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Date Created: 10/07/15
Activation DNA has to be folded 0 DNA long but really thin allows it to be folded 0 Diameter 2nm 0 Length 034 nmbp 0 Human haploid genome is approximately 1m long of DNA unfolded O Diploid genome 2nm 0 Sometimes tetraploid 4m long of DNA 0 DNA divided into 23 chromosomes 435cm of unfolded DNA per chromosome avg I But diameter of a nucleus is 310um 8700x smaller than the DNA per chromosome 0 By volume though the DNA can fit 0 Volume of nucleus 65um3 0 Volume of DNA 3pm3 Packing Ratio 0 RR unfolded lengthfolded length 0 Should be greater than 1 I 1 unfolded 0 Packing ratio of DNA 8700 has to be 8700 to fit Problems With DNA Folding I Repulsion of negative charge on the backbone of DNA gives resistance to folding 0 To fold DNA must 0 1 Neutralize the charge on the DNA backbone with histones O 2 Fold bend backbone by wrapping around a protein nucleosome Composition of Chromatin 0 DNA 30 0 Protein 60 30 histones 30 nonhistone proteins NHPs 0 RNA 10 not structural leftover pieces of mRNA Histones 0 Basic Comprised of a larger than expected amount chance 5 each amino acid of basic amino acids lysine and arginine I 5 categories of histones based on function some variation ex more than one type of gene for H1 but same function not sure Why variation overall sequences are remarkably conserved 0 H1 H2A H2B H3 and H4 I Ratio of 1 H1 to 2 of each of the others I 2 moles of H2A H2B H3 and H4 join to form an octamer 0 H1 Largest H4 smallest histone Histones assemble into a nucleosome Nucleosomes 2rld level of Packing Discovered by isolating interphase nuclei lysing the nucleus centrifuging and observing under an electron microscope Originally called nubodies Visualized as beads on a string under microscope O 10nm fiber DNA is wrapped around the histone octamer approx 2 times H1 in contact with nucleosome not part of octamer in contact where DNA is entering and exiting Nucleosome actually a thick coin shape not a sphere Nucleosome H1 octamer linker 200bp Chromatosome H1 region octamer 166bp Core particle octamer 146bp Packing Ratio 0 Between 3 and 12 avg 6 usually in the PR 6 form 0 Packing Ratio decreases when more stretched out O P R 3 when linker DNA stretched out I 11nm nucleosome width 115 linker length 2256nm68nm 3 O P R 6 pulled together but narrow sides touching 39 68nm11nm 6 O P R 12 when narrow sides outward 39 68nm55nm 12 Solenoids 30nm fiber 3rd level of packing 6 nucleosomes in a circle sets of 6 set on top of each other DNA wraps to the nucleosome to the side of it and then down H1 necessary for solenoid structure holds it together 0 Packing ratio 68nm x 6 408nm11nm 37 Looped Domains Folded loops 300nm700nm 0 Euchromatin loop 0 Associated with expression 0 Replicons correspond to looped domains can open up independently to 10nm fiber to be transcribed 0 Solenoids looped 0 RR 1000 Radial Loops 0 Heterochromatin mitotic chromosome 0 Attaching together the attachment sites 0 Highly packaged form associated with genes not being expressed or DNA with no genes 0 Facultative heterochromatin can be switched during activation to allow expression 0 Constitutive heterochromatin always in highly packaged form not expressed 0 ex telomerescentromeres Packing Ratio Summary Level Name PR Length Width 1 DNA 1 44 cm 2nm 2 10 nm fiber 6 73mm llnm 3 30 nm fiber 37 12mm 30nm 4 Euchromatin Loop 1000 44pm 780nm 5 Heterochromatin mitotic chromosome 8000 55um 14um Trends 0 At each packing level length is decreasing and width is increasing 0 DNA per micron is increasing packing ratio increasing Modifications 0 Ends of the histones tails are on the outside of the octamer and exposed to chemical modification 0 4 types of modifications to the amino acids of the histones O Acetylation ubiquitylation methylation and phosphorylation 0 Modifications happening at specific locations modifications are very conserved consistent I N terminus beginning end C terminus end Acetylation 0 H3 and H4 0 Acetylation loosens the chromatin structure allows genes to be transcribed activation 0 Histone Acetyltransferases HAT acetylate 0 With increasing acetylation the positively charged histone lose affinity for negatively charged DNA and the 30nm fiber loses H1 and changes to 10nm fiber 0 Deacetylation strengthens the chromatin structure prevents genes from being transcribed Deactivation 0 Histone Deacetylation Complexes HDAC deacetylate restore 30nm fiber 0 Both HATs and HDACs are multisubunit complexes 0 Acetylation is a signaling enzyme that recruits nucleosome remodeling complexes NRC O NRC are ATPdependent multiprotein complexes that remodel by using the energy of ATP hydrolysis ATP ADP Pi O NRC can slide the nucleosome along the DNA to expose sites for DNA binding proteins restructure the nucleosome in place to facilitate binding of a DNA protein or transfer the nucleosome from one molecule to another I SWISNF a type of NRC Mechanism of Remodeling 0 HAT works together with NRC to remodel chromatin 0 Involve 4 major machines NRCs HATs RNA Polymerase and Preinitiation Complexes PIC O PIC RNA Pol II Transcription factors such as swi5p or TFIID which contains the TATA binding protein I 3 different ways to accomplish remodeling O NRC before HAT I A transcription factor of protein PIC has access to the solenoid I PIC recruits NRC which burns ATP to open up the complex then HAT comes along and acetylates which keeps it openkeeps it from refolding 0 HAT before NRC I HAT acetylates first and then recruits NRC which comes in and acts as a snowplow to open the complex which is followed by RNA polymerase which opens it further 0 HAT and NRC at the same time 0 Generally either activator recruits HAT which acetylates to make promoter accessible or activator recruits NRC Which remodels to make promoter accessible Epigenetics 0 Outside of genetics 0 Genetics Mutation I change in genotype I change in phenotype Natural selection over generations Environmental in uence Adapting to environment 0 Epigenetics O 1 Environmental effect causes a change in the gene expression not a change in the gene I Activation in some cases can be controlled by the environment some changes may be permanent change in expression of somatic cells 0 A histone modification 0 B DNA methylation I C noncoding RNAs involved in regulation 0 2 Epigenetic change is heritable transgenerational epigenetics I A Epigenetic change in germ line gametes I B Epigenetic change can be maintained during replication gives it its permanence 0 Environmental in uence puts onremoves tags that turn genes on and off that can be transmitted to offspring 0 DNA methylation is associated With turning genes OFF I Not sure how germ line cells escape epigenetic reprogramming scrubbing all tags may be similar to how imprinted genes do 0 Possibly could methylate histones Which 90 are rid of during spermcell formation and instead DNA wraps around protamines but 10 retained could pass on info 0 Other studies indicate short RNA microRNA molecules altering expression that are passed on 0 Majority of traitassociated DNA variations occur in the previouslycalled junk DNA that does not code for proteins these regions control gene expression 0 Histone code hypothesis specific combinations of histone tail modifications epigenetic markers are associated With transcription factors that increase or decrease gene expression 0 Different nucleotides have different affinities for transcription factors