electronic cigarette Emission spectrum Translation <Genetik> Vancomycin Protein Ribosome Chemical structure
Stop codon Ionenbindung Biosynthesis Separation process Residue (chemistry) Ester Wursthülle Acid Island Cell (biology) Protein Gene expression Amino acid Sea level Setzen <Verfahrenstechnik> Nucleotide Concentrate DNS-Synthese Walking Stockfish Electronic cigarette Molecular biology Combine harvester Amine Ribosome Base (chemistry) Alanine Messenger RNA Basenpaarung Biomolecular structure
Sense District Sense District Nucleotide DNS-Synthese RNA Wursthülle Stockfish Peptide sequence Methionine DNS-Synthese Man page Translation <Genetik> Protein Transcription (genetics) Protein Amino acid Sea level Biomolecular structure
Acyl Aldehyde Burn Glutamic acid Enzyme Amine Aminopeptidase Protein Emission spectrum Phosphate Posttranslational modification Glutamin Peptide Protein biosynthesis Walking Chemical reaction Methionine Wine tasting descriptors Electronic cigarette Glutamic acid Vancomycin Phosphate Protein Azo coupling Thermoforming Biomolecular structure Stereoselectivity Sense District Ionenbindung Bearing (mechanical) Reaction mechanism Activity (UML) Action potential RNA Hydroxyl Lightning Acid Man page Protease Cell (biology) Gene expression Amino acid Active site Hydrolyse Carboxylierung Selenite Heterodimere Proline Formaldehyde Antibacterial Amine Functional group Chemical structure Base (chemistry) Carboxylierung
Intergranular corrosion Activin and inhibin Cell growth Binding energy Stem-loop Enzyme Cupcake Ribosome Aminopeptidase Protein Morse-Potenzial Inhibitor Organic food Enzyme inhibitor Trauma (medicine) Chemist Cancer Feed (film) Chemische Biologie Translation <Genetik> Tyrosin Protein Enzyme Cobaltoxide Biomolecular structure Blood vessel Recreational drug use Reaction mechanism RNA Hydroxyl Nutrient Wursthülle Acid Man page Appetite Meat analogue Volumetric flow rate Lactitol Human body temperature Cell (biology) Controller (control theory) Mortality rate Amino acid Initiation (chemistry) Active site Tool steel Genregulation Sea level Monoamine oxidase Naturstoff Area Potenz <Homöopathie> Tumor Biogenesis Ethylene-vinyl acetate Meat analogue Biochemistry Amine Functional group Genregulation Messenger RNA Pharmacy Basenpaarung
Übergangszustand Sense District Ionenbindung Biosynthesis Kohlenhydratstoffwechsel Epidermal growth factor Erdölraffination RNA Wursthülle Nucleic acid sequence Cross-link Man page Mercury (element) Lake Cellulose Enzyme Afterburner Cell (biology) Amino acid Library (computing) Translation <Genetik> Mixture Glycosylation Process (computing) Area Nucleobase Mixture Tumor Glykolsäure Proline DNS-Synthese Walking Sulfur Chemical reaction River source Electronic cigarette Wine tasting descriptors Protein Functional group RNA Amino acid Covalent bond Ultraviolettspektrum Polymer Thermoforming Stuffing Biomolecular structure Stereoselectivity
Translation <Genetik> Peptide Protein Covalent bond RNA Topicity Thermoforming
OK I will come back to back with a pick up where we left off last time last summer talked about on all things are interrelated and in particular today we're going to be talking about translation of messenger RNA to make proteins will be but looking at time the intricacies of that Howard's regulated and 2 other aspects and then I will look at the incorporation of unnatural amino acids and proteins dissident imported frontier this is an important frontier in chemical biology because it allows us to expand the palette of what's available for doing experiments involving proteins and proteins I do a lot of them but they only have 20 functionalities available to them and in recent years chemical biologists like Peter Schulz had been inventing ways of expanding their talent to go beyond the naturally occurring 20 will talk a little bit more about that in a moment and then finally will end today by talking about on libraries OK next week will be on 2 Wheat 6 will be on 2 Chapter 5 protein structure and a gentle be 2 lectures on marketing structure and there will be on 2 Chapter 6 protein function and again I'll be 2 lectures from that and work will just keep rolling month ago so many questions that were going things like that are it's the OK so
announcements the Kerry whatever these and and have to go over them
again I have office hours today Her due to come by a alternatively come by the tears office hours my office our next week will be on Wednesday I believe it's 245 to 345 OK I really
talked about letters recommendation
that some last-minute announcements on the book on the Journal article reported that this is going to be due next Thursday a week from today at 11 AM it is essential that you submit but they hard copy to be and also an electronic version through the tournament dot-com website and along those lines it's not really turned and officially until the hard copy is received an electronic version I will not accept any e-mails submissions it is hunted 20 of you enter I get 120 PDF staff to print out OK so no e-mail submissions must be received as a hard copy I'm OK so very briefly living review the requirements article choice is a good chance for you to think and make sure that your following directions only research articles you know it's a research article that has a method section of some experiments described in it and experimental section that discusses how the experiments were done now sometimes those experimental sections are found in the supplementary material that's on wine to accompany the paper so nowadays when papers published and typically is published this kind of the abridged version and then there's a 2nd supple that that's also published on online and concurrently and that supplement includes a lot of details that are too voluminous to fit in the paper carried journals journals have a requirement that you can exceed a certain number of words and it can include ceramic figures but there doesn't seem to be any requirements on the supplement so what people typically do nowadays is have these monster size supplements so and last year for example I published a paper that was 4 pages long and that it had like a 25 page supplement of single-spaced unit 25 pages with like an additional 15 figure something like that so that's not all that unusual but and so if you can find in that supplementary materials and methods experimental work he could find in actual journal article then you know that you're looking at a research article not a review on news and views again and again and I hear the journals that we're going to be using for this but 1 thing I need to caution you about is that this is the nature of nature the magazine or nature the Journal not nature Pharmaceutical reports appear there's probably 25 to journals that have the title nature not only 2 of those are acceptable for this for this report but project 1 them is nature of another 1 is Nature Chemical Biology all of the other variants on nature will not be acceptable OK so Macmillan which is the publisher nature again has a large number journals and they might say /slash nature them flight unless they were actually published in Nature or Nature Chemical Biology they're not acceptable for this project OK again if you give me something and you follow directions understand handed back to you on graded and tell you to redo it and give it interviewer late great for that assignment so it's essential to get the Journal article cracked and yes West every last year I so it yeah it did anyone had trouble during the dot-com your troubles of several had trouble doing would do it successfully now are a picture of pointing out for me I will have to take a look at that very minute OK thanks and thanks for letting me know that's good to know that in the last 2 points must have been published in the last year but it needs to have the number 2012 or 2013 on it and it must clearly focused upon chemical biology so has the ear of chemical biology article in the definition of chemical biology that using for this class which all of you know that thanks for appointing an arbitrary and any other issues that are coming up on any issue that came up my office hours how do you find a journal article that's relevant to your interests I'm hoping you all know about public that there's ways of restricting pub then searches to specific journals and I encourage you to use the OK now if your interests are exceedingly obscure like and you're only really interested in dermatology it's possible there were no chemical biology articles that are covered you know EP adorable cells in the last year has so it's possible that you won't find any chemical biology stuff going on in that field and in which case you might want to pick another topic and expand your interests OK but if you're interested like each idea something they're probably a dozen Chemical Biology relevant articles published in HIV last year OK maybe even more I don't even know OK so it's possible you might have to change around here you're topical events and to see what's available and again I highly encourage you to choose a topic for this assignment that will then meet you at your proposal right now in your reading a state art paper and when it comes time for you to propose something you can basically take what was in the paper apply that a go 1 step beyond OK that's a really good way to be creative read something it's really cool get inspired by it and bring in some new technique or something like that and then before you know it you're on your own it makes sense to have any other questions about the tough assignment like that OK
