MOLECULAR BIOLOGY MCDB 3500
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Chapter 2 The Molecular Nature of Genes II How does DNA act as a genetic information carrier Page 2435 NF N gt 539phosphae Ni1 Qw Hiioi CH2 0 O 339 gt NH N N k C o k L O N O u N39 o o ProicH OPOCH2 Di 539 O 5 39 Of 0 0H 53 Phosphomesler bonds 5 end a 5quotph05phate H O NH k m o N 0 orFl rorCHz 5 3 NH2 N x O N o OF 7 OCH2 3 o NH2 Fhosggrr esier N N lt I J A o N N O L 0CH2 8 0 Figure 210 OH anhydroxw 3 end How does DNA serve as a genetic information carrier Polynucleotide sequence can carry complex information n2 16 different possible sequences 4x4 n3 64 different possible sequences 4x4x4 n4 256 different possible sequences 4x4x4x4 n5 1024 different possible sequences 4x4x4x4x4 n6 4096 different possible sequences 4x4x4x4x4x4 ny 4y different possible sequences Chargaff s Rules TABLE ADAPTED FROM CHARGAFF39S 1949 PAPER DNA SOURCE ADENlNE THYMINE GUANINE CYTOSINE r 1 139 Calf Thymus 17 16 12 10 s j 39 Beef Spleen 16 15 13 10 Yeast 18 19 10 10 I frquot 3 L Tubercle Bacillus 11 10 26 24 Erwin Chargaff observed in 1950 that the content of purines was always roughly equal to the content of pyrimidines Furthermore the amounts of adenine and thymine were always roughly equal as were the amounts of guanine and cytosine AT CG but what does this mean Xray structural information Figure 212 Rosaline Franklin 1952 Courtesy Pruisssnr M H Wiikins Biophysics Dept Kings39 Coilega London What does this interesting pattern mean The DNA is a simple regular structure with repeated units The distinctive quotXquot in this Xray photo is the telltale pattern of a helix le lt a 5 If T 1 The helix must have the same width In an Xray diffraction pattern the closer the spots the larger the actual distance So the horizontal bars actually correspond to helical turns The vertical distance between the bars 34 Angstroms is a measure of the height of one helical turn MAW re gV W 343 34A I From the Xray diffraction pattern Crick also deduced that DNA should be a double helix with the phosphate groups on the outside and the bases on the inside A M79 How do the helies t together How are the nitrogen bases arranged 4 7 GT A V AC T C L 7 A r GA In a moment of part insight and part luck Waston realized that adenine could pair closely with thymine and that guanine could pair closely with cytosine Moreover the AT base pair was about the same width as a GC pair V GC 97 AT More importantly this base pairing agreed with and explained Chargaff s observations AT and GC Figure 213 H o H N G H C Sugar N H O S 39 39 39 39 ugar H Hydrogen bond H N H 0 CH3 Sugar Francis Crick agreed with Waston s hypothesis He also pointed out that because of certain bond angles and the proximity of the base pairs the two helices had to run in opposite directions The helices are antiparallel to one another o o O c m phosphateester chain 0 c amp N In bases 0 P Watson and Crick submitted a 900 word manuscript on their DNA structure prediction to Nature In their paper they wrote It has not escaped our notice that the speci c base pairing we have proposed immediately suggests a possible copying mechanism for the genetic material This basically serves as a guideline on how DNA can be faithfully replicated or copied into the next generation Figure 215 2 Replicate gt 1 2 Finish Unwind Replicate Replication gt gt gt 9 55 a 5 0f M QJO M 0 Q 5 Q Q 5 m fOfQO Francis Crick wig Maurice Wilkins James Waston Rosalind Franklin 339 w b The 5 Double rd nHelix J Y NOBEL PRIZE VINNEB JAMES DWATSON v ION Fitch Form NWP R esidues Pitch A per turn 246 1 1 