Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Slide 44Slide 45Slide 46Slide 47Slide 48Slide 49Slide 50Slide 51Slide 52Slide 53Slide 54Slide 55Slide 56Slide 57Slide 58Slide 59Slide 60Slide 61Slide 62a bStructure and binding of tetrameric lac repressor proteinhinge + helix-turn-helixIPTG (isopropylthioglucose)CH2SOHHOHOOHOtruncation at hingetruncation at hingetruncation at point of hinge attachmentTetramer, with two of the tetrameric units selectedB. Rotated 90o around core axisHeadpiece (hinge + HTH motif)A. Looking down DNA helixLac repressor dimer bound to operatoranti-inducero-nitrophenylfructose (ONPF)hingeLac repressor + IPTG truncated at hinge.Lac repressor + ONPF truncated at oligomerization domainContrast inducer-bound and active lac repressorOOOOHPONNNH2NNOcyclic AMP (cAMP)E. coli CAP +cAMP + DNA HTH motifcAMPTFIID =TAFs +TATA box : is a conserved A · T-rich octamer found about 27 bp before the start point of each eukaryotic RNA polymerase II transcription unit; it is involved in positioning the enzyme for correct initiation.A TATA-less promoter : does not have a TATA box in the sequence upstream of its start point.The initiator (Inr) : is the sequence of a pol II promoter between –3 and + 5 and– has the general sequence YYANWYY. (C/T C/T A N A/T C/T C/T)– It is the simplest possible pol II promoter.Human TBP complexed at TATA box of DNAGGCATATATATC5'3'In general. Promoters contain different combinations of TATA boxes,CAAT boxes, GC boxes, and other transcription factor binding sites.Saturation mutagenesis of the upstream region of the β-globin promoter identifies three short regions (centered at -30, -75, and -90) that are needed to initiate transcription. These correspond to the TATA, CAAT, and GC boxes.Eukaryotic PromotersAn enhancer contains several transcription factor binding sites (indicated below the sequence. The histogram plots the effect of all mutations that reduce enhancer function to <75% of wild type.DNA binding motifsZn fingersHelix-turn-helixHelix-loop-helixLeucine zippersC-terminalN-terminal-Cys-X2-4-Cys-X3-Phe-X5-Leu-X2-His-X3-His-The zinc finger motif is:Zn finger fragment bound to DNAN-terminalC-terminalSTEROID RECEPTORLeft: Zn-finger binding domains of dimer complexed with DNA.Right: Ligand binding domain with activator and co-activator bound.DNA bindingDNA sequence spacingGLUCOCORTICOID RECEPTOR FRAGMENT BOUND TO DNAN-terminalC-terminalfingerZn binding motif -Cys-X2-Cys-X13-Cys-X2-Cys-Hormone binding and dimerization regions vary from 5-15%homeodomainHelix-turn-helix binding motif#3, C-terminal#1, N-terminal#3#2#1bHLH DNA binding motif of transcription factor MyoDhelix 1helix 2Heterodimer of HIF2 alpha and ARNT C-terminal domainsCREB-DNA complexLeu selected in zipper domainArg selected in DNA-binding domainChains: blue = c-Fos, green = c-JunResidues: red = Leu, purple = ArgHuman protooncogenes c-Jun:c-Fos heterodimerHNNNNOH2NNNNNOH2NHNNNNOHH2NNNNNOHNHHN NOOHHNNNNOH2NNNNNOH2NHNNNNOHH2NNNNNOHNHHN NOOHHNNNNOH2NNNNNOH2NHNNNNOHH2NNNNNOHNHHN NOOHH3CKeto-enol tautomerization of Guaketo form (normal)enol formpairing properties of enolPoint mutation, transitionpyrimidinepyrimidinepurinepurinePoint mutation, transversionpyrimidinepurinepurinepyrimidineG-TG-C+A-TG-CC-G,T-AA-T,G-CT-Anonsense mutation occurs when a termination codon is generatedmissense mutation occurs when a different amino acid is codedG-TG-C+A-TOne round of replication on mismatched strands “fixes” the mutation.3'-G-p-G-p-G-p-G-p-G-p-G-5' 3'-G-p-G-p G-p-G-p-G-p-G-5'5'-C-p-C p-C-OH 5'-C-p-C-p-C-OHp-CG-pMechanism for frameshift mutations+1 frame shift ATA GCA ACC GGC CTT ACA AAT TCwild type ATA GCA ACC GCC TTA CAA ATT CChange of reading frame following an insertion of Ginsertion deletion[2] Note that this suppressor tRNA can still recognize the wild-type Trp codon as well as the UGA codon.[1] The suppression efficiency varies for different sites because the mRNA sequence following the nonsense codon influences how well the suppressor tRNA works. 0.1-30%[2]trpTtRNATrpCCA --> UCAOpaltrpTglyTtRNAGlyUCC --> UCAOpalglyTtyrUtRNATyrGUA --> UUAOchresupMtyrTtRNATyrGUA --> UUAOchresupClysVtRNALysUUU --> UUAOchresupNlysTtRNALysUUU --> UUAOchresupLglnUtRNAGlnUUG --> UUAOchresupBtyrUtRNATyrGUA --> CUAAmbersupZ11-100%tyrTtRNATyrGUA --> CUAAmbersupFtrpTtRNATrpCCA --> CUAAmbersupU6-54%serUtRNASerCGA --> CUAAmbersupD30-100%leuXtRNALeuCAA --> CUAAmbersupP0.8-20%glnVtRNAGlnCUG --> CUAAmbersupEEfficiency[1]GenetRNAAnticodon changeTypeSuppressorSuppressor tRNAs from E. coliOOOOPOOOHNOOOOCH3CH3HNOthymine-thymine dimerT THHHHHFp teerrorHuman homolog hMsh2Human homolog hMlh1Proteinsthatinteractwitheachothersufficientlystronglytoformisolatablecomplexes,andproteinsthathavesimilarfunctions,havebeengroupedtogetherandprovidedwithacommonbackgroundcolor.Particularlynoteworthyisthe10-proteincomplexcalledTFIIH(backgroundcolor=green),whichisessentialforDNArepairandtranscription.Global genomic nucleotide excision repair pathway (GG-NER)Transcription-coupled nucleotide excision repair pathway (TC-NER)Completion of nucleotide excision repairhTDG glycosylase bound to abasic site remaining after removal of T in a T●G mismatchAbasic sugar, blueOpposing G, purpleContacts giving hTDG specificity for GpC context are R275 (black) and A274 (green)human 8-oxoguanine glycosylase (hOGG1)Complex with DNA containing 8-oxoGua8-oxoGuaHNNNHNOH2NO8-oxoguanineLysine at 249 displaces 8-oxoGua from C1’ (arrow)H2NOglutamineH2NlysineC1'HUMANMITOCHONDRDIALURACILGLYCOSYLASE(UDG),WITHGLYCOSIDICLINKAGEHYDROLYZEDhuman alkyladenine glycosylase (AAG)complex with DNA modified by tricyclic base 1,N6-AdeNNNNNdR1,N6-dAdoAAG catalyses deglycosylation by a water bridge to GluHolliday junctionEukaryotic homologues to RecA are characterized in yeast: DMC1 and RAD51BACTERIAL REPAIR BY