I want to talk to you very briefly about scientific writings as we've already discussed this is a major portion of the grade and I it's really essential for your future career and I believe very passionately in the ability of the of the portents of effective writing so I want to give you a few guidelines these aren't hard and fast rules rather guidelines that if you follow I guaranteed to your writing will be significant substantially better than just everybody else's writing a case of the 1st of these is strive for simple direct clear sentences think of your job as being like a journalist a reporter you want to have like a Hemingway s style meaning really short declarative sentences were each sentence is clear at this point and your goal is to make your writing as clear as possible located the absolute clear as possible and are the best way to do that is have short sentences if your sentence goes past about line and have it's simply too long but it is a good chance that the reader is going to be reading these things very quickly right that's the way everyone reads nowadays and will probably not have a chance to keep track of that and so Dechy clue you in that it's time to break the sentence up into something short and every sentence needs to have on a subject and verb and if you're choosing a verb choose 1 that involves an active voice use the active voice if you don't know an active voice means please go see I and someone on campus who can help you with writing there's a writing coordinator who can help you about this business about active voices totally mystifying to you get it checked out OK you need to know what that means and also along those lines if the earlier thing I mentioned about pub doesn't make sense to you go see the librarian in the Science Library OK there's there's people are expert at doing searches for the kind of thing that you're doing OK so you know whatever I'm telling you to do if what I'm telling you is totally foreign to you and wholly unfamiliar then I'm it's incumbent upon you to seek out resources that will help you if that's a and I'm I can help you a little bit during office hours but others people who were even better than I am writing on campus and even better than I am doing searches and ID should seek them out and use their expertise as well as on double-check your explanations for understand ability a comprehensive Billy this is really important you should be able to take your journal article reported that after it's written and then handed to the person sitting to the right of you and the person should be able to understand so make sure that it's understandable that's really that the true test that's 1 of the things I'm looking for a good writing I should be able to understand what's written on and then this is really important as well and avoided pronouns that are unclear this happens a lot of in this assignment it's very important that you specify precisely the objects and subjects of your sentences OK so by pronouns I mean things I words like but it's in you know things like their them the use those types of words are inherently unclear so what happens is you'll have some sentences like you know bile acid drives up production of immune cells or something like that and the next sentence they will say these these are terrible effects and what I don't know is whether these refers to then he themselves for the bile acids it is not clear to me and I know what you're thinking anything healthy spent a little bit more time on it will be clear but that's not the way you want to start communicate you want to communicate so that the reader has 1 and only 1 interpretation of your writing and again and if you avoid pronouns when it's unclear exactly what's what's being referred to you can make your writing much more precise and that's 1 of the things you strive for good science writer have questions about US science writing about style this is the style that I want you to follow up when you turn this and this is how all be thinking about when I signed grades to the written section of your report questions about the style a it's about time I want to touch you finally about plagiarism again this is 1 of those things that drives me crazy every year no matter how many times to talk about this will be the last time I discuss it there and the reason I'm going to discuss it with you now is I'm aware that not everyone knows what plagiarism is or certainly everyone gets caught doing plagiarism claims that they don't know so we're going to talk about it and define it very precisely case of plagiarism is borrowing someone else's words and other relevant question is how many words do you have to borrow before counters plagiarism case in science writing obviously you're going to be barring some words OK because you're going to be discussing the same sort of thing but what I'm interested in is your own thinking about those words OK so for example if you're writing about the evil kind case and were thus able protein that and I'm expecting you to borrow that were able to it's unavoidable you can't get around it without barring that but what I'm interested in is how you think about people your own thoughts about this protein and your own spin on that's OK so for example if a particular area and clever sentences something like although compounds that are effective in Beecher proved to be so to cytotoxic I says therefore inhibitors provided proof of concept for the efficacy of disrupting able OK so that's the example that he found in literature and you and you agree with this this makes sense to you and you want to have a sentence like this in your own report on let's talk a little bit about what plagiarism would be if you borrow that's OK so what happens is what students will avoid are what students will attempt to do is still go through and will do all of map to Mac version of the same sentence up here but but in their own report and this is what I call plagiarism so for example the replace compounds so small molecules and all place effective with acceptable activity and I'll say instead of in the trouble outside cells to cytotoxic proved toxic and cellular assets in the event of the reportedly had the support of molecules .period demonstrate proper concept effective efficacy of inhibit disrupting inhibited effectively that's plagiarism Acadia basically stolen someone else's fault appear now admittedly you have use different words you've done a one-to-one mapping of different words but you haven't told me anything new and I don't care about someone else's thoughts I care about your thoughts the goal of this assignment is for me to learn about your thoughts became the reason why I'm telling you this is not that I don't know that the whole world is all about ripping off stuff off the web and you know putting a new name on it and stuff like that that doesn't bother me that's not my concern here my concern here is that I learn how creative you are and how effective you are at reading something and then interpreting it in a new way in a way that hasn't been interpreted before that's the goal of this assignment so the goal assignment is not too simply recapitulate someone else's ideas the gold assignment is for you to tell me your own ideas and that's what I want to create I want to you and not someone else and so that's why I care about plagiarism OK so let me show you how to do this so that you avoid plagiarism OK so