and thus affect Whether the histones Will be modified or not 0 Transcription factors bind directly to DNA and have different affinities for different sequences 0 Methylation Off 0 Lysine 9 on H3 I Maintained by bonding to methylated HP1 heterochromatin protein 1 O Suv39H binds to HP1 and methylates the K9 position of the subsequent nucleosomes until stopped by the boundary element I Boundary element separates active from inactive chromatin may or may not correspond to attachment sites between looped domains 0 Acetylation On 0 Acetylase acetylates active nucleosomes Replicating Chromatin 0 When DNA is replicated histones must also be doubled which involves synthesiszing new histone proteins and assembling new nucleosomes 0 Nucleosomes disassemble when a replication fork passes through 0 Each parental nucleosome separates into an H32H42 tetramer and 2 copies of an H2AH2B dimer 0 The H3H4 tetramer immediately goes to one of new strands of DNA and starts assembling new nucleosomes 0 H2AH2B dimers enter pool with newly synthesized dimers 0 There are also new H3H4 tetramers one of which will go to the other strand and start nucleosome assembly 0 H2AH2B dimers then join the tetramers to form the nucleosome and may be new or parental I The process of nucleosome assembly is directed by histone chaperone proteins 0 Maintaining genetic markers 0 The state of the previous nucleosomes are propagated through replication 0 Upstream of the replication fork on the newly synthesized DNA HP1SuVar methylates nucleosomes adjacent to already methylated H3H4 tetramers I transmission of inherited characteristics Basal Level Transcription Eukaryotic RNA polymerases I Universal to almost all eukaryotic organisms 0 Major difference from E Coli In E Coli there is ONE RNA polymerase most similar to eukaryotic RNA Pol II it may have a different 039 factor and thus recognize different promoters but it still has the XZBB core 0 Hard to identify which RNA polymerase was which sorted out by sensitivity to a toxin made by a mushroom Polymerase Genes Transcribed Location Otamanitin sensitivity RNA Pol I rRNA 188 288 5 SS Nucleolus Low RNA Pol II mRNA Nucleoplasm High RNA Pol III tRNAs SS rRNA snRNA small Nucleoplasm Moderate nuclear involved in splicing I All three polymerases are multisubunit complexes sometimes multiple dozen subunits O Subunits divided into large catalytic or BB activity units that are heavily studied and highly conserved and smaller units involved in modulation of RNA expression 0 Some subunits are common to 2 or all 3 of the polymerases RNA polymerase Promoters BIND Transcription Factors I Not promoters in the same sense as prokaryotic I Were looking for eukaryotic equivalents of the lO 35 promoter sequence I There are some common sequences that they thought were at first but aren t I More accurately cisacting DNA sequence elements 0 Important difference from E Coli in E Coli not much happens more than 35 nucleotides upstream of the start of transcription in Eukaryotes important regulating sequences may be lOOslOOO nucleotides upstream of initiation site 0 Methods for identifying sequences differed from classical genetics 0 Classical Genetics Random mutant phenotype I Genotype 0 Reverse Genetics Genotype I In vitrovivo mutation I Phenotype I Ex in vitro lyse cell to form a lysate centrifuge to get soluble proteins on top and membranes on bottom I purify to get just supernatant cleared lysate which included RNA polymerases I add DNA and see if transcription occurs I Ex in vivo Inject DNA to cell and see if transcription occurs I One method of in vitro mutagenesis used to identify the consensus sequences was linker scanning I Linker scanning involved changing 8 nucleotide sequences of the DNA added and seeing if transcription occurred If transcription did not occur this tells you that the sequence that was mutated in the added DNA was important to transcription 0 ie Experimentally determining consensus sequences 0 RNA Pol II Promoters Name Sequences Location TATA box TATAAA 25 CAAT box CCAAT 75 variable GC box GGGCGG Variable I Results of linker scanning for RNA Pol II in thymidine Kinase tK O tK Herpes simplex virus thymidine kinase promoter 0 25 pS proximal sequence experimental proof of TATA box 0 47 to 61 dSI distal sequence 1 a GC box 0 80 to 105 dSII distal sequence 2 experimental proof of CAAT box GC I Could not identify CAAT and a GC as separate because they were less than 8 nucleotides apart 0 Beforeafterand between these sequences nucleotides could be changed with no effect on transcription I Other Pol II sequences 0 In SV40 TATA 6 upstream GC boxes 0 General form TATA box upstream element CAATGC relatively fixed an enhancer a DNA sequence not dependent on location or orientation that is important in binding regulatory transcription factors 0 P01 I promoters 0 CORE 45 to 20 Overlaps site of transcription initiation O UCE Enhancer Shown 107 to 156 can move space and relation to CORE and still works 0 Pol III promoters O In SS RNA I Used deletion mutagenesis or deletion mapping independently delete upstream or downstream nucleotides of different sizes and see if it affects transcription I Didn t lose transcription until deleting all but 5 nucleotides in almost the middle of the gene 0 I ICR internal control region I Actually 3 regions Box A an intermediate element and Box C O In tRNA I Linker substituted mutations within gene I 2 regions that when substituted prevented transcription I Box A and Box B later extended and called Box B 0 ICR If gene is transferred to another organism it takes its promoter with it General Transcription Factors GTFs 0 DNA sequence elements promoters bind GTFs 0 Different GTFs for each polymerase I Upstream of Pol II 0 Pre Initiation Complex PIC is assembled on TATA I Assembles into 2 lobes Globe and Plobe O 1 TFIID with TBP Basic assembly complex initiation factor I Transcription Factor for RNA Polymerase 11 Unit D I TBP TATA binding protein 0 One of the subunits of TFIID 0 A position transcription factor 0 AKA commitment complex because it recognizes and commits to transcription 0 Binds at minor groove and bends DNA approx 80 0 Present for all polymerases even though only upstream Pol II has a TATA box I D multiple subunits called TAFs TBP associated factors 0 Has inhibitory and positive subunits in terms of initiating transcription 0 Inhibitory units keep transcription from starting until everything is assembled have to be removed for initiation to occur 0 0 2nd TFIIA and TFIIB I Recruited through protein interactions not directly binding to DNA I A barely upstream of TATA 0 Activates D by pushing offremoving a major inhibitory TAF 0 Remember A Activates I B barely downstream of TATA 10 to 10 0 Defines direction of transcription 0 3rd TFIIF and Pol III I F is recruited I Heterotetramer 4 subunits 2 small and 2 big 0 Large subunits ATP dependent helicase that makes open complex by unwinding the DNA 0 Small subunits bind to Pol II like sigma interacts With P01 and bring it into the complex I In eukaryotes Pol cannot recognize TATA no 039 recognition factor it is recruited in by the 2 small subunits of F 0 Pol II starts making RNA even though it can t move yet makes RNA in place 0 4th TFIIE and 5th TFIIH and TFIIJ I E H and J involved in promoter clearance Which involves moving the polymerase I E binds at approx 30 helicase activity I H large complex factor With 3 types of activity 0 1 ATPase activity 0 2 Helicase