332 10 456 12 Inclination of base pair from horizontal 19o 12 90 Although GC and AT are true for every organism the amounts of GC contents vary from organism to organism Figure T23 Table 23 Rela ve G C Contents of V DNAS Sources of DNA Percent G C DiciyosieI um slime moid 22 Streptococcus pyogenes 34 Vaccinia virus 36 Bacillus cereus 37 E megateriu as Hemophius influenzae 38 Sacchammyces cerevisiae 39 Caif thymus 40 Rat llver 40 Bull sperm 41 Streptococcus pneumoniae 42 Wheat germ 43 Chicken liver 43 Mouse spieen 44 Salmon sperm 44 39i39s 44 T1 bacteriophage 4E Escherichia C0 51 17 bacteriophage 51 T3 bacteriophage 53 Neurospora crassa 54 Pseudomonas aerug nasa GE Sarcina urea 72 Micrococcus Iysode kticus 72 Herpes simplex virus 72 Mycobacler um phlei 73 Sou12 From Dexmeow ma Brnoremisnyuiiie Nunie cAziris am Hmiio mew v Adams 9 2i Lumencon Two DNA strands can be separated simply by heating a process called DNA denaturation or DNA melting Figure 213 H o H N The temperature at which the DNA j H c strands are half denatured is called 39 the melting temperature or Tm N H 0 Sugar 0 CH3 Tm of a DNA is largely determined A T byitsGC NZ Sugar 0 Sugar The amount of DNA strand separation can be measured by the absorbance of the DNA solution at 260 nm light Relative absorbance at 260nm 10 i 7 70 75 80 85 90 95 Flgure 23917 Temperature The process of reuniting the separated DNA strands is called H annealing or renaturation Temperature 25 C below Tm DNA concentration the higher concentration the better the annealing Renaturation time the longer the time the better the annealing The process of annealing a DNA strand with a complementary or nearly complementary RNA strand or DNA strand from a different origin is called hybridization Denature Doublestranded DNA RNA E g annealing W Figure 224 DNAs are of various sizes and shapes Figure T24 Table Source Molecular weight Base pairs Length Subcellular Genetic Syslems SV40 mammalian tumor Virus 35 gtlt10s 5226 17 pm Baclerlophage X174 doubleistranded form 82 X 1t6 5386 18 pm Bacteriophage A 33 x105 5 X10 13 um Bacteriophage T2 or T4 13 x10E 2 x105 50 lm Human mitochondria 95 gtlt1O i 16596 5 pm Prokaryotes Hemophrlus rn uenzae 12 x 109 183 x 106 620 um Escheri his coi 31 X109 465 x 106 16 mm Salmonella typhimurium 8 X 109 11 X 107 3 8 mm Eukaryotes content per haploid nucleus Sass aromyces cerevisae yeas1 79 X 109 12 X 107 4 1 mm Neurospora crassa pink bread mold 13 x 10 0 727 X 107 92 mm Drosophfa melanogaster fruit fly 12 X 10 718 X 105 430 cm Rana pip39ens frog 14 gtlt10 3 23 x 10 0 77 m Mus muscuus mouse 15 gtlt10 z 22 gtlt 10g 75 cm Homo sapiens human 23 gtlt10 2 35 X 109 420 cm Zea mays corn or maize 44 gtlt10 z 66 X 109 22 m Lil11m long39 orum My 2 X 1014 as X 1011 N100 m Chapter 20 DNA Replication synthesized strand Each daughter duplex has one parental stand and one newly OOOO0Ok Finish Replication gt 00000Ob 000000b liVUiDVVOSlViO VVLDVQLLSOVLV E Figure 215 a Semiconservative I b Conservative I 0 Random dispersive I httpwwwdnaftb0rg dnaftb 20 concept indexhtml Figure 2039256 era on H D D L L Expected densiiy gradientresu s D 3D at Consewative I D D D D L L D D Firstv Secoan b generanon generanon Semxconservalive H l DD D L L D D L L L L L L D Expected densny gradiennesuns D I 1 I DD DL LLDL First Second generation generauon c Randomdlspersive l I Mixed Mixed Allmixed Expected density gradientresults P 3 3 D D D L 25 D 75 L a Continuous g r Replication fork 3 3 5 5 9 W5 5 3 3 b Sem idiscontinuous 5 l st d 5 Replicationfork aggmg ran 3 b I 3 y 3 E 539 5 5 Leading strand 5 3 3 c Discontinuous Okazaki s model