this 1 down here this would be OK OK so what you do is you take this sentence and you think about it a little bit of head and you and you start to say Well you know what the problem here is 1st the sentence is Cluj it's a mess it violates the rule about too long a sentence right it's complicated short declarative sentences are better suited gonna break it up you're going to say the compounds reporters paper toxic Procell studies that's unavoidable at a right this is a fact there is no way that you can escape not being able to state the facts you could put the facts in your you're reported that you need to it's the 2nd part that interests me more which is interpretation and what want this said here here's the report however advances cancer therapy by describing a novel load a small molecule inhibitors in addition disruption of Abel a so what you've done here is you put the report you this report this scientific discovery in the context of the the larger field which is cancer research and then that's your spin on it OK that's what you've done to help me no about your creativity and that's really where that's the the value added that I'm looking for in a good scientific communication OK I know that you don't have to restate the facts you might even have to restate some of experimental methods that doesn't bother me what I'm really interested in the show it's how you interpret those facts how you spin the facts how you put them in the context of chemical biology and in the field and in cancer research backs the part where you get the acreage appear that's the part that interest they appear that's the part that I can say allow this person is thinking in a unique way that's part that really I'm looking for in this assignment it is that makes sense and I'm not trying to scare you but this plagiarism stuff but it is scary because of a leader in your career you can get fired from your job for even small amounts of plagiarism the the great historian Doris Kearns Goodwin was caught out of your barring something like half a sentence half of the sentence was enough to tarnish a a lifelong of work where she had achieved so much and I don't let that happen to you that's not it's not a it's not fair for all of your hard efforts OK do not want to be in that position and so now would be a time to try to resolve not to let that happen at any other other thoughts are questions about plagiarism does this make sense I'm not giving you a definition and give you an example hopefully the example makes total sense if it doesn't pass now OK so again and I will be searching for oil habit is actually doing Google searches and searching for this it's very easy for us to spot and if we do we do come down very heavily on this very hard because this is an academic integrity issue we will report people to the dean of there will be serious consequences I don't want to happen and so if we manage to have a whole year where we have to assignments was 0 plagiarism that I'm going to bump the grades up there on the the interface between these and these indecency cases that will happen for the whole class so there's a stick stick is the dean's office in the Securities procured higher grades and help me I get to the carrot side I'll tell you that offering the carrot now for many years and I've never ever been able to deliver this could be the year company I know it depresses me that's lady talking about this stuff because every time it have my office like I did think that was later will now you go In the at any questions about this concept OK only after the Oh I'm so so glad you asked that OK so this is brilliant OK so the question I got was but what if you use this 1st salads and right after that you put a number 2 and that's the reference countered that the paper that this was cut this came from the answer is no that would not be acceptable because you'd still be claiming that these words are your own words appeared the way that would be acceptable to use this would be to use the 1st sentence of published sentences put it in "quotation mark "quotation mark designate that you borrowed it from someone else and then put the reference back down to the citation OK that's really really important everyone of plagiarize as includes references not everyone but 90 per cent of the people put references to the stuff that the plagiarizing from a a doesn't have that still counts as plagiarism even if the reference the stuff that the sausage EU barred the stuff from so I let's see are you here just visiting here for the class of I'd welcome here which have a seat so that your be comfortable so I just don't you look so uncomfortable for all those who are willing to worry about it you're here so they get the hot seat OK any questions about announcement in the questions so it's a good question but let's move on and here's what
we saw last time what we saw last time is far away this is malleable polymer that folds upon itself as it forms Watson Crick and hoops the base pairs and this malleability and is a really fantastic property because it gives this biopolymer lots of different shapes to allow it to access a different structures and the structures as we're going to see today and confer function peso 1 of the themes of the class is that structures of biopolymers leads to the function form follows function in biology not always but most of the time and we talked a little bit about different base pairs and I also want to emphasize that the molecules were talking about the transcription factors the enzymes these are dying on dynamic molecules these are molecules that lives and breathes that had motions associated with them that have kinetic and dynamic parameters associated with that 1 of the dangers of teaching a class like this is that I show you a bunch of pictures of beautiful molecules in a case like going to the zoo or something but on instead of being in a position to Europe is the where everything is frozen in place in you know that's not really the way animals exist you know animals like to move around and they like to be roaming around the Savannah or their cages or whatever it is that doing biomolecules similarly move around they have dynamics when I talk about something like the transcription factor and I describe it riding the rails of the Posso di there's backbone I really needed that is exactly what it's doing it is cruising on that idea neighbor highway as it looks for the correct base pairs to grab on to this is essential you must start thinking about these molecules as having a 4th Dimension of having motions associated with them on and this is 1 of the frontiers in chemical biology and it's an area that we need to continue to push an explorer and understand better because in doing so we creating a much richer view of how things are happening inside cells OK I'll try to continue to emphasize this point and we talked a little bit about how transcription factors skin DNA sequences at very high speeds and then they form a distinctly different interactions upon finding their specific sequence that they want to buy in other words the error of zooming along these fostered Esther rails and when they find that particular of correct structure baby Spanish French down and they form interactions either directly with the DNA bases or indirectly through water molecules with the DNA bases and that's what allows them to bind to a particular sequence of DNA recruited the other props factors that are required for transcription and eventually recruited on a plenary and kick off transcription at the end of Tuesday's lecture we introduced you to this piece to hybrid screen this is a very powerful tool that allows us to test protein-protein interactions themselves it's used pretty ubiquitously I would say on the last few years its use has fallen off a little bit but it's still 1 of the major of tools that are used in and say biochemistry molecular biology laboratories and even chemical biology laboratories I told you about the variant that had to binding partners there are however variants that have 3 mining partners where you can have stayed 2 proteins that are kind of like that at the bread in a sand wedge and then a small molecule in the middle it's kind of like the meat in the same way and the 3 these things have to come together before the transcription takes place and then it's also possible to look for things that push apart the interaction if you're turning on say a toxic gene and so that's called the reverse to hybrid and so there is you know half a dozen or so different variants of the seized to hybrid available this hybrid idea they're available and but they're all based upon the idea that you could seperate out the activation domain from the DNA binding domain and in doing so and you end up with something that can be read capitulated that can be reformed upon interaction upon formation of interaction OK any questions about what we saw on Tuesday questions about anything like that I well and I want to move on and I want to talk next about translation and a acidic have just a little bit more to talk about in terms of transcription and a messenger RNA and the branch translation OK so we get to where we left off last time the case the last time I ended with the observation