Activity 0 3 Kinase Activity 0 Phosphorylates adds phosphates to the tail of the large subunit in the RNA polymerase 0 Doesn t phosphorylate all but a lot of serines and threonines Which gives the official signal for promoter clearance O CTD LSU Pol II CYSPTSPSn n26inyeast50inmammals 0 Rides along With Pol 11 during elongation I Upstream of Pol I O UBF upstream binding factor is now the assembly factor Drives assembly I Can bind to sequences in UCE or CORE 0 SL1 initiation factor With TBP I Recruits the polymerase and allows initiation I TBP no TATA box for it to bind to but still necessary for transcription Pol III tRNA PIC O O O O 2 ICR A and B are also transcribed 1St TFIIC recognizes A and B acts as an assembly factor I Recruits TFIIIB 2nd TFIIIB with TBP initiation factor I Doesn t bind to DNA proteinprotein interaction binds upstream of C I Recruits Pol 111 3rd Pol III binds to B Pol 111 SS RNA PIC O O O 2 ICR box A and box C 1St TFIIIA recognizes and binds to box C 2nd A binding to C facilitates TFIIIC recognizing and binding to box A I Remember A binds C C binds A 3rd TFIIIB W TBP initiation factor binds I Recruited by assembly factors C and A 0 Binds to other TFs not to the DNA I B recruits and positions RNA pol III on the gene Pol 111 can transcribe Without TFIIIC leaVing even though it looks like it would be in the way can move around to other side of the DNA Eukaryotic Gene Regulation Summary 1 Activation Provides the potential for transcription Doesn t necessarily mean that it Will be expressed but the DNA is set up for that potential Prokaryotic always has that potential Eukaryotic extra step Putting DNA in the euchromatin form giving it access to polymerases etc 0 Chromatin Remodeling and other Epigenetic factors 0 Nucleosome Remodeling Complexes 0 Histone Acetyltransferase HAT and Histone Deacetylation Complex HDAC 2 Basal Level Transcription 0 General Transcription Factors preinitiation complex 0 RNA Polymerases 3 3 High Level Transcription 0 DNA control elements enhancers 0 Regulatory Transcription Factors 0 Activators Repressors Silencers O Combinatorial gene regulation High Level Transcription I Enhancers Cis Acting DNA sequence elements 0 Orientation and position independent I Can move upstream OR downstream even after promoter or change direction 0 If the promoter orientation was ipped the gene would transcribe the other way if the position was changed transcription would initiate at a different position I Can change position or orientation via in vivo or in vitro mutagenesis and transcription still occurs 0 Ex SV40 a eukaryotic virus one of the first enhancers identified I 72 bp element enhancer when deleted transcription dropped to basal level 15 of completely activated level I Experimentally ipped moved 1Kb upstream still worked 0 Unique to SV40 not universal to all enhancers could be moved to another gene and worked as an enhancer for a new gene 0 Transcription Factors 0 2 domains binding and activation I 1 activation domain contacts from a distance PIC or proteinTAFs to give the high levels of transcription I 2 Binding domain Binds to enhancer 0 Binding mechanism in prokaryotes helixturnhelix 0 In eukaryotes 3 different binding mechanisms 0 1 zinc fingers Amino acids pulled into a fingerlike structure by the coordination of zinc to 2 histidines and 2 cysteines One side is an alpha helix and one side is a beta ribbon Alpha helix goes into the major groove and interact with the backbone of DNA which allows for it to be sequence location specific In TFIIIA 9 zinc fingers coordinate to the major grooves of the ICR O 2 leucine zippers Mostly leucines hydrophobic interactions between leucines hold together 2 alpha helix subunits Positively charged amino acids interact with the negative charge on the DNA backbone major groove Coiled coil 0 3 helixturnhelix occasionally more of an exception than an actual form 0 Experimental proof of 2 domain functions I Removed activation domain no high level transcription ruled out hypothesis that binding of the protein was causing activation I Changed binding domain and inserted a matching operator from a prokaryote so that the TF would bind to a different area and transcription occurred I Control Prokaryote binding domain on prokaryote operator without activation domain no activation 0 Example of EnhancerRegulatory TF system 0 In Yeast Gall system I PIC assembles on Gall promoter Regulatory transcription factor binds to an enhancer called UASG Upstream Activation Sequence for Gall I UASG can have an effect on the Gall promoter from a distance 0 Transcription factor on enhancer interacts with positively acting TAFS in the PIC to give remaining 95 of transcription 0 Enhancer ProteinPolymerase Interaction 0 How can the enhancer work so far upstream Action at a distance 0 How can it contact the PIC O 3 proposed mechanisms I A Sliding some examples have been found but not usual 0 Factor with activation domain binds at the enhancer and then slides down to contact TAF I B Looping 0 DNA between enhancer and PIC forms a loop that allows the factor to bind the enhancer and remain on the enhancer while still contacting PIC 0 Explains how if the enhancer is moved farther away it can still work makes a bigger loop or if orientation is changed twists the loop I C Oozing not main mechanism 0 Other transcription are recruited until they can contact PICI AF 0 Experiment to prove looping mechanism with Pol 1 I Put short sequence between enhancer and initiation site I 60bp worked 70 bp worked I 65 or 75 bp didn t work when DNA is that short it isn t as exible and can t turn 5 nucleotides 12 turn Had to be 10 nucleotides to be a full turn so they could contact I Regulating Activation Global Regulation 0 Hormoneregulated set of genes 0 Hormone brought into cell and binds to receptor Receptor has another protein connected to it that prevents it from entering the nucleus When the hormone binds the receptor it releases the protein which allows the receptor and hormone to enter the nucleus and bind at the enhancer I Ex Glucocorticoid binds at GR which releases Hsp90 heat shock protein that keeps GR from entering nucleus and also keeps it from degrading 0 Signal Transduction O O Ligand ex hormone small molecule able to bind and activate receptor binds at receptor receptor kinase RK phosphorylates the receptor which then phosphorylates another kinase which can phosphorylates another kinase called a kinase cascade the final phosphorylated kinase can then enter the nucleus and phosphorylate the initiation factor which makes it active now called an activation factor and activates transcription Signal transduction amplifies and transduces the signal can happen multiple times and thus amplify the action of a hormone 0 Combinatorial Gene Regulation 0 0 Multiple ligands and receptors any of which can activate one gene or one ligandreceptor can activate multiple genes Often multiple enhancers upstream of genes enhancers may be involved in multiple regulations Transcription 0 Activation O Chromatin Remodeling 0 Acetylation Modification of histones I Basal Level Transcription 0 General Transcription factors binding promoters TAFs to form a PIC 0 High Level Transcription 0 Regulatory Transcription Factors RTFs binding to enhancers and contacting PIC from a distance by looping preRNA I mature RNA 0 mRNA 0 Spliceosomal introns I Spliceosome U1 U2 U3 U4 U5 U6 0 5 cap I Phosphotransferase Guanylyl transferase Methyl