makes two predictions 1 At least half of the newly synthesized DNA 2 appears rst as short pieces One can then label and catch them before they are stitched together by allowing only very short periods pulses of labeling with a radioactive DNA precursor If one eliminates the DNA ligase responsible for stitching together the short pieces of DNA you can then easily detect these short pieces of DNA Pulse labeling experiments x 39 3HT X gt pulse W 3HT long j Radioactivity 103 cprnfO ml Okazaki fragme ts Long DNA pieces 4120 sec i Sh0i1 DNA pieces 1 Relative distance from top of tube Oi Short DNAs Long DNAs Radioactivity 103 cpmO1 ml 6 Short DNA pieces 0 1 2 Ligase mutant 3 Relativa distance from top of tube 0 III Short DNAs How does DNA synthesis start A DNA polymerase With all nucleotides and other necessary molecules cannot start DNA synthesis on single stranded or doublestranded DNA templates It needs a primer to start making DNA sFigure 208 1 2 M13 phage DNA replication in E coli is inhibited by the antibiotic rifampicin Which inhibits the activity of E coli RNA polymerase DNase cannot completely digest Okazaki fragments leaving small pieces of RNA with 1012 bp long MCDB 3500 Lecture 8 2602 Figures 67 69 611 616 610 611 613 621 623 536 620 537 530 151 Ed 68 610 6 11 612 614 617 629 631 531 620 532 621 Transcription in Prokaryotes Reading Chapter 6 E coli RNA polymerase S 39OOL2Holoenzyme ocz Core enzyme 13 beta 150 kDa Binds nucleotides 3 betaprime160 kDa Binds DNA 0 sigma 70 kDa See below 1 alpha 40 kDa Recognizes UP elements 039 a speci city factor Helps the holoenzyme recognize the promoter sequence Destabilize nonspeci c polymeraseDNA interactions Stimulates transcription initiation but not elongation Crystal structures of Holoenzyme vs Core enzymes Fig 69 C0rec10sed hand H0100pen hand Once sigma falls off forms closed hand around DNA RNA polymerase needs to be highly processive Promoter binding af nity Holoenzyme gtgtgt Core enzyme Only the l l J 39 the r Promoters Polymerase binding site Closed promoter c0mp1ex100se binding of holoenzyme scanning model Open promoter complex requires sme1ting of DNA Minimal promoter consensus sequence 35 box 5 TTGACA339 10 box 539TATAAT3 339AACTGT5 3 ATATTA539 Transcription Initiation O stimulates initiationmakes contacts at 10 amp 35 Melting of approx 10 bp near the transcription start site Polymerase Binding Sequence speci c binding Exp Gel mobility shift Assay Gel retardation assay Fig 536 Used to identify the fragment to which a protein complex binds Exp Exonuclease III rotection Assa Fi 620 Used to de ne a region of the DNA bound by protein Exp DNase Foot rintin Assa Fi 537 Used to identify the sequences to which a protein complex binds Exp Primer Extension Assa Fi 530 Used to identify the 539 end of a transcript Which of the following DNA is more stable assuming all has the same number of base pairs A DNA With more A T pairs B B type DNA C A type DNA D DNA With more G C pairs E Equally stable Sl l l Although GC and AT are true for every organism the amounts of GC contents vary from organism to organism Figure T23 Table 23 Rela ve G C Contents of V DNAS Sources of DNA Percent G C DiciyosieI um slime moid 22 Streptococcus pyogenes 34 Vaccinia virus 36 Bacillus cereus 37 E megateriu as Hemophius influenzae 38 Sacchammyces cerevisiae 39 Caif thymus 40 Rat llver 40 Bull sperm 41 Streptococcus pneumoniae 42 Wheat germ 43 Chicken liver 43 Mouse spieen 44 Salmon sperm 44 39i39s 44 T1 bacteriophage 4E Escherichia C0 51 17 bacteriophage 51 T3 bacteriophage 53 Neurospora crassa 54 Pseudomonas aerug nasa GE