Fall 2014 LS4 Midterm Review

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<ul><li><p>8/10/2019 Fall 2014 LS4 Midterm Review</p><p> 1/4</p><p>Translation</p><p>Large, small subunitrRNA, proteins</p><p>RNAs play key role in almost every step of translation </p><p>Mediate interaction btwn mRNA and tRNA during initiation, catalyze peptidebond formation in elongationKnow the three sites: A, P, E and whre they are located wrt the large subunit and themRNA</p><p>Initiation:IF-1, IF-2 (G), -IF-3Know which translation factors are the G proteins and which arent </p><p>IF-2 is the only G-protein; when the large subunit binds IF-2 hydrolyzes GTPand IF-1 and IF-2 are released</p><p>Shine Dalgarno seq- recognized by base pairing with 16s RrnaThe AUG isnt enough for prokaryotes to initiate translation; they need theshine dalgarno located in the 5 UTR to distinguish btwn start codon andinternal AUG. the small subunit goes along looking for the SD sequence, H-bonds to it, and if AUG is situated correctly in the P site, the small subunitbinds</p><p>Initiator tRNA and formylated METFormylated MET resembles peptide bond so can fit right into P site</p><p>Ternary complex formation: total ribosome, all of initiation factors are gone, readyto go into elongation</p><p>Elongation:EF-Tu, EF-GBinding of aaTRNA to A site: bc our initiatiortRNA is in the P sitePeptide bond formation- know the chemistry- who is attacking who</p><p>N of aminoacyl tRNA attacks carbonyl of peptidyl tRNATranslocation- facilitatedby the EF-G; EF-tu brings in the tRNABoth of these are G rpoteins</p><p>TerminationRF1, RF2, RF3, RRFEF-G-GTP and IF3 play roleEF-G-GTP helps in translocation; IF3 helps dissociate two subunitsStop codon always in A site, release factors along with EF-G mimic the tRNA bc theyhave to fit into the A site- anything that has to fit in the same site has to look thesameWhole process begins with hydrolysis of polypeptide from the tRNA</p></li><li><p>8/10/2019 Fall 2014 LS4 Midterm Review</p><p> 2/4</p><p>Translocation- ribosome is moving relative to the mRNA. It is a relative movement.The trnas stay associated with the codons that they came in. the tRNAs and mrNAsare hydrogen bonded they will stay fixed. The tRNA is still bonded to the mRNA moving relative to ea/other</p><p>Prokaryotic gene regulationBasal, repressed, activated levels of transcription</p><p>Basal is neither activated nor repressed; no transcription factors binding ornon-functioning transcription factors (ex. Is Gal4)</p><p>Effectors regulate the regulators the proteins are sensitive to the environment sothey are there to regulate in response to something else. Effects allostericallyArabinose regulates AraCcAMP regulated CAP</p><p>Transcription factors all need to bind to DNA bc that is how they know what Gs toregulateThey have seq. UPSTREAM of promoters in prokaryotesSeq. identified by dna binding motifsRun a gene thru a database and see if it has a dna binding motifIn porkaryotes helix turn helix is most commonHomeodomain found in eukaryotes which is slight variation2 dimeric types: basic leu zipper and basic helix loop helix-=- BOTH HAVE TO BEDIMERS TO BINDZn fingers are largest class; can be dimeric or monomeric</p><p>Dont memorize details of structure but know generalities of how they interactw/DNA and how they interact w/themselves if they are dimers</p><p>Bacterial gene regulation</p><p>Lac opronInducers vs substrates (Xgal, IPTG, lactose) know why we can separate inducer fromsubstrate. LACTOSE CAN BE BOTH. Inducers are the allosteric effectors. Substrate isconverted to a productIn most cases there is two diff proteins involved inthese processes</p><p>Experiments leading to model of lac regulationLac repressor- inducerIsolated mutation in repressor protein that bound the inducerLater found out there was positive regulator -- CAPCAP-cAMP- glucose sensor, DIRECT interaction w/RNA pol</p><p>Ara operon</p></li><li><p>8/10/2019 Fall 2014 LS4 Midterm Review</p><p> 3/4</p><p>First operon that was discovered that loops so u can have sites far away that areregulating promoter regionAraC can act as