Transferase SAM O 3 poly A tail I CPSF and CstF CFI and CFII PAP and PABII 0 rRNA 0 Group 1 introns sometimes I Organellar 0 Group I introns Organellar some rRNA I Prokaryotic excision of rRNA subunits and tRNAs 0 RNAase III RNAase P RNAase E endonuclease I Eukaryotic selfcatalyzed double transesterification I Selfcatalyzed I linear intron 0 Group II introns Organellar I tRNA processing 0 tRNA intron removal cleavage and ligation endonuclease and RNAase Ligase O 5 cleavage RNAase P O 3 cleavage endonuclease 0 CCA Addition tRNA terminal transferase 0 Base modification RNA editing Translation 0 1St genetic code antiparallel matching of codon in mRNA to anticodon in tRNA 0 Wobble hypothesis gives degeneracy to genetic code one anticodon can recognize multiple codons 0 Illegitimate anticodons those that would recognize stop codons or would match with codons that specify two different amino acids 0 2nd genetic code matching of tRNA to amino acid 0 tRNA synthetase different synthetase for each amino acid I Recognize identity elements in tRNA 0 2 reactions to charge tRNA Translation initiation 0 Prokaryotic 7OS initiation complex 0 ShineDalgarno sequence UGGAGGA in mRNA 0 Eukaryotic SOS initiation complex 0 Ribosome binding at 5 cap 0 Scanning I Kozak sequence around AUG context Translation Elongation 0 Charged tRNA into empty A site 0 Prok EF Tu EF Ts O Euk eEF1a eEFlB 0 Peptide bond from new amino acid and amino acid that was in P site 0 Peptidyl transferase 0 Translocation move peptidyltRNA to P site empty tRNA to E site and then out 0 Prok EF G O Euk eEF 2 Translation Termination 0 No tRNAs recognize stop codons RFs Win 0 Prok RFl or RF2 recognize stop 0 RF3 RRF 0 Euk eRFl only 0 eRF3 Levels of Gene Expression Control in Eukaryotes 0 In eukaryotes RNA is processed from a primary transcript to mature this step is almost completely absent in prokaryotes 0 Another level of control unique to eukaryotes is transport control transport from nucleus to cytoplasm not necessary in prokaryotes because there is no nucleus 0 Translational and protein degradation control occur in prokaryotes and eukaryotes Difference between Prokaryotic and Eukaryotic mRNAs Bacterial mRNAs Eukaryotic mRNAs Transcription Unit Polycistronic Usually monocistronic Initiation RNA pol binds DNA RNA pol recruited to promoter Elongation Rate 40 nucsec 40 nucsec 5 end Triphosphate Methylated cap methylated G nucleotide 3 end defined by Termination Cleavage RNA processing 3 end Last base of mRNA Poly A tail Translation Rate Similar to elongation rate because often happen at the same time Slower than elongation synchronization not necessary Half life Very short rapidly turn onoff genes Longer Bacterial Animal 0 Unique to eukaryotes intronsexons 0 Selective advantages of introns O 1 Regulates production of transcripts O 2 Gives versatility to eukaryotes that missing in prokaryotes RNA Processing cleavage of RNAs modifications of bases on the RNA and splicing 0 tRNA 0 Made With a 5 and 3 extension that has to be removed 0 Base modification 0 Intron removal tRNA introns 0 rRNA 0 Separation processing 0 Base modification 0 Intron removal Group I introns 0 mRNA 0 5 Capping O 3 poly A addition adenylation 0 Intron removal Splicesomal introns 0 Organellar RNAs Mitchondria and Chloroplasts 0 Group I and Group II introns I Group I almost exclusively in organelles sometimes in rRNA RNA Processing in general DNA gene Primary RNA Transcript m Mature RNA Transcript 0 Primary Transcript RNA made directly from DNA 0 RNA Without any processing 0 Should be identical to DNA except to Us and Ts 0 Mature Transcript Modified altered primary transcript that is now ready to be translated 0 Exons translatable material Splicesomal Splicing in Pol II mRNA Transcripts 0 Splicesomal Introns GUAC introns 0 What defines the boundaries between intronexon Looked for consensus sequence 0 Consensus sequence in intron and part of exon O GU in DNA GT in RNA 0 GU is exquisitely conserved because it s part of the splicing mechanism 0 Proof of importance of intronsconsensus mutation in intron causes a large effect O Gle mutation in the intron causes splicing to occur in different places because other places are now a better match to the consensus O In some cases part of the exon is spliced out and in some cases part of the intron is retained translated 0 Some splicesomal introns break this rule because different mechanism 0 EX AUAC introns exception to rule Mechanism of Spliceosomal Introns What is recognizing the consensus sequence 0 With preVious consensus sequences recognition by proteinNow RNA 0 Cleavage not a cut and glue mechanism 0 Transesterification Changes an R Group 0 In RNA Double transesterification both catalyzed by RIBOZYMES 0 Diester and an alcohol I different diester and an alcohol all in one step 0 Reversible reaction catalyzed by a spliceosome 0 A A little distance from 3 end has a 2 OH Which is the attacking group in the transesterification reaction l Splicesome Splicesome 0 A ribonucleoprotein complex 0 Approx same size as ribosome big multisubunits RNA and proteins components are defined by 5 different RNAs 0 Small ribonucleoproteins RNP Nucleoplas RNA Polymerase Structure m RNP Transcripts U1 II Cruciform has sequence that is complementary to consensus binds at consensus U2 II Section that is complementary to internal bulge A U4 II Complementation to U6 U5 II Stemloop structure U6 IIII snRNA Complementation to U4 0 U3 was discovered then later realized to not be a part of the spliceosome 0 All very abundant 0 Commitment step where U subunits are bindingcommits to splicing 0 U1 binds to 5 consensus sequence and recruits U2 which binds to A causing it to bulge 0 U1 and U2 interact with each other causing the RNA to fold 0 Then U4 and U6 that are connected and work as an RNA duplex bind U4 essentially works as a carrier to bring in U6 0 Rearrangement occurs U4 is displaced and U6 binds to U2 and displaces U1 U5 binds and works as a structural subunit that holds the exons together 0 The U2 U6 duplex catalytic compleX not a protein a ribozyme 0 In the new rearranged form with the exons closer the RNA is now ready for the double transesterification 0 U5 brings together the Gs of the two exons that form hydrogen bonds a non watsoncrick base pairing interaction 0 Almost all steps require ATP to help release linkedligated exons Introns removed and Dbr1 a debranching enzyme causes it to unloop at the A and then can be degraded 0 Spliceosome subunits are released from the intron and are used repeatedly to remove introns generally not consumed in the process cycle when released from intron to act on other premRNA 0 They do turnover over time but generally pretty stable Isoforms 0 Human haploid genome approx 3x109 bp 0 Early estimated to be about 100000 genes revised and lowered multiple times 0 Current estimate 26000 not that different from yeast 0 Of the 26000 genes 0 23000 protein coding genes 0 3000 RNA coding 0 1200 pseudogenes mutated 0 6080 alternative splicing isoforms 0 Can get more than 1 approx 6 I 12 different protein products from 1 gene 0 These different possible protein products from identical genes are called isoforms 0 Example of Isoforms Sex Determination in Drosophila 0 Male no le sex lethal 0 Female le product from sex lethal gene regulates alternative splicing O The tra protein tratransformer effects ultimate splicing of dsx double sex Which produces either dsxF female or dst male 0 Different