Sarcina urea 72 Micrococcus Iysode kticus 72 Herpes simplex virus 72 Mycobacler um phlei 73 Sou12 From Dexmeow ma Brnoremisnyuiiie Nunie cAziris am Hmiio mew v Adams 9 2i Lumencon Two DNA strands can be separated simply by heating a process called DNA denaturation or DNA melting Figure 213 H o H N The temperature at which the DNA j H c strands are half denatured is called 39 the melting temperature or Tm N H 0 Sugar 0 CH3 Tm of a DNA is largely determined A T byitsGC NZ Sugar 0 Sugar The amount of DNA strand separation can be measured by the absorbance of the DNA solution at 260 nm light Relative absorbance at 260nm 10 i 7 70 75 80 85 90 95 Flgure 23917 Temperature The process of reuniting the separated DNA strands is called H annealing or renaturation Temperature 25 C below Tm DNA concentration the higher concentration the better the annealing Renaturation time the longer the time the better the annealing The process of annealing a DNA strand with a complementary or nearly complementary RNA strand or DNA strand from a different origin is called hybridization Denature Doublestranded DNA RNA E g annealing W Figure 224 DNAs are of various sizes and shapes Figure T24 Table Source Molecular weight Base pairs Length Subcellular Genetic Syslems SV40 mammalian tumor Virus 35 gtlt10s 5226 17 pm Baclerlophage X174 doubleistranded form 82 X 1t6 5386 18 pm Bacteriophage A 33 x105 5 X10 13 um Bacteriophage T2 or T4 13 x10E 2 x105 50 lm Human mitochondria 95 gtlt1O i 16596 5 pm Prokaryotes Hemophrlus rn uenzae 12 x 109 183 x 106 620 um Escheri his coi 31 X109 465 x 106 16 mm Salmonella typhimurium 8 X 109 11 X 107 3 8 mm Eukaryotes content per haploid nucleus Sass aromyces cerevisae yeas1 79 X 109 12 X 107 4 1 mm Neurospora crassa pink bread mold 13 x 10 0 727 X 107 92 mm Drosophfa melanogaster fruit fly 12 X 10 718 X 105 430 cm Rana pip39ens frog 14 gtlt10 3 23 x 10 0 77 m Mus muscuus mouse 15 gtlt10 z 22 gtlt 10g 75 cm Homo sapiens human 23 gtlt10 2 35 X 109 420 cm Zea mays corn or maize 44 gtlt10 z 66 X 109 22 m Lil11m long39 orum My 2 X 1014 as X 1011 N100 m Quiz True or false The GC and AT base parings are important for 1 holding the two DNA strands together 2 replicating DNA 3 the temperatures required for denaturing and renaturing of two DNA strands 4 Keeping the DNA heliX With the same Width 5 the length of the DNA molecule 6 Whether a DNA should be in A form or B form Chapter 4 Molecular Cloning Methods Page 5969 How do we know which DNA region is important for a particular biological function For example which gene decides the eye color Which gene decides the height of a person Which genes decide the sexual identity Whether a gene is mutated in a cancer patient Identify separate and manipulate a speci c DNA fragment Molecular Cloning The process of inserting a piece of DNA molecule of interest into a DNA carrier vector in order to make multiple copies of the DNA of interest in a host cell such as bacteria Pilfl mi f of us39ylwulzu cloning Separate a gene from the other genes Ampli cation of modi ed forms of genetic materials Manipulation of a piece of DNA for further experiments Vector DNA carrier Plasmids Cosmids YAC Bateriophage Virus l mm Wm M Lk m 535 quotWNW Restriction Endonucleases uTl Ie Molecular Scissors Host enzymes that prevent the invasion of foreign DNAs such as Viral DNA by cutting them up These enzymes cut within the foreign DNAs rather than chewing them away from the ends Endonucleases These enzymes recognize a speci c DNA sequence 412bp which is twofold symmetry and cut both DNA strands Some enzymes make staggered cuts GAATTC CTTAAG