that the bacteria and you carry loads and have very different levels of complexity in terms of their M RNA processing right where bacteria had DNA that's transcribed and then this summer on a leads directly to translation where as nuclear have on DNA that's transcribed and then I introns or inserts a cut out that Sonza rejoicing and then MRT is modified at 1 and there's a poly detail added at the other end there's a cap and all this must take place before translation can actually happened so what we dive right in and take a look at it back a little bit closer at the the chemistry of you Quranic in translations were sorry on the chemistry
of and Moroney processing before translation case so
here is that you know here's a little short justice and water history through literature from short summary of what the I'd changes look like OK so again DNA needs transcription get was already transferred thorny transcript his 1st capped at the prime and and there is this GE Mathilde cap that's added and this is kind of a weird looking the right has found a trifle also Esther died yesterday on the back bone and it has some weird linking this is pipeline to fight crime and have this cap over here but this evolved in a way to allow them on a date to be on the shuttle very quickly to the Rivasseau appear cable talk a little bit more about how that works in a moment and at the other end of the 3 prime and the messenger RNA is tagged with a long sequence of EIS so this is called the poly a tail at the 3 crime and and then finally the introns box placed out there actually chopped out from the reader chopped up by an active process involving other proteins or sometimes just spontaneously and then finally leftover stuff the Exxon's are actually expresses protein has so there's a lot of modifications that takes place after the messenger RNA is synthesized and when we take
a closer look at best let's start with this GTP cap and this is the triphosphate there's the triphosphate over here but this is a we're looking at a sequence notice the extra metals 1 here there's 1 here and other than that it looks kind of like a G on this has the function of helping to load the 5 prime and of the messenger RNA onto the ribosome OK so it gets things going the
way this bond is formed for the methylation of that is a distinct from I'd say 99 per cent of carbon-carbon bond forming reactions in biology it but it's also number 2 in terms of its importance so for that reason we should take a moment to talk about this 1st and let me just as I digress for a moment and we'll talk later about how 90 percent-plus of carbon-carbon bonds are formed biology there formed using Algol reaction this actually is a rare example of a carbon-carbon bond or of sorry is actually not covered is a carbon I had around but this is a rare example Though forming a bond the comment cannot 3 2 and the novel reaction come back OK this is actually uses an S reaction and it's a straightforward nucleophilic attack by the lone Peronist nitrogen notice at this moment here is not involved it not involved in Eramet history so it a very good nucleophile to attack the level proof of this presidency findings as the Dennis methionine has the role of delivering methyl groups can actually tell anything about it this is that a freeze up there's not so helpful to us the time so apologies now that's
on the 5 pride and the messenger RNA on the the 3 primary and there is an appendix of a playoff series of areas that are appended and the number exact number varies but it can be really locked it goes between 50 to 200 bases of just Pollyanna better simply stuck on there and I know what you think you're thinking this is a total waste of energy .period why would it bother doing us a K what it's what is up with that and I know this is useful because it binds to a poly a binding protein is a protein shown here that evolved to blind to these hauliers and that helps direct the the an ace to the writers of the present turns out that's actually useful things so these 2 ends of messenger RNA actors specialized handles where they have a direction aleady and directionally as you know matters a lot in the sequences of our day right was only 1 direction that leads to a correct sequence of other directions leads to gibberish ,comma now because all of the messenger RNA is are appended with this ah Polly a tale there's a really effective way that we can use to isolate all messenger RNA is Moselle and throw away everything else so what you can do it you could set up a solid support that has a bunch of cheese down to a pay and then hybridized that to all the stuff found themselves the only things that will stick by the messenger RNA is which have a poly a tale so in practice the way this works in his arm will use the but DCC and which we previously saw offered forming bonds backing him 51 but here we're going to use it the form of fossil dinosaur bonds and of which you do is you simply add an excess of the of the Oxy are sorry but this is achieved motto phosphate and 2 With this DCC and then in the presence of cellulose this will not react with the cellulose the primary hydroxyl of the cellulose notice that this is cellulose cellulose of course is the BTG glucose as polarized and here's the primary height 6 hydroxyl of the glucose is and that will react with 1 of the city's amenities will polarize with each other and in the presence of this coupling agent DCC mechanism here is exactly like what we saw when we saw formation of mn bonds using GCC back are soft organic chemistry backing can 51 and on it that mechanism is not apparent to you please go back to your SUV organic chemistry textbooks and look it up again that mechanism that's a useful work OK in any case which you end up with then is basically paper that has a budget cheese covalently linked to an apology to just sequence just kind of hanging out there in space and on you then solubilized says you don't get water and you flow over this extraction itself so almost everything in the cell washes them past the that the paper except for the messenger RNA because the message on days are now going to form what a great base period these duties to Napoli a on the messenger RNA Poly TO the cellulose in the 2 of these hybridized to each other and that allows you to isolate all of the messenger RNA days and wash away all the stuff that's found itself makes sense OK so this is very routinely used but I don't think most people have that sent to much time thinking about how this is synthesized sounds pretty straightforward OK let's talk about
the next steps in the processing of message or a days so after army after the year of after the capped on 1 and with the GDP caps and on at the other end of the Paleaae of the introns have to be spliced out there's a bunch of In a short nucleotide will arrive in nuclear tied repeat that RE are that sort of thing piled on introns brings stuff together and set up a tree and spouse correlation reaction because this is where you get a transfer apostle Deuster born from here to here so it's just a simple exchange and that has the effect of cutting out the enshrined in this interesting Lariat structure details here are not so important for us on this
