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Central DogmaDNARNAProteinDNA is the master blueprint, set of directions that everyone has to follow.Transcription and translation are happening at the same time in prokaryotic cells.Transcription and translation does not happen at the same time in eukaryotic cells making proofreading happen.- Add more steps- Everything is moving in a single directionTranscription (Txn)- DNAmRNATranslaiton (Tln)- mRNApeptide- DNARNAPeptide(s)Quaternary structure, modification (phosphorylation)ProteinTranscription and translation is a one-way process, Central Dogma of BiologyGenes can and DO sometimes overlap – splicing of RNAPre-mRNA is unprocessed, a raw “photocopy” of DNA code.Prokaryotes: naked DNATranscription and Translation are concurrentEukaryotes:- Txn inside nucleus- Tln in cytoplasmRNA processing occurs in Eukaryotes only4 nucleotides20 amino acidsIn Bacteria:Polyribosome: complex of ribosomes that go through transcription and translation at the same timeDNA Replication – single origin of replication in a genome- Compact that triggers transcription and translationWhat if a mistake is made?- Codon changes can be made duringo DNA Replicationo Transcription- Point Mutations:- Single Base Changeo May be silent: “Wobble” – redundancy in the 3rd nucleic acido May change AANow the detailed tour of transcription and translation- Transcription:1. Initiation2. Elongation3. TerminationInitiation- Promoter: site where the work beginso Eukaryotic Promoter: TATA Box (does not have to be a TATA box) Total of all three classes of players (many transcription factors) = Transcription Initiation Complex Spacing of TATA box and start codon is important (generally -25bp; or 25bp upstream of ATG) Not all promoters are TATA boxes Tells RNA polymerase which way to go – upstream and downstream- Two Common Promoterso Eukaryotic 5’ TATAAAA 3’ 3’ ATATTTT 5’o Prokaryotic 5’ TTTTATA 3’ 3’ AAAATAT 5’o When vertical, left strand = mRNA goes from top to bottom, right strand = mRNA goes from bottom to topo Left strand = mRNA going from top to bottom, transcription starts after promoter, right strand = mRNA going from bottom to top, transcription starts after promoter. – not right after, just a close distance- Worker: RNA polymerase – making RNA off of the DNA templateo Elongation RNA polymerase adds nucleotides to the 3’ end of the growing RNA strand. It doesn’t have to complete its work before a second RNA polymerase starts working.- Terminator: site where the work is stoppedo Termination RNA polymerase transcribes a terminator sequence and keeps on going in eukaryotes Eventually, the RNA molecule will be cut free from the polymerase just pastthis terminatorProkaryotes: mRNA is ready to be translatedEukaryotes: pre-mRNA is exported into the cytosol- Introns are cut out and exons are spliced together- 5’ cap and 3’ poly-a-tail is addedPre-Translation Processing- In eukaryotes, the mRNA is not ready to leave the nucleus immediately after transcriptiono 5’ Capo 3’ Poly-A-TailThe mRNA is finished…now what?- It is sent out of the nucleus and into the cytoplasm, there it will need to meet a ribosome.- Also required:o Amino acidso tRNAso Energy (ATP)Amino Acid Attachment Site- Two-dimensional Structure- Certain parts are connected by hydrogen bonds- Contains anticodon areaAminoacyl-tRNA Synthetase (Enzyme)- AMP charges the tRNA that will make an active tRNA and amino acidSchematic model showing binding sitesA Site (Aminacyl-tRNA Biding Site)P Site (Peptidyle-tRNA Binding Site)E Site (Exit Site)Initiation – 5’ cap and other sequences help ID where- All starts with a methionine (Met) – Initiator tRNA binds to the mRNA binding site - GTP converts to GDP- Large ribosomal subunits comes to make up the translation initiation complexTranslation- Initiationo Activated tRNA, mRNA, two subunits of rRNA come togethero rRNA recognizes leader segment of mRNA, assembles around ito Initiation codon tells rRNA where to add the first AAo Assembling this complex requires energy! Supervised by initiation factors (proteins)- Elongationo Addition of each AA to the growing polypeptide chain (dehydration synthesis)o “Ratcheting” of ribosome so it continues to move along mRNA, requires energyo Loading of new AAs to A site- Terminationo STOP codon read by rRNA. Water consumed in hydrolysis reaction that breaks bond between protein and P siteo Polypeptide released from ribosomeo Ribosome subunits separateTypes of Modifications:- Proteolysis – protein cutting- Glycosylation- PhosphorylationPossible destinations marked by “zip codes” which is part of N-terminal AA sequenceRibosomes are not permanently attached to rough ER. They dock at receptors that recognize the N terminus of the new peptide chain.TRANSLATIONAL ERRORS: FROM YEAST TO NEW THERAPEUTIC – pittcatPrimary Literature- Original findings, including materials and methods- IMRAD formato Abstract – What was done in a nutshell?o Introduction – What was the problem?o Materials and Methods – How was the problem solved?o Results – What did they find out?o Discussion – What does it mean?o Other Elements Citation – Whose word did they refer to? Title Abstract Acknowledgements (Optional) – Who helped them out? References Appendices (Optional) – Extra information Good papers are in peer-reviewed journals!- Authorso First Author – most of the grunt work, undergraduate/graduate studento Middle Authors – doing a portion or some part of the labo Last Authors – head of the lab, primary investigatorTitle and Abstract- Information dense- Details instead of dramaIs Bambi killing the forest?Introduction vs. Abstract- Introductiono Context, perplexity, resolution in funnel formato Sets the reader up for the rest of paper by defining the topic area and specific problemo Contains references to other sourcesMaterials and Methods- Reagents- Times, temperature, organisms- Techniques used- ControlsResults- Just the facts. What was seen.- NOT what it means- 50% greater mortality at Dosage X when compared to the control”- Figures, tables galoreTables- Data grids- Self explanatory when combined with titleFigures- Images, maps, charts, graphs, anything that is not a data grid- Get captionsWhy yeast?- Saccharomyces cerevisiae- B20-22 in back or textS. cerevisiae- Eukaryotic- Unicellular- Replicate asexually or sexually depending on conditions, can


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Pitt BIOSC 0160 - Lecture notes

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