isoformslimitation of rearrangement have to include first exon A and last exon E Can only cut outexclude internal exons cannot rearrange ex ACE cannot be ABC 0 From 8 possibilities ABCDE standard splice sitesdefault splicingcutting out all the introns and leaving all the exons ACDE ABDE ABCE ABE ACE ADE AE 39 8 possible transcripts Sample Test Problem I How many different RNAs are produced from this preRNA transcript I How many different proteins Group I Introns 0 Mostly organellar sometimes rarely in ribosomal RNAs if they are it s usually in lower eukaryotic organisms 0 Group I introns were discovered in rRNAs 0 In rRNA not really considered splicing because the processing of the prerRNA cleaves exons from introns but does not ligate them back together they are separated into parts that become the independent subunits of the ribosome black area is ligated 268 in euk 0 Important process in prokaryotes and eukaryotes 0 Prokaryotic prerRNA O O O 2 promoters 2 sets of lO 35 consensus Gene is an operon Primary transcript is called 308 transcript although it is more theoretical than something you can isolate because it is being processed at the 5 end before the 3 end is transcribed never really exists in whole form Products 168 RNA small subunit 23S RNA SS RNA large subunit 2 tRNAs rRNAs have a stemloop structure in the preRNA while tRNAs have a clover shape RNAase III cleaves 168 and 238 to excise the subunit while SS is cleaved and excised by RNAase E tRNAs excised by RNAase P at 5 end and endonuclease at 3 end This process is repeated different times in different ways by different enzymes which mostly differ in transcript by removal of the tRNAs or lack thereof 0 Eukaryotic prerRNA O rRNA is transcribed by Pol I 0 Theoretical primary transcript called 45 S 0 Products 188 588 288 large subunit I Also part of the ribosome is the 5S subunit but the 58 is made by Pol III 0 58 is unique to eukaryotes because it is its own subunit that interacts with 288 after transcription I In prokaryotes there is a similar sequence but it is connected intermolecular interaction instead of intra molecular but the sequence is still there 0 The mechanism of cleavage involves a double transesterification but does not require a spliceosome the intron is selfcleavingselfsplicing the ribozyme catalytic activity is in the intron itself this process does not require ATP 0 Functional intron internal mutations in the intron will affect its ability to splice itself out 0 POH on a G nucleotide instead of the bulged A attacks the phosphodiester bond between 3 of exon 1 and 5 of intron so that G and A form a new phosphodiester bond This puts the G on the 5 end of the intron and OH on the 3 end of exon 1 O 2 The OH then attacks the 5 end of exon 2 which puts the OH at the 3 end of the intron and 3 end of exon 1 connected to 5 U of exon 2 O 3 This leaves the 2 exons connected by a phosphodiester bond between U and U and a linear intron I At the level after 2nd transesterification that the other introns are stopped at group I DOES NOT form a lariat structure 2 branch 0 4 In a 3rd transesterification a G nucleotide with the OH at the 3 end of the intron attacks a phosphodiester bond in the middle of the intron which creates 2 pieces of the intron one circular The circular part retains the 3D structure of the ribozyme and can act as a transacting enzyme 0 Secondary structure of the group I intron folds into a 3D spherical shape 0 SR PQ EE are conserved consensus sequences and are diagnostic of Group I introns O Mutations in these sequences affect the splicing function of the intron 0 Group I organellar introns NOT rRNA GI introns have maturases Which are proteins produced from the intron that have an open reading frame that stabilize the ribozyme O Maturases have no catalytic function only work to stabilize function to remove own gene Group II Introns 0 Also selfsplicing intron no spliceosome required 0 Secondary structure is a 6 radial spokes on a Wheel structure 0 Radial spokes 5 and 6 are small and very conserved 6 contains the bulged A nucleotide 0 Each spoke is double stranded RNA the center of these spokes provides space Where you can have proteincoding genes in them 0 These proteins are reverse transcriptase that give GII introns the potential for mobility I Proposed that the mobility of GH introns is why we have so many introns GII moved and then evolved to spliceosomal introns 0 Group II introns following an almost identical mechanism to spliceosomal introns form a Lariet intermediate structure and have similar consensus sequences at the 5 end 3 end and the branch although the branch consensus sequences in GII introns is closer 57bp to the 3 consensus than the nuclear 2237bp 0 There is evidence that GH and nuclear introns are evolutionary relatedcommon origin 0 Same mechanism as spliceosomal introns O Bulged A 2 OH attacks exon 1 3 G I Lariet intermediate branched RNA 0 OH now on 3 end of exon 1 attacks 5 G of exon 2 0 I Phosphodiester bond between exons and Lariet structure I Only difference is that it is selfsplicing intron exists for the purpose of excising itself 0 Proposed that it is perpetuated by getting replicated along with DNA and can jump to other sites but for DNA to be able to replicate it has to be able to excise itself so that it doesn t disrupt Mechanism Summary Comparison Group I Group 11 Nuclear mRNA Spliceosomal Introns 1 OH on G attacks 3 exon 1 1OH on bulged A attacks 3 lOH on bulged A attacks 3 exon 1 creating a Lariet exon 1 creating a Lariet 2 OH on 3 end of exon 1 2 OH on 3 end of exon 1 2 OH on 3 end of exon 1 attacks the 5 end of exon 2 attacks the 5 end of exon 2 attacks the 5 end of exon 2 This leaves connected exons This leaves connected exons This leaves connected exons and a linear intron and a Lariet intron and a Lariet intron 3 In a third transesterification the intron is circularized tRNA MaturationProcessing 0 Involves cleavage of a 5 extension 5 leader sequence cleavage of a 3 extension intron removal base modification and a CCA addition 0 1 5 extension cleavage is catalyzed by RNAase P a ribozyme sometimes this ribozyme will have a protein attached to it but the catalytic activity is in the ribozyme 0 2 3 processing is almost exclusively done by an endonuclease 0 3 Intron Removal 0 Introns are not present in every tRNA even within an organism 0 Usually small introns 1416bp 0 Introns almost always in an anticodon loop 0 These introns work as a kind of temporal control deactivating the tRNA until it should be active 0 Intron removal follows a different mechanism than GI 11 and nuclear I It is a 2step cleavage and ligation 2 different reactions I Endonuclease comes in and cleaves the intron connected sites then RNA ligase ligates the 3 exon 1 A to the 5 exon 2 A O Assayed by heat breaks into 2 pieces generates half tRNAs that can be detected 4B ase modification different chemical groups are added systematically to sites in the tRNA ex methylation m dihydrouridine DHU pseudouridine LJ or TU 0 Approx 120 different kinds of modifications that can happen on tRNAs 5 Enzyme called tRNA terminal transferase or sequential tRNA terminal transferase adds CCA to 3 end of tRNA 0 tRNA terminal transferase does not use a template but adds nucleotides to the end Where amino acids attach Without the CCA end the tRNA is useless Intron Comparison Summary Type of Intron Catalysis Mechanism Functional Structures Relevant Proteins mRNA nuclear Spliceosome