does however bring up the really interesting observation that RNA is cable capable of catalyzing reactions and this is kind of our 1st example of this that we're looking at it some detail so I want to show you a more canonical example of an RNA acting as a catalyst and that example is the classic and hammerhead right beside OK so here's the structure of the hammerhead right designed in green this is a on naturally occurring already sequence and in red this is a sequence of masses of arms RNA that's targeted for cleavage by this time by this hammerhead-were right I and what the hammerhead I'm does is it Orient's of bases close fight to the 2 prime hydroxyl to deeper-than-expected I'm hydroxyl is also a magnesium bound and that sets up a nucleophilic attack on the phosphorus of the phosphate and this is starting to look really familiar right we've seen ways to cleave already before and you know what this is identical to it the only difference here is that the I'm polymer organizing this this this dark catalyzing relayed this attack on happens to be an honest and so whenever we see it as a catalyst that is an on that's catalyzing some reactions were just going to call it a right beside so like an enzyme except it's made out of work on a case from recently my colleague entree look tack discovered that these cells that these aren't are these writers lines are very widely dispersed across all all the creatures found on the planet and he's found them in humans he found them starfish and a whole series of other organisms from again all of these require magnesium magnesium is playing a key role as Lewis acid it's stabilizing but on the negative charge that surrounding this phosphorus and making a better electrified OK so
I'm in the cell the cell has a message on and then I and it has to eventually degraded so the cell happened and furthermore this cell it is uh you know constantly coming up with stuff that you know that's getting about murder was a viral RNA so there has to be a mechanism of destroying on after it's finished the peso after its time has come after the translation is taking place there needs to be away of degrading the messenger RNA and for that matter it's useful to be able to degree and RNA that's coming in from Idaho viruses and things like that so but this has been taken aback OK so the way this works from 1 way to target messenger RNA for destruction is to use it in the sense that DNA so the antisense DNA will work recruit which will after hybridized is to the messenger RNA it will work crews ribonucleic H and this will then destroyed the the orange OK so this idea of using antisense DNA as a way of on targeting specific messages sent out by the cell from would be amazingly powerful right we have always say shutting down cancer if we can target specific style of messenger RNA is that are associated with cancer this would be very very powerful OK so In recent years now and there is no reason years is going on for like 20 years there's been a attempts to develop in the sense therapies these are therapeutics that will do something exactly like vessel delivered a sequence that hybridized to specific messenger RNA is and then recruits rival nuclear states to degrade that message and prevented OK this is distinct from conventional pharmaceuticals which to which often feature small molecule that inhibits some enzymes case of a standard way to do this would be to allow the messenger on to be translated resulting in an enzyme and then destroyed the error not destroyed but disrupt the enzyme by inhibiting it using some small molecule inhibitors OK we saw examples of this right we suffer example chloramphenicol targeting chloramphenicol a single transferees right and so in this case and will instead of like targeting the enzyme the results from translation ridicule the message itself prevent translation and in this way prevent this enzyme from doing its functions packed so it's really distinct mode of therapeutics and it's I would say a couple of years ago up until the 2 3 years ago I was deeply skeptical about the whole thing but there's been recent progress I believe there's no to drugs and approved by the FDA based on this principle and things are starting to look a lot stronger and cake here's what the
problem was here's why this took so long OK so here's 1 example of they are FDA-approved drugs that I use this this and this principle of the drums called of form of yourself and it targets of CMB cytomegalovirus and RNA and it does this by lots of your support of your centre-forwards perfect Watson Crick base pairing with the CMB and that in turn recruits on a stage at which the scissors to chop apart this Bob sequence and that the real problem here is that these these on sequences that 80 cents sequences notice of called antisense because they have to have the Watson Crick base pairs so instead of having it that it's so there has to be season GEC isn't he is lining up along looking now and it doesn't look so the from missiles but you know I made bright so here's Stephen isn't Jesus sees lining up so on that's why they call it he but a major challenge is delivering in these biopolymers in a way that they can actually get inside the cell and the effect of challenge number 1 is that the DNA and RNA are pretty short lived outside the cell we've already discussed already says there's plenty of on cases that are circulating there's also plenty of Indian aces on those tend to chop apart wayward strands of DNA urinated that happened to be floating around OK so from what people have been doing is modifying the backbone so instead of Oslo that Esther backbone of this into sense therapeutic instead 1 oxygen says we replaced with the sulfur and that backbone modification prevents the degradation of the US targeted sequence OK so that's 1 thing that's happening
here are some examples of other backbone modifications in 1 of those on phosphorus is replaced entirely with family bonds in a peptide nucleic acid and perhaps the most effective examples of these are these more fully know only nucleotides that I have this weird more fully in type of a backbone of these tend to work really well pays more fully elegantly tides are used routinely in chemical biology in biology laboratories as a way of knocking out specific messages seeking take some and Lorene Take that sequence converted to an 80 cents and then order up a more fully know only nucleotide which incidentally is not cheap but it can be done and I you can then use this directly in your experiments notice that the big change here is a change from having lots of negative charge on the backbone to have a neutral backbones OK that helps quite a bit in terms of delivering the therapeutic inside the cell mate negatively charged things have trouble passing through the fossil lipid membrane layer that surrounds cells right we talked about how this is there has it outside that's polar inside this hydrophobic charge things don't like sitting through that pull that off hydrophobic region of the fossil lipid .period plasma membrane and so for this reason these neutral things that are are more effective yes
I would say earlier OK yes means it doesn't happen anymore really just be a big here but they stressed in the right I've said
before this is useful laboratory straight example of this OK so what we're doing here is on more interested in targeting a particular message on a that poses the Menton and the Manton protein is produced it will be staying using it body an antibody happens to be dyed red so you will see it under fluorescence Mike microscope wrapped image and I think I'm turned on the lights even more because it's a little hard to see but it looks a little bit better on my serve disk attorneys or very briefly and so here's cells in blood this is the nuclear speak staying with the floor for DAB that happens to combine well to do you know I think we might have even seen a little bit about it earlier but structure earlier and again in red this is the lamented protein OK so this is basically a beacon negative control this is on short interfering RNA that does not target the mountain chain essential that you do these controls OK so you've treated the cells with of RNA but in this case is on a the doesn't have the antisense necessary to target the message encoding the mountain looking over here cells that and targeted using this as irony which again is this RNA interference mechanism that we've been talking about but now the antisense targets the emirate codes fermented and notice that there is very little red this might be a little bit here but for the most part it's totally clear of the red yet you can still see the nuclei of the cells rating is still see these blue nuclei which is the DNA of in the nuclei being stains take you once you have tasted this works really well right now and again on the on the way this is going to work in this case it is so you