Some make even ClltS CCCGGG GGGCCC S leoxyribose P phosphate groups P P P P P P P P OH S S S S S S S S I I I I I I I T I9 T T 6 5 C T A C A A T T GI A I I I I I I I S S S S S S S S HO P P P P P P P P Bluntend cleavage P P P P 3bH P5 P P P OH S S 5 S S S S S I I T G T T A A C T A C A A T T G A I I S S S S S S S 5 HO P P P 3P H03 P P P P a gt Wan gt an ng rm McGuwHiilcumpaniag m Pevmiism mm ierleunduciicmaruimhy Table 41 Recognition Sequences and Cutting Sites of Selected Restriction Endonucleases Enzyme Recognition Sequencequot Alul AGiCT BarnHl GiGATCC Bglil ALGATCT Clal ATiCGAT EcoFil GlAATTC Haelll GGLCC Hindll GTPy i PuAC Hindlll A AG CTT Hpall CLCGG Kpnl GGTACLC Mbol LGATC Pstl CTGCAiG Pvul CGAT LCG Sal GiTCGAC Smai CCC GGG Xmai CLCCGGG Notl GCLGGCCGC Oniy one DNA strand written 5 3 lei to right is presented but restriction 4 hi a m v Ewn The cutting site for each enzyme is represented by an arrow The rst cloning experiment done by Boyer and Cohen EcoHl Tetracychne39 svpmycin39 Sulfonamide iEGDFU lECDFH LIT Transform bacteria Telracycline Slreptomycin Vectors the DNA carriers Capable of replicating in bacteria an origin of replication Allow the vector as well as the foreign DNA to amplify in the host cell 1 Plasmids cm Hinle EcoRV j 2 Phages ECO Origin of replication Antibioticresistant genes Allow the host to grow on selective media Can selectively amplify this speci c vector in the host cell Multiple cloning sites Allow insertion of foreign DNA Vectors the DNA carriers Plasmid as a vector Host E coli Vector size usually about 3kb Insert size up to 20kb usually below 5 kb Insert select inactivation of the ability to resist an antibiotic F39st PSH Ps DNA ligase Tiansiorm baclelia Tev Amps Figure 45 Original tetracycline Replica ampicillin DNA ligase covalently links two DNA strands to 3 Restriction enzyme MCDB 3500 Lecture 13 22002 Figures 527 Sltechnique 1025 1026 1027 1028 1029115 1112 1117 11271133 ls39Ed523 Sltechnique 1025 1026 1027 1028 1029 116 1112 1117 112511261131 Pol II Transcription For mRNAs How to identify the 539 end S1 mapping Class II Promoters 1 TATA Box Around 25 Tissue speci c genes have TATA box Mutation in this element causes variable Tnx initiation or reductionloss of expression TA TA less grammarsquotHousekeeping genes many developmentallyregulated genes 2 Upstream elements upstream of TATA box Examples GC Box GGGCGG or CCGCCC GC boxes Can have several GC boxes Bound by Spl transcription factor CAAT box Bound by CTF and CEBP 3 Initiators Transcription start sitePyPyANTApyPy 4 Downstream elements Unusual No concensus sequence MCDB3 500 02 Weaver Figures 201 202 203 204 206 207 208 20112015 Lecture 3 DNA Replication What we ve learned from E coli 1 Consider the speed and the sequence of the usage of PolIII Poll and Primase Semiconservative Q What type of experiments would you do to show this Semidiscontinous a Leading strand b Lagging strand c Okazaki fragments Q What type of experiments would you do to show this 5 to 3 Direction on newly synthesized DNA Q What type of experiments would you do to show this Bidirectional Q What type of experiments would you do to show this Uses RNA primers by primase RNA polymerase DNA replication ALWAYS requires a PRIMER Q What type of experiments would you do to show this Chapter 4 Molecular Cloning Methods Page 5969 Quiz True or false The GC and AT base parings are important for 1 holding the two DNA strands together T 2 replicating DNA T 3 the temperatures required for denaturing and renaturing of two DNA strands T 4 Keeping the DNA heliX With the same Width T 5 the length of the