Double 1 Bulged A spliceosomal introns Transesterification 2 Lariat rRNA nuclear Group I RNA catalysis self Double Guanosine cofactor introns splicing Transesterification 3rd G With 2 OH transesterification in intron tRNA nuclear RNAase P and 1 Endonuclease Endonuclease cleavage and 2 RNA ligase ligation Group I organellar RNA catalysis self Double Guanosine cofactor Maturases splicing Transesterification 3rd G With 2 OH transesterification in intron Group II organellar RNA catalysis self Double 1 Bulged A Reverse splicing Transesterification 2 Lariat Transcriptase mRNA Processing Last part of mRNA processing aside from splicing involves addition of a 5 cap and a 3 polyA tail Cap and tail are added before the gene is even done transcribing so often seen as part of the premRNA as well as the mature RNA added before introns are spliced out but included in RNA processing from DNA I mature mRNA 0 Cap is important because it defines mRNA as information that needs to be translated ribosome binds cap to translate UTR untranslated region at 5 and 3 end of premRNA note genes are transcribed but NOT translated O UTRs are important sequences especially at the 3 end 0 3 UTR called a polyA site Which includes the signal to cleave the RNA terminate transcription 39 UGA Stop codon is in the exon though not in the UTR 3 Poly A Addition 0 Poly A site in UTR at 3 end AAUAAA consensus sequence VERY conserved 0 Downstream GU rich site serves as binding site but not very conserved 0 Complex assembles O CPSF cleavage and polyadenylation specificity factor binds to consensus O CstF binds to GU rich site downstream of consensus O CPSF and CstF then bind to one another causing the DNA to bend 0 CPSF and CstF recruit CPI and CFII cleavage factors I and II which bind to the RNA between the AAUAAA site and GU rich site and cleave it 0 The area downstream of the poly A site is degraded 0 Oligoadenylation PAP Poly A polymerase uses ATP and Mg2 to add As without using a template 0 This addition occur very slowly minutes 0 Once enough As have been added PABII Poly A Binding Protein 11 is recruited 0 Poly A extension PABII catalyzes the A addition adding about 200 in seconds 0 Number of As added differs between organisms but generally about 250 in the tail 0 The number that can be added is about 250 before everything is released unknown mechanism 0 Polyadenylation doesn t always occur at the same site there can be multiple poly A sites 0 Through alternate splicing an alternate poly A addition entirely different proteins can be formed from the same gene in two different tissues 0 Posttranslational cleavage that activates the protein creates different peptide hormones 0 How many would be possible from alternative splicingpoly A addition in the Calcitonin gene 5 Methyl Capping At the same time as poly A addition or even before before the gene is finished transcribing the 5 end can be capped Before cap is added 5 end has a triphosphate then the first nucleotide phosphodiester bond second nuc etc 1 Phosphotransferase makes the triphosphate into a biphosphate 2 Guanylyl Transferase then adds GTP guanosine triphosphate Gppp to the mRNA 0 The alpha phosphate from GTP is retained and adds to the biphosphate at the 5 end in 5 to 5 linkage 3 Methyl Transferase aka guanine 7 methyl transferase catalyzes the addition of a methyl group With cofactor SAM Sadenosyl methionine a methyl donor to methylate at the 7 methyl position 4 Methyl Transferase aka 2 0 methyl transferase adds SAM to methylate the 2 position Total Additions Cap 1 Major 2 O 2 position on oxygen on first nucleotide Cap 0 Major 7 methyl 7 position on G from GTP Cap 1 Minor Amino group on nucleotide Cap 2 Minor 2 methylation on second nucleotide 0 OOO Translation Overview 5 3 5 N Met His Asn Leu Gly 0 DNAEIRNA through WatsonCrick base pairing adds nucleotides to the 3 end until termination in prokaryotes or poly A site in eukaryotes that gets cleaved 0 Amino acids made in a NI C direction How does a specific codon correlate to a specific amino acid 0 Proposed adaptor molecule by Watson Later I tRNA 0 Codon on the mRNA base pairs not always Watson Crick bp With an anticodon on a tRNA 0 This codonanticodon base pair interaction is called the first genetic code 0 The other end of the tRNA puts in an amino acid if it is charged With the right amino acid on the 3 end 0 An enzyme called tRNASynthetase recognizes the specific identity of a tRNA different tRNAs have different identities Translation Machinery 0 Ribosome 0 One major ribosome remove Mg2 with EDTA 2 subunits Urea I parts of subunits proteins involved O The catalytic activity in the ribosome comes from the RNA thus the ribosome is a ribozyme but it also includes assorted proteins that surround the RNA and serve to stabilize it Organism Ribosome Subunits rRNAs Prokaryotic 70S Large 50S Large 23S and 5S Small 30S Small l6S Eukaryotic 80S Large 60S Large 28S 58S 5S Small 40 S Small 18S 0 Each subunit has between 21 and 49 associated protein 0 S Svedberg unit that indicates size I Greater S larger but not additive 0 5S subunit 120 bases 0 Large subunits can be 20004000 bases 0 rRNA I SSU small subunit 16S in E Coli 0 4 domains that are very conserved not necessary in actual sequence but in secondary structure implies structure related to function 0 Folds into sphere I Mutations that effect making proteins map back to alterations in the rRNA structure I Decorated on outside With SSU ribosomal proteins catalytic center almost pure RNA tRNA structure 0 Clover leaf structure With 4 and sometimes 5 leaves I Accepter arm antiparallel duplex contains 5 and 3 end 0 Amino acid attaches to the CCA at the 3 end I D Arm DHU dihydrouridine arm 0 Stem almost always 4 0 Loop size varies I Anticodon arm 0 Anticodon at bottom of loop 34 35 36 0 Almost 7 bases on Darm side of anticodon and 5 bases on other side I TLJC arm 0 VERY conserved sequence in loop GTLJCPuA that is diagnostic of a tRNA 0 Interacts With D arm in some nonWatsonCrick base pairing to form an L shape folded structure I Some have an extra arm that compensate for the varying size of the Darm loop to put the same bases as the anticodon I L Shape tRNA Reading Frames 0 Reading frame counting by 3s defined by where the tRNA starts 0 Where you start from changes the amino acids I starting point is critical 0 Each strand 3 possible reading frames 0 Open reading frame frame that reads to produce amino acids without a stop codon 0 Usually 100 codons or AA without stop is considered a significant open reading frame a lot more than you would expect by chance 0 When looking at a gene don t know which DNA strand was used as a template or which reading frame was used to translate on which strand I 6 possibilities O The reading frame without a stop codon or with the longest amount of AA without stop is assumed to be the strand of DNA and reading frame that made the protein 0 In example RNA shown closest to the RNAlike strand of DNA farthest from the DNA that it used as a template RNA 5 DNA 5 DNA 3 RNA 3 1 2 Degeneracy Redundancy 0 Means that multiple codons code for the same amino acid 0 64 different codons I 20 different amino acids 0 61 codons are sense codons code for an amino acid includes start codon AUG met 0 3 codons are nonsense UAG UGA and UAA stop codons chain terminating codons I No tRNAs in normal cells carry the matching anticodons for these Wobble Hypothesis 0 The same anticodon on a tRNA With the same amino acid can