have a lot of plasmid that includes this mail prepare and furthermore it's even more complicated than that from what you do is you actually set up so you have this plasmid remember recall that plasmids are circular DNA from the plasmid encodes the this sequence that's going to be the antisense sequence and end up in a practice is actually encodes not something that simply an antisense sequence rather it echoes both the center and the antisense sequence into a hairpin OK so over here this upper strand is this sequence the Ambonnay it looks kind of like a modern-day out that encodes the mentor gene it but it's just a little fragment of that and then but there is a little late but here right that's a loop that we've seen before and then down here on the Lower Austria and this is the antisense sequence so sense indecency case now what happens is on this short hair .period is now a section of double-stranded RNA and then activates a mechanism the cell called Dysart and Dicer goes through and systematically looks for any sense strands of messenger RNA there have this sequence and catalytically goes through and starts chopping those apart a panic chops 1 after another apart state and if you want to learn more about Dicer and and other proteins of you can read about it in the text at and next start
switch gears so as to any other questions about the MRA processing questions about the topic it turns out it's a really active area of research and it's always been active always fascinated by the few surprises the costly coming along I want switch gears that I want to talk to you a little bit about what happens next and the messenger RNA is eventually delivered to the right in procuring the ribosome binding site or RBS is something called a shine Delgado's sequence and its of a guideline than a sequence of you can actually get away with some variations on this shine Delgado sequence but it turns out that if you don't program at but nothing happens every so often you know someone new joins laboratory designs their proteins you know they're they're constructed be expressed and nothing happens the cells refused to take it up is because they've forgotten the shined up on a sequence so it is essential new carrots and there's something called the Kozak sequence and this idea is the same there's this area where the messenger RNA is bound by the ribosome and iconic gets everything going OK now the
actual ribosome catalyzing stammered bond formation is a pretty straightforward reaction simply consists of the means of attacking esters OK so I recall from backing him 51 that if you mix together amino acids and you boil them for a long time you can form armored bond but deficiency was very low and you have control so much over on which him bond was going to be made pay so we talked about why it was important to activate the carboxyl 8 of the amino acid to form enamored bond with greater specificity right and if you're a 51 C with me we had this conversation and again if this conversation about activation and DCC is totally foreign totally confusing go back and take a look in your textbook from some organic chemistry said so DCC I've alluded to twice in this class and the times I told you if you don't know what it is to go back and look at that and so in this case the cell doesn't have access to DEC said OK instead its activation agent is on warming amino acid into an activated Esther OK so here's an amino acid happens to be the finding and all over here is the transfer on it and so what's going to happen is the arms this will form an Alan Bond with a N terminus of an peptide bond that will attack this Astor his this'll be the 1st mn bonds and on the 2nd 1 will be the next amino acid delivered to attack transfer RNA of the star of this threonine etc OK so the ribosome is stringing together these transfer RNA is that have activated amino acids attached to them so I'm going to be referred to these activated amino acids as amino easel T on it were a soul refers to the fact that these have swarmed into Esther functionalities that makes sense OK and furthermore it makes sense that we have is activated Esther and another way of thinking about this is that hydroxide is a terrible leaving groups and so instead of having hydroxide is a leading group we have I'm Alcock side that happens to be a special Cox cited the catalysts but on that's idea aren't so chemically what's happening here is the on incoming amino acid is attacking disaster cases illustrate to attack of the low-carb meal and then but you worn the stature he dual intermediate that attaches digital intermediate then collapses and the results it is on the formation of an Emmitt Bond and I have 2 possible mechanisms here and 1 that could take place under basic conditions on the top and 1 that takes place under acidic more acidic conditions on the bottom either 1 of these is legitimate both of them need to formation of enamored by the straightforward mechanism it's 1 that I'm hoping you're familiar with from back in the day and it if not go home tried a couple times herself it should be pretty straightforward OK so let's take a
look at the ribosome itself the ribosome is really a mega machine it's a huge machine that has upwards of 20 different parts to its constituent parts and these include both proteins and which are shown here in blue and also RNA sequences that are all kind of put together a case so here's the message on aiding red eye and then here's the peptide being spit back out of the ribosome but notice that the the actions sigh the site of action called active site is in the very center of the ribosome and if you look at the center of the ribosome it's mainly on a case so in fact the ribosome it is the right design it actually it relies upon on a to catalyze this amino license mechanism that I showed you earlier OK we
see if I got everything right here OK it's and sure of proteins etc. OK but here's how it
turns out that because it plays such a key role in the cell a protein translation it's also a major focal sites for antibiotics target and it's hard for antibiotic resistance to with this 1 because on it's hard and not mess up the ribosome without losing its catalytic efficiency which is to a quarter of the cell to start messing around with and so on many antibiotics target of the riders on and 1 of these for example is the antibiotic Texas cycle and attach cycle binds directly in the the active site up here and also as a lower affinity binding site on down here but and is shown in purple OK so attentive cycle routinely given Antioch medication effective way of killing off bacteria it happens to have slightly higher affinity for war and the bacterial ribosomes than the human right is known but the differences are fairly subtle OK
so here's the structure of Texas cycling down here and has 4 rings hence the name and again this targets the ribosome there a whole series of different molecules target the riders things like Hannah Meissen Eritrea said this is another 1 that should be familiar to those of you who have bacterial infections at some point you like you probably can refer it's a Mac relied antibiotic will talk more about these public he tied about exit amounts in a in a few weeks partly to and the class but also it targets the ribosome streptomycin also targets the ribosome totally different structure and this is enemy don't like idea about 1 and then they're antibiotics the target not the ribosome prosaic but the machinery that helps to load Tr ace up onto the ribosome and as the 2 ways of doing this 1 is targeting you have to you shown here I carry son and another 1 is targeting another protocol DFG which is this 1 over here and in the case all 5 of these molecules operated by a common mechanism they all operate by shutting down protein translation for the cell is 1 of those areas where that's just really rich with lots of lots of different antibiotics and will see this time and again when we talked about the molecules the target DNA we talked about molecules that target of the writers of the sort of like Achilles heels for the cell areas the real choke points that it about and get in and that's a pretty readily and do it in a broad spectrum way where the killing lots and lots of different species really of bacteria this case OK
so let's talk about translation so translation starts with a stock code on in you carry out sister code on encodes the amino acid methionine and so the end terminus Of all the time all proteins synthesized you carry out so starts off with a formal fighting notice that this format man and that's been appended to it and it's just another way of getting things going on in so in 0 sorry this is the bacteria case bacteria starts the formal methionine you carry no formal methionine let's take a closer look at the Turin