DNA molecule F 6 Whether a DNA should be in A form or B form F Enzymes that can cut DNA Enzymes thalumjoin DNA t 1139Z Z71 39 39 quotKL 0 3 l mm Wm M Lk m quotWNW Restriction Endonucleases uTl Ie Molecular Scissors Host enzymes that prevent the invasion of foreign DNAs such as Viral DNA by cutting them up These enzymes cut within the foreign DNAs rather than chewing them away from the ends Endonucleases These enzymes recognize a speci c DNA sequence 412bp that is twofold symmetry and cut both DNA strands Some enzymes make staggered cuts GAATTC CTTAAG Some make even ClltS CCCGGG GGGCCC S deoxyribose P phosphate groups m gt I m m4 OA Ln m gt I m m gt I m m cgtm m Igt m m 3 m mgt m Bluntend cleavage P P P P 3bH P5 P P P OH S S 5 S S S S S T T T G T T A A C T A C A A T T G A T T S S S S S S S 5 HO P P P 3P H03 P P P P a gt Wan gt an ng rm McGuwHiilcumpaniag m Pevmiism mm ierleunduciicmaruimhy Table 41 Recognition Sequences and Cutting Sites of Selected Restriction Endonucleases Enzyme Recognition Sequencequot Alul AGiCT BarnHl GiGATCC Bglil ALGATCT Clal ATiCGAT EcoFil GlAATTC Haelll GGLCC Hindll GTPy i PuAC Hindlll A AG CTT Hpall CLCGG Kpnl GGTACLC Mbol LGATC Pstl CTGCAiG Pvul CGAT LCG Sal GiTCGAC Smai CCC GGG Xmai CLCCGGG Notl GCLGGCCGC Oniy one DNA strand written 5 3 lei to right is presented but restriction 4 hi a m v Ewn The cutting site for each enzyme is represented by an arrow The rst cloning experiment done by Boyer and Cohen EcoHl Tetracychne39 svpmycin39 Sulfonamide iEGDFU lECDFH LIT Transform bacteria Telracycline Slreptomycin Recombinant product can be selected using antibiotics ECDRI EcoFil Slreptoycin39 Sulfonamide LECDFU ECOFH x Tetracyclme39 Transform bacteria Telracycline39 Slreptomycin Vectors the DNA carriers Capable of replicating in bacteria an origin of replication Allow the vector as well as the foreign DNA to amplify in the host cell 1 Plasmids Clal Jj Hinle EcoRV j EcoRI Origin of replication Antibioticresistant genes Allow the host to grow on selective media Can selectively amplify this speci c vector in the host cell Multiple cloning sites Allow insertion of foreign DNA Vectors the DNA carriers Plasmid as a vector Host E coli Vector size usually about 35 kb Insert size up to 20kb usually below 5 kb Insert select inactivation of the ability to resist an antibiotic F39st PSH PSI DNA Iigase Tiansiorm baclelia Tequot Amps Figure 45 Original tetracycline Replica ampicillin DNA ligase covalently links two DNA strands to 3 Restriction enzyme It is really a pain to do replica plating an322 DNAngase Tipsquot 23 Figure 45 LL G G gt Original Replica Original tetracycline Replica ampicillin Transform bacteria pUC IB pLICTQ OLpeptide 0r Nterminus of 3 galactosidase l 2 3 4 fMET TIHH MET ILE ATG ACC ATG ATT l 2 3 4 IMET THH MET ILE Multiple cloning sites 160239 MCS quotx ON I I II I I a a 5 6 T THFI ASN SER ser ser val pro gly asp pro ACG AAT TCG AGE TCG GTA CGG GGG GAT OUT I I I I I II I EcoRI 5er Kpnl I IBamHI Smal Xmal 5 THFI pro ser leu his ala c315 arg ser thr ATG ACC ATG ATT ACG CCA AGC TTG CAT GCC TGC AGG TCG ACT I l I I Hindlll Sphl Psrl Sail Accl Him 8 9 Ian glu ser Ihr cys arg his ala ser leu ala LELJ ALA CTA GAG TCG AGO TGC AGG CAT GSA AGO TTG GSA GTG GCG I I I I I Xbal II Pstl Sphl Hindlll Sail Accl Hincll E3 7 8 9 law glLI asp pm arg val IprQ ser ser ASN SEFI LEU ALA CTA GAG GAT GGC CGG GTA COG AGO TCG AAT TCA CTG GOD I II I I I I Xbal BamHl Kpnl Sstl EcoRl Smal XmaI ocpeptide when combined with the carboxyl terminus of Sgalactosiase can make a functional Bgalactosidase Bgalactosidase OLpeptide Carboxyl Terminus apeptide