interact With multiple codons in sometimes nonwatsoncrick base pairing 0 0 Ex tRNA charged With Phe has anticodon GAA which can interact With codon UUU or UUC therefore each of those codons codes for Phe This means that there are less tRNAs than there are different codons 61 but at least one tRNA for every amino acid I Wobble position 5 base in anticodon and 3 base in codon 0 These positions are the only positions that do not have to be WatsonCrick I Wobble rules in E Coli A as a 5 base in anticodon never exists Will always be modified I A is modified to I inosine I I is a Glike structure only differs from G in the removal of an amino group I Al I change amino group to a carbonyl U is usually modified Wobble rules are not actually absolute have preference to certain bases but generally consider absolute Combination of I C can cover all codons and U G can cover all codons I What defines a WatsonCrick base pair versus a Wobble base pair is how many hydrogens are bonding between the bases and the position of the amino group that is bonding with codon 0 In WatsonCrick there are 3 O In Wobble base pair there are 2 I IC is Watsoncrick LIKE because the middle amino group is bonding with the nitrogen on the codon but nothing bonding with codon base carbonyl like it is with the bottom amino group in G the 3rd hydrogen bond I IU more Wobblelike because the codon base is shifted up and the middle amino group is now bonding with the carbonyl in the codon U The Genetic Code I Standard Usually shown with codons and associated amino acids 0 Usually doesn t show tRNA anticodons 0 Some amino acids are 2fold degenerate 2 codon families 2 codons code for one amino acid 4fold degenerate 4 codon families etc 0 Minimum Number of tRNAs 0 Remember One tRNA can match UP TO 3 codons because of I 0 Two lcodon families Met and Trp I Each has to have one specific tRNA 0 Nine 2codon families I Each family will require one tRNA because one anticodon can cover 2 G U or I 0 One 3codon family isoleucineile I One tRNA 1 0 Five 4codon families I Each family will require 2 tRNAs IC or UG 0 Three 6codon families I Each family will require 3 tRNAs I Think of 6 as 4 requires 2 and 2 requires 1 0 Total 2x1 9x1 1x1 5x2 3X3 Maximum possible number of tRNAs 53 Difference between number of codons and max possible tRNAs is due to illegitimate anticodons O Called nonsense suppressor tRNAs arrive by mutations 0 These are anticodons that cannot be used by E Coli wobble rules Arises when there are anticodons that would match with 2 different codons that specify different amino acids Ex 5 IUU3 anticodon would match with 3 codon base pair U C Iasn or A El lys therefore this is an illegitimate anticodon Ex2 5 UAU3 anticodon would match with 3 codon base pair A I ile or G 0 met If a mutation arises that creates one of these illegitimate anticodons it ll probably be lethal Termination codons 0 There are no legitimate normal anticodons that recognizes a termination codon thus defining it as such 0 The codon is recognized by a protein called release factor tRNA types 0 O O O 20 different isoacceptors accept one type of amino acid 20AA 20 isoacceptors 3153 different anticodons Same anticodon with the same sequence from a different gene Same anticodon w same seq from the same gene that is modified differently Implies that the same gene may be doing different things in different tissues depending on how it s modified 0 Same anticodon w different sequence tRNA genes 0 Must be greater than minimum number of tRNAs 0 If greater than maximum possible number of tRNAs then you know that you have at least some genes that are coding for the same anticodon tRNA Methanococcus 37 E Coli 62 Physarum Polycephalum 319 Drosophila 600 Humans approx 1000 The genetic code is pretty universal to all organisms but the wobble rules vary between organisms 0 An increase in wobble freedom ability to nonwatsoncrick bp means a decrease in the minimummax number of tRNAs 0 Ex Yeast decrease in wobble freedom I 45min61max Mitochondria 0 Own ribosomes mitoribosomes tRNAs anticodoncodon interactions I different wobble rules I different genetic code 0 Increased wobble freedom 0 Min 22 tRNAs animals 24 tRNAs yeast 0 Wobble rules 0 4codon families can be recognized by a single tRNA I predicted 23 tRNAs 0 In animals one tRNA for met and no tRNA for AGA AGG termination so 22 tRNAs 0 Mitochondrial genetic code not all but most variation 0 No single codonspecific recognition 0 Some tRNAs are missing I Ex UCU tRNAArg in humans Second Genetic Code 0 1St genetic code codonanticodon interaction wobble rules 0 Necessary but not sufficient 0 2nd genetic code other end of the tRNA end that accepts the amino acid 0 If a tRNA has the same codonanticodon interaction but has the wrong amino acid the wrong amino acid will be put into the protein no backup Mechanism of amino acid attachment 0 tRNA synthetase attaches the amino acid to the 3 end of the tRNA 0 20 different amino acids and 20 different tRNA synthetases no degeneracy 0 The tRNA synthetase has specific recognition ability of the tRNA by looking at different regions of the tRNA that are unique and define the identity of the tRNA 0 recognizes identity elements 0 Identity elements expected to be just the anticodon and in some cases it is but also other elements and often doesn t even include the anticodon region 0 EX 370 base pair nucleotide 73 called the discriminator often part of identity can be in DHU arm many identifiers in acceptor arm 0 Extra arm not detected to be part of identity 0 2 reactions occur that add the amino acid to the tRNA 0 1 amino acid ATP I amino acidAMP PPi I High energy reaction requires ATP to occur I PPi pyrophosphate I Intermediate molecule aminoacidAMP aminoacyl adenylate carbonic phosphate anhydride 0 Very reactive unstable 0 2 amino acid AMP tRNA I aminoacidtRNA AMP I AminoacidtRNA aminoacyltRNA I Reaction occurs at the 2 or 3 oxygen of the A on the 3 end of the tRNA 0 Remember CCA end on the 3 end of tRNA necessary for adding an amino acid 0 These are called coupled reactions because the product of l is part of the reactants of 2 the concentrations of one reaction affects the other 0 Cycle of charging a tRNA 0 Amino acid and ATP independently bind to the empty tRNA synthetase I gt Involves the recognition of the specific amino acid by the enzyme 0 Enzyme catalyzes reaction 1 amino acid now connected to AMP 2 phosphates lost 0 Uncharged tRNA then binds to the enzyme I gt Involves specific recognition by the enzyme of the correct tRNA The enzyme transfers the aminoacylAMP to the tRNA reaction 2 0 The enzyme releases AMP and the charged tRNA is now an empty enzyme and is recycled to catalyze another reaction 0 Peptide Bond Formation 0 The bond between each amino acid in a protein 0 Amino group N on the amino acid of the incoming charged tRNA A site attacks the carbonyl group on the amino acid of the P site tRNA with attached peptides O This leaves the P site tRNA with an OH group and disconnects it from the peptide chain it is now an uncharged tRNA and is released 0 The peptide chain is attached to the tRNA in the A site 0 Proteins then grow by addition of an amino acid to the cterminus end 0 Formation of peptidyl transferase a ribozyme catalyzes this reaction 0 Associated with the rRNA of the large subunit of the ribosome 0 Used in prokaryotic and eukaryotic organisms highly conserved Prokaryotic Initiation of Translation I Ribosome starts out as multiple