a tyrannies again bring amino acids the ribosome has activated esters as needle insult urinates OK and at 1 end of the right is a sorry 1 end of the tiara today the 3 prime and the amino acid is loaded in as a Esther we down here at the other end there's 3 weeks called the intake hold on which will try to hybridized to the messenger RNA in if they hybridized that tells the ribosome that's the correct sequence of the cracked amino acid that's being loaded in 4 and the bond formation this is really essential this base pairing between NT coat on and put on loops this is what allows the and corrects the synthesis of the correct sequence right otherwise you begin at DNA appear messenger endanger proteins this 1 this is the last step really in the US central dogma of molecular biology this is what gives you the correct sequence that was encoded by DNA in the 1st place OK now but here's the way this works
so the the messenger RNA His read out in 3 base pairs sequences called code arts take each 1 of 3 bases leads to a different amino acid in I'm showing you what the amino acids are of the 20 amino acids on this genetic code diagram In here's the way you read this genetic code diagram you start in the center and this tells us let's just start with G. OK so if the 1st of residue is key and the 2nd 1 is and the 3rd 1 is this season's GCC would lead to alanine K C H leads to collaborate the UGA however at least stops cases to possible stop codons 3 possible stock could islands that are useful and those tell the ribosome you off you know cacophonous Lorene you're done OK and that stops the sequence OK so there's 64 possible combinations there's only 20 amino acids blossom starts so on what this means then is that several code lines and code for the same amino amino-acid in practice there is some slight preference for summoned codons over others and this preference is dictated by the levels of T R so there is some tyrannies that are present in higher concentrations in the cell and in practice when you design a preview of expression you look for I couldn't there are more popular then the less popular 1 there's some code of ones that are exceedingly rare inside the cell and if you have a choice but said they are 4 different code islands in the case of 380 down here will choose the most popular ones and I don't remember what it is but you choose that ACC rather than ECU because it's represented more often than not you OK so the
years so it was that here's what it looks like on DNA has a sense strand antisense strand in during the transcription a copy again is made of the sense strand and this copy is translated out on this sequence up here results in on the amino acid protein sequence down here so for example ATG we've seen is a stock code on call a circuit on but we know it encodes the finely cut
a T G OK methionine right case of this encodes methionine and over
here ATG as a code on at the DNA level results in a finding down here OK similarly GTG G T G and so
over here results severely gain seeking do this pretty readily family's genetic code you know we'll diagrams that provided that it is in the book to him very readily figure out what is a sequence of protein will result that makes sense focus on now
crucial step at some point you have to load the correct amino acid onto the tiara if the the amino acid is mismatched with the anti code on down here this cell is in big trouble right this is essential to get the correct sequence out and so on In symbologist debated for a very long time how the molecular recognition of the T on a would work with jet with recognizing just 3 bases of the I of the anti code on loops of the cheering and practice what we found is actually the enzyme responsible for this loading an enzyme that I called amino lethal Turin hastened the taste of this enzyme is a monster OK so it forms a dimer it showed hearing Green these are To urinates 1 on left side 1 on the right side and that is how this thing is just grabbing onto both of these so it's interacting not just with the ENT code on down here to read out the sequence but with lots of other places along the T and furthermore appeared this is the active site where on the amino a soul of the amino acid forms of Esteban street crime hydroxyl also you in a moment with the mechanism of that reaction it's but again I noticed that the new wasteful Tierney said the taste in goal the whole Teran it's at a fair and so there's more interactions just the intercom and furthermore earlier urging Member I told you how TRA is especially were very heavily modified back when we were looking at say the cloverleaf structure of during a I said how heavily modify the hour that heavy modification helps direct the correct your way over here and to the correct amino acid up here and it's being read out by this summer protein by this enzyme that's checking it over a OK makes sense are so let's take a closer
look at the mechanism and in practice the mechanism involves activating the carboxyl late because again carboxyl later very ignorant they don't like to be so they don't like to form of bonds already hydroxide is about leaving group and so in practice this is activated by forming day on an easel phosphate intermediate using ATP as activating agent peso phosphate is kind of like nature is postulated these late it's some super meeting group that's ubiquitous that's found all over the place and in biology and this is going to work by forming a readily high delights of 1 of so for example on the Tiny all Arnason details on starts with glutamine "quotation mark glutamic acid glutamate and the activates this through a nasal phosphate intermediate and so on then the good miltiary said that this asked OK is the easel fostered intermediate available glutamate and it's not glutamate then at high July says this intermediate and if it is then it adds the the amino acid to the 3 prime hydroxyl of the Turin OK so it's a little bit complicated is actually on a couple of steps where things are checked and the other tyranny is bound and is gripped in a big bear hug where it's actually making sure that has the correct urine making sure by testing antiprotons on but also looking along the length of Turin and then on different intermediates easel phosphate intermediates are brought up to the active site and the enzyme is this the correct 1 is this glutamate and if it's glutamate that it forms a bond if not then it kicks off and when it kicks off that actually hide realizes the I'm lost state of the art of the the prostate intermediate OK the questions so far yet with back it's insanely wasteful right burning 18th review this so undersell invested enormous amount in protein synthesis care which is 1 of the reasons why cells hate doing over expression if they can avoid it that is a huge selection against you know when we do protein of expression allowed and turned cells into factories for producing proteins they would love to deal to avoid doing that effort if they could at you the huge amount wasted effort here ATP is getting Burke had requested the questions OK I'm so I told you that In bacteria in precarious and they all end with an informal group found at Appendix and terminus there's an enzyme called peptide tidy formalize that hides is all this informal groups and in on you carry out humans and often times the start methionine is high July off using methionine amino pet today's this is simply a Prodi's that kind realizes the and the bond here pesos gets in there Hi July says that I am bond but doesn't specifically on enter my its turns out this is also a potential target for antibiotics and so
for example the answer about the the natural products human tumor Guillen inhibits NGO Genesis which is the growth of blood vessels in the Indian human bodies and on it does this by using a very interesting mechanism so the natural products naturally has a three-member during an epoxy side that is precisely positioned next to a nucleophilic and that is all functionality recall that all functionality we talked about it on Tuesday in the context of Onasis here we're seeing it again in in an enzyme a different I'm not decide it's also neutral and it's also has this has again the PKK of 7 that we saw and so therefore it here is likely be available to act as a nucleophile and be covalently modified when it attacked this a Parkside cases this is an example of a suicide inhibitor it's suicidal because it gets in and then and it would work well in this case is sort of reversible but oftentimes irreversibly modifies the enzyme active site and in doing so kills the enzymes actually personally hate that word suicide inhibitors and I actually prefer a Trojan horse inhibitor which is a better word it is claimed by ,comma blocked user will be 1 of my heroes so enamored but it's caught on so it's hard to OK now why would you want to inhibit injure Genesis blood-vessel growth is great if you're at the gym working out you certainly want to have a blood vessel growth to feed those muscles that your building right of cake now the problem is when I tumors start to grow they have of gracious appetite they are desperate for everything they need more nutrients they need more oxygen they are and they're really hungry OK and so they will attract blood-vessel growth to them to feed the resultant tumor so important anti-cancer strategy targets that blood-vessel growth and prevents the blood vessels from growing and those drugs are called intake NGO Genesis drugs they inhibit injured Genesis and for some reason inhabiting the finding in pet today's is a strategy for blocking and you Genesis to block the feeding of tumors preventing the growth and hopefully getting them to shrivel up and it turns out that section effective strategy when it's combined with other anti-cancer therapeutics OK