F Carboxyl Terminus upeptide when combined with the carboxyl terminus of galactosiase can make a functional galactosiase O a O O Cterminus of gahclosiase u pe tide K 39 an C Cterminus of galactosiase u peptide Phages as DNA carriers Bacteriophages are natural vectors that transduce DNA from one bacterial cell to another A bacteriophage can be regarded as a virus for a bacterial cell A bacteriophage cannot live or reproduce Without getting inside a bacterial cell Oh Goodness My E coli has a Virus Viruses and Bacteria T4 bacteriophage is a virus that looks like an alien landing pod With its six legs the bacteriophage attaches to the surface of the much larger bacteria Escherichia coli E coli Once attached the bacteriophage injects DNA into the bacterium The DNA instructs the bacterium to produce masses of new viruses 80 many are produced which then burst out of the bacterium and kill it Now THAT39s a NASTY virus mg W 39 A single phage can infect and clear out many bacterial cells and creat a plaque on a bacterial lawn A plaque contains a homogeneous population of a phage MCDB 3500 Lecture 12 21802 Figures 1154 1032 1030 1033 1152 1036 1St Ed 1152 1032 1030 1033 1150 1036 Eukaryotic Transcription Polymerases compleX with many subunits Evolutionary relationship to prokaryotic core polymerase RNA polymerase I Makes rRNA Not sensitive to aamanitin RNA polymerase IIMakes mRNA and most of snRNAs not U6 Very sensitive to aamanitin RNA polymerase IIIMakes tRNA and 5S rRNA U6 Somewhat sensitive to aamanitin Pol I Transcription rRNA genes Has high GC contents 60 Repetitive10020000 copies Found in nucleolus Class I Promoters Variable from species to species Two promoter elements Core element between 45 and 20 binds SL1 Upstream Control Element UCE between 156 and 107 binds UBF UCE binding factor Exp Identify the promoter elements by mutation analysis deletion linker scanning etc Class I Transcription Factors TFsL Promoter UCE Core UBF Upstream Binding Factor Binds to UCE Stimulates transcription Not speciesspecific SL1 TBP 1 TAFs 3 TBP TATA binding protein TAF TBP associated protein Binds to the Core element Can direct RNA Pol Ito initiate correctly Speciesspecific Plays a role in assembling the P01 I preinitiation complex Required for transcription UBF SL1 Pol I Tnx Pol III Transcription For tRNA SS rRNA U6 snRNA and other small RNA genes Class III Promoters Internal promoters The entire 539 from 1 can be removed without affecting the full promoter activity The 539 of the gene itself up to 50 can be removed without affecting the full promoter activity Class III TFs TFIIIA Required for SS rRNA transcription not for tRNA transcription ZincFinger Transcription factor TFIIIB Required for Pol III genes Binds near the transcription initiation site aided by TFIIIC Contains TBP TAFIIIs TFIIICRequired for all PolII transcription Binds to Box N B Transcription 1 Initiation a RNA polymerase binds to promoter b Flrst few phosphodiester bonds Iorm 2 Elongation 8 Termination pp 0 Fig 314 Bacterial promoter m 35 box 10 box Transcription TTGACa TnmaT AACTG ATaTA l Unwound region Fig 68 E coli RNA polymerase re 913 o09 0 9 B 8 C C l3 o 0t 1 2 3 Fig 61 Transcription initiation in bacteria 1 Forming the mosey promoter Complex 2 Forming the open promoter complex 3 Incorporating the first few nucleotides 4 Promoter clearance Fig 611 Sigma factor is needed for promoter binding Cupyngm T y mo IvauuuLuuH or msp ay 100 g Holoenzyme U 1 E CU 9 lt1 10 z D x 39 8 a 0 Core g o C I m 20 40 60 H Fig 65 Specific domains in Sigma bind the 10 and 35 elements in bacterial promoters Dwight