repressor or activator; this is dependent on arabinose effectorIt co-activates with the cap-cAMP so this is sensitive to level fo glucose in the mediaInvolves looping of the DNA</p><p>Ara and Lac are both combinatorial because they both are regulated by TWODIFFFERENT REGULATORS. ANYTHING THAT IS REGULATED BY MORE THAN ONEPROTEINCombinatorial is found in prokaryotes but eukaryotes are more complex</p><p>Eukaryoteic transcriptionEukaryotic promoter (TATA box at -30). AT rich sequence; this is recognized by TBPos this is really the first element that aws discovered in eukaryotes to define thepromoterTBP was the first protein to be recognized to bind to promoter</p><p>Formation of preinitiation complex at Pol II promotersSteps in initiationRole of TFIID (TBP) nad TFIIH</p><p>Know F comes in with Pol IID nad H have specific roles</p><p>TBP-DNA interaction bc it is so different than all the other ones we have talkedabout which are alpha helices interacting with the major groove and TBP was one ofthe first proteins to interact w/minor groove using Beta sheets osvery diffinteractionCTD and phosphorylation 2 sites that are phospholryalted on 7 aa repeat; thesediff sites of phosphoryaltino are correlated w/diff steps in initiation and processingStructure of 5 cap, poly A function and general structure it is added on aftercleavage of the message, add 200 As to end of message Know function of cap and tailTorpedo model of termination</p><p>Poly A is important for translation initiation bc of the circular transcriptcAP important for translation initiation bc it is recognized by eIF4</p><p>Splicingin prokaryotes we can have polycistronic messages with multiple ORFs but since wehave SD sequences, ruibosomes can find internal AUGs but in eukaryotes they doscanning mechanism which means u can only translate one open reading frame andthe later AUGs you wont find. So messages made with intron/exon have to bespliced to one reading frame</p><p>hnRNA vs mRNA (pulse chase experiments)showe that we reduce since of hnRNA in this processing and later</p><p>experiments looking at electron microscopy show that size is reduced bc introns aretaken out</p></li><li><p>8/10/2019 Fall 2014 LS4 Midterm Review</p><p> 4/4</p><p>snRNPS are involved in recognizing splice sites; where they bind, what they do</p><p>formation of spliceosome once final spliceosome is formed we can catalyze the 2transesterfication rxnssplice sites (5 , branch, 3)be able to one a diagram know where is 5, 3, etc </p><p>what would happen if you knowcked one of these outtwo transesterication reactions what is attacking what?Structure branch A (A point with three phosphodiester bonds know what the bondsare), lariatAlternative splicingExon shuffling</p><p>Transaltion in eukaryotes</p><p>5 cap recognized and small subunit plus tRNA scans for first AUG this is why u cant take prokaryotic rNA and get it to be translated, bc it doesnt have</p><p>a capsmall subunit is actually already bound by tRNAicap and polyA to form circular template so we can get re-initiation to occur quicklyno formy-Met, no RBS</p><p>kozak- ppl say ATG bc talking about sequence in DNA so AUG and ATG are usedsynonymously it is the ATG of the coding strand not the template strand</p><p>regulation of eukaryotic transcriptionenhancers: sites where activators and repressors bind</p><p>initially they were all having positive effects but later found u can haverepressors binding to these regions too</p><p>anything that binds to enhancer has to have speicifc DNA binding domain and has torecognize a sequence but there are enzymes used in this process of activation orrepression that dont bind DNA coactivators and corepressors recruited toenhancers by activators and repressors; the co ones can modify chromatin orinteract w/general transcription factors or the mediator they can be interactingw/other things and are BROUGHT IN BY ACTIVATORS/REPRESSORS</p><p>co-activators and co-repressors: no DNA binding domains, recruited by activatorsand repressors, frequently involved in modifying chromatin</p><p>Eukaryotic examplesGal gene controlGal4 UAS, Gal</p></li></ul>