subunits E Coli SOS and 308 but has to be separated for initiation 0 1 IFl initiation factor 1 separates the subunits of the ribosome 0 2 IF 3 binds to the small subunit 308 to prevent reassociation of subunits O IF3 also helps bind the mRNA 0 3 IF 1 and IF 2 with GTP bind to the small subunit 0 4 Formylated tRNA comes in and interacts with IF 2 0 Before it comes in tRNA is charged with methionine by the mettRNA synthetase O Transformylase an enzyme that uses lOformyl THF a formyl donor to put a formyl group on the amino acid 0 This formyl groups blocks the amino group don t want anything on it because it is the first one in the chain 0 This step of formylation only occurs in initiation I 5 mRNA comes in at the same time as the tRNA 0 Binds through RNARNA interaction displaces 5 end of rRNA that was bound intramolecularly to the 3 end and binds antiparallel to the 3 end of the rRNA O Consensus for ribosome binding site on the mRNA UGGAGGA I This consensus is used to position AUG in the correct spot I This consensus sequence IN THE mRNA is called the ShineDalgamo sequence or SampD or ribosome binding site I Occurs approx 57 nucleotides upstream of the AUG I Not always super conserved but generally AG rich 0 At this point the 308 initiation complex has formed includes 308 subunit mRNA IF12 and 3 the formylated charged tRNA in the P site and an empty A site I 6 The 308 subunit then releases IFl and IF3 which at the same time allows the large subunit to come back on I 7 GTP hydrolysis occurs GTP I GDP Phosphate which releases IF 2 and the attached GTP I Now have 708 initiation complex with tRNA in P site close to the AUG codon Eukaryotic Initiation of Transcription I Ribosome starts outwith subunits associated 608 408 I 1 eIF6 dissociates the subunits by interacting With the large subunit 0 2 eIF3 binds to the small subunit aids later in mRNA binding I 3 eIF2 with GTP brings in MettRNA nonformylated even though initiation I 4 tRNA interacts at the P site by interacting directly with rRNA no mRNA yet I 5 eIF3 and eIF4 bring in mRNA 0 CBP cap binding protein is a subunit of eIF4 that is What really brings in the mRNA 0 CBP recognizes the 5 cap on the mRNA and brings it in I 6 the small subunit scan the mRNA to find AUG 0 No SampD sequence to bind at binds at the 5 cap and then slides down scanning the mRNA until it finds the start codon IN CONTEXT I Context Small subunit not really looking for AUG by itself looking for Kozak sequence GCCACCAUGG around the AUG I The ribosome pauses When it hits the consensus sequence I If the leader is long enough several hundred nucleotides often multiple small subunit ribosome Will be lined up behind the first I Experimental proof of scanning If there is a secondary structure hairpin loop that is too close to the cap site for the small subunit to fit no initiation even if AUG is exposed If there is too large of a secondary structure for eIF 4B to break down no initiation even if AUG is exposed can t unbind and bind past it I Often multiple AUGs If some in bad context the ribosome will skip over and go to another one but if the AUG in good consensus context is blocked by too big of a secondary structure the ribosome will stall and start at the worse Kozak I 7 eIF5 brings in and positions the large subunit 0 At the same time as bringing in the large subunit GTP hydrolysis occurs and eIF2 GDP and eIF3 are kicked off I 8 eIF2GDP then with the help of a factor loses GDP and regains GTP regenerates eIF2 GTP so that it can be used in another reaction 0 The SOS intiation complex is now formed Elongation 0 Very similar in prokaryotes and eukaryotes but different names for elongation factors I Start of elongation tRNA in P site empty A site 0 1 Charged tRNA comes into the A site 0 Step aided by EFTuGTP eEF10t I EFTuGTP brings in the aminoacyltRNA and then GTP hydrolysis releases the EFTuGDP I EFTs eEF1 B then binds to EFTu displacing GDP I GTP then binds to displace EFTu to regenerate EFTuGTP complex to bring in another tRNA in next round I 2 A peptide bond is formed between the amino acid on the new A site tRNA and the amino acid methionine that was in the P site 0 This disconnects the methionine or any AA from its tRNA 0 Uses peptidyl transferase I Experimentally proven to be catalyzed by peptidyl transferase because antibiotics chloramphenicol and carbomycin specifically target this enzyme and when taken activity inhibited I Peptidyl transferase is a ribozyme rRNA I 3 Translocation movement of the peptidyltRNA into the P site moves one codon down drags mRNA with it deacylated tRNA moves into E site and exits A site now empty for next charged tRNA 0 Uses EFG eEF 2 and GTP I GTP hydrolysis O EFGGTP complex binds to the ribosome GTP is hydrolyzed and the ribosome moves one codon down and displaces the now uncharged tRNA that was in the P site I It is possible that GTP hydrolysis changes the structure of EEG to facilitate this event 0 The uncharged tRNA binds to the E site which blocks the next aminoacyltRNA from binding to the A site until the peptidyltRNA is correctly bound to the P site I After the peptidyltRNA is correctly bound the uncharged tRNA is released I EF G eEF 2 is released and reused Now have tRNA with attached peptide chain in the P site and another charged tRNA can come in and add an amino acid to the chain The complex between the mRNA and the ribosomes that are translating it is called a polyribosome or a polysome EFTS GTP Hydrolysis Initiation hydrolyzes one GTP every round addition of each amino acid hydrolyzes 2 GTPs and termination hydrolyzes 1 GTP RNARNA Interactions tRNA interacts with mRNA through codonanticodon interactions The subunits of the ribosome interact with one another The top of the tRNA interacts with the large subunit Prok23S EukZSS and the bottom of the tRNA interacts with the small subunit of the ribosome Prok16S EukZSS Prokaryotic Termination of Translation Same stop codons in prokaryotes and eukaryotes UAG UAA UGA that signal termination of translation When termination codons go into the A site no tRNA wanticodon will recognize it RFs go in instead 0 RFs are always competing with tRNAs when a tRNA matches it wins when there is no tRNA for the codon stop codon the RF wins RFs have shapes that mimic that of a tRNA RFl or RF2 release factors recognize a stop codon O RFl UAA UAG O RF2 UAA UGA The binding of RFI or 2 to a stop codon triggers peptidyl transferase to cleave the polypeptide from the tRNA in the P site The polypeptide then leaves the tRNA RF3GDP then binds to the ribosome which releases the RF from the stop codon and the ribosome GTP then replaces GDP RF3 hydrolyses the GTP which allows RF3 to be released from the ribosome RRF ribosome recycling factor is shaped like a tRNA then binds to the A site Then EFG binds causing translocation of the ribosome and moving RRF to the P site and the uncharged tRNA to the E site The RRF releases the uncharged tRNA and EFG releases RRF causing the two ribosomal subunits to dissociate from the mRNA Only difference in eukaryotes is that a single release factor eRFl recognizes all three stop codons and eRF3 stimulates the termination events Ribosome recycling occurs in eukaryotes but there is no equivalent of RRF A polypeptide folds during the translation process 2 polypeptides With identical amino acid sequences can fold to produce polypeptides With different structures and functions
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