so in addition to what I've shown you there are higher levels of regulation taking place inside the cell better regulating and translation and on these are things in years my favorite understand described 1 of the many possibilities of my favorite is a on a messenger RNA that at 1 and has its own ribosome binding site it's obvious hit in 1 inning here European when the temperature in the cellars increased the Watson Crick base pairing of this hairpin breaks apart exposing the ribosome binding sites and then allowing the message to to be translated that's really elegant OK that's the kind of elegant design that I really love and in theory I'm everyone in the class could design temperature sensitive sequences that would get turned on civic temperatures knowing for example the Wallace rule OK now and we talked about how did how this happens in the cell we chemists and our creative what constantly looking for new ways to to tinker with staff and tried it and they get better control over things inside the cell and 1 really exciting area that has been that has been really taken off in last few years but has been applied for roughly 20 years or so on idea of incorporating unnatural amino acids into proteins and so do this and work out what chemical biologists have been doing is hijacking the naturally occurring amino acids the cases that are found in different organisms and then co-opting them into noting specific amino acids unnatural amino acids oftentimes these amino Wiesel said that cases are on the modified their mutant proteins so the modified to accept unnatural amino acids This is an analog of amino acid tyrosine that would usually have a hydroxyl over here but now has an immediate and that's really cool experiment because now you can test what happens when I put a better based in place of the hydroxyl by putting out functionality in place of a funeral functionality and it turns out this is really a powerful it's something my own laboratory applies we apply just as a tool because other laboratories that are trying to extended to other areas and it's really something I encourage you to using your proposals against basically bread-and-butter technique using chemical biology laboratories that eventually will spread to biochemistry that's as well the thing is you can do
all kinds of stuff if you can incorporate unnatural amino acids for example you can cope incorporate middle populating amino acids and amino acids that form covalent bonds in the presence of UV light form cross links this is an example of the affinity tag amino acids that will come react specifically of carbohydrates and amino acids that will form across lakes in the presence of other functionality such as it is I so this is enormously powerful and inorganic urge you to just use it now and it's a very routine technique at this point that this is something that actually works well enough I have an undergraduate in my laboratory former can 128 student is doing it as we speak OK and it actually works pretty well and that really impresses me but there are no he's basically taking a technique that uh that is described in literature that are laughter never applied before and getting it to work OK any questions so far right it would switch gears again but we talked about translation we talked about corporation of natural nonsense I next 1 ends with a
discussion of afterburners and RNA sequences that are blind or catalyze reactions OK so it turns out that you can make a very very large libraries of on I mean I'm talking enormous you could make underwater attend to the 13 to tend to the 14 different sequences is that the 1 followed by 14 0 it's OK and you can have it all those different sequences in a little tiny at the north to small tested and and from there you can do all kinds of experiments on a case of for example you can identify the RNA sequences that might catalyze this reaction pretty readily obtained the way you would do this is on you'd set out so in this case you're looking for work on something that will will catalyze the guy glycol glycosylation of this Amin over here and so that we will do this is unreal have some sequence appended and then you look for all of the ones that have a soul for incorporated using mercury as a attract OK OK so that's kind of overview let's look at the details said the key concept here is that sulfur and mercury form a very strong bond and you can pull out specific sequences that happen to have sulfur in sequence case here's the way this actually works with that what you do is you start with some random DNA sequences work and is used any of the 4 DNA-based theirs OK see start with 4 DNA base pairs ACT attendances in the sea duty in this for the ACT year-to-year Interpol wondering how do you possibly synthesize tend to the 14 different sequences will turns out it's very easy at every step in the process you injected and all 4 DNA bases during the synthesis of the DNA a rather than just adding it is you had a mixture of 25 per cent is 25 cent seized 25 % Jesus cetera so that gives you all random DNA sequence of order of 10 to the 14th as you might tend to look for different DNA sequences you then use RNA polymerase we happen to favor 1 that's used by a virus viruses are very good at getting their stuff to the head of wine they're very aggressive enzymes which makes sense they have all they can to be really aggressive like that and so you can use this G-7 on a polymerase that will then converted DNA sequences into random RNA sequences and and then you can look for on a sequences that incorporates sulfur the paper and so here's your your compound that you're looking for a reaction when it could incorporate sulfur by some transition state then you can isolate that sulfur using a bond between mercury and sulfur and the Mercury is attached up to some solid support like the carbohydrate that we saw earlier like the cellulose that we saw earlier we talked about the Poly T called the exact same idea OK so now the only on a cell get isolated are the ones that have sulfur cooperated that every act specifically with this compound go from 10 to the 14th just down to add another 20 of 30 they're doing something this is really powerful because if you get tend to give you a Chilean 100 Chileans different sequences together there's a good chance that you can find 1 or 2 that do something special in your sequence and you can imagine evolving as you could take that sequence of mutated further make changes down here the selection and then do it a bunch of times in practice we often go for like 10 rounds with these on a libraries and all these are often called out tumors there are any sequences that blind to some target the inventor of this whole idea is going to be here at UC Irvine next week OK so some of the pioneers in this area are famous here he hereby and a guy who's the president and CEO of a company that set up around this concept will be here is you're going to be a seminar next week also send you the details I encourage you to go to a seminar its candidate and is kind of a heavyweight in the field OK
last thoughts and there's an antibiotic called pure Meissen which manages to sneak into the riders on and on form covalent bonds by mimicking the mean wasteful to urinate OK I'm I don't know why I have this year it doesn't look so intrigued but skipped
that OK let's just end here activists so when we come back next time we'll be talking about my favorite topic proteins


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AV-Portal 3.21.3 (19e43a18c8aa08bcbdf3e35b975c18acb737c630)