Iii 39i39i ie iiiieGreiiriiill Cempenieer Irie iPerrriieeieri required for repreduetieri er display i 2 3 4 HEN E more Depiitiee containing highiy CDHSEWEd regions of the ireneeriptiem initieiieri ieeter 870 I92 Celi Preeei Cambridge MA Fiepririied by permission Copyright The McGrawHill Companies Inc Permission required for reproduction or di ssss y HOOC 39 1 NH2 0 r U Q43 0 H R588 07 R084 07 T440 gto7 R96 0 T100 0 r39 m TATAAT 3 35 10 140 AMP incorporated anIiU1 mL Cupyngm y 07 D J O WWMW m mspray v32P ATP y 32P GTP 05 10 dug01 mL O 39CO 3 D O y32PNTP incorporated pmolim mL Fig 612 The RNA polymerase melts the region around the transcription start site Copyright g inn iepiuluuwiicrdispiay HSRS R s GA R s 7 Fig 619 Copyright mpvuuuLnuH or dispiay Ori in a Transcription g initiation produces short aborted transcripts XC BPB on 6 MER l9 The alpha subunit of RNA polymerase can bind upstream UP elements in strong promoters Fig 629 q PiOCH 0 quotp F o 0 DH OH Reagenll b D 0 HS NPOF UF L 03907 039 O o u OCH lt Nucleotides crosslink to the A x 0 4 OH H t 1 we NiProiPir reinfom J tiUTP V A U O H t O Tqu OCH U 0 RNA pol Beta subunit Copynght 9 The McGraWH 2 3 4 5 6 Figs 632 and 633 Snuvce Gvachev e at Sludwesonthemncuonatmpogre The RNADNA hybrid is gt8 nucleotides Copyngm r9 3 mo wulunuuuwmwuy 1 2 3 4 5 6 7 89 1o1112131415161718 Protein m DNA l M U position 2 3 5 6 7 1o 141 24 3 6 7 8 1o 13 18 I RNA 92 039 RNA 339 end 20 22 2223 1251 261 271 301341 44144 47 150quot51quot152 541 571 62 Fig 637 Model of the transition from closed RPc to open RPo promoter complex Copyngm N Inc WWW m u may Extended rTOetement F gweme DOW wewmg n3 DNase d t eam may 3 rec On 20 25 5 Source Murakaml et al Scrence 296 p 1288 2002 byme AAAS Fig 647 Model for Rho independent transcription termination in bacteria J a Hairpin begins to arm i m Hairpin arms an n estabues n 1 p00 i mane mwmm W m megm WWW mu a Fiho binds 12anscnpl at rho leading sue and pursues polymerase dependent transcription term i nation in bacteria E coli changes gene expression to utilize different nutrient sources Lactose used Glucose used Bacterial density cellsmL O 2 4 6 8 10 Time h The E coli lac operon a Na Iacmse vepressmn d Operaor rac lacZ IacV ram Promolsr mHNA Repressm monomer Tetramer m lactose deveplessicn Transcnpuon wrmT BGalacosxdase Permease Transacetyzise F1 g 73 The lac repressor dissociates from the operator sequence upon IPTG inducer binding Input DN A bou nd Cupynght The McGrawHIII Compames no 39 reproduciion or dIspIay I I 40 O IPTG quotI 30 20 10 IPTG fl A I I t I o 01 02 03 04 Repressoi grin L I r Fig 76 Figure 75a b mhywlnnm mmncnmt n Mmmummwuwtuuw Merodiploid wilh one wudtype gene and one a Mutant tepressur gene uquot l P 0 2 Yquot A Hepressar 39 I 0 2 w A N o repressol b Mutant aperaor tot l l 0 2 w A RawTessa I 1 F 0c 2 y N t t t lac ulnducts Results No lac products in absence cl lactose No lac producls KI39I absence 0 lactose Na lac producls In absence at lactose lacpmducts m absence ut Izmase Conclusinn Butn lac a perans lepressible39 mumth is sswe One lac operan nonrepresstb mutatton is cisdnmtnam Figure 750 d calm1mme muean ee m veneeewuuem ea mmummmu sy Mercaiplom with one WINtype gene and one o Mutanl repressor gene us lnducerV 2 W Fe 0 net n39mncle Results N lac pmducs m presence or absence uv actase ND ac prod ucs m presence or absence 0 ac ose m prunucls in absence 0 IacIcse Lac momma in absence 0 laclose ConI usian Bum ran spawns unlnducible mutalmn 5 215 and trans aminanl Ealh Vac opemns nonrepressxble mulzmn Is dommam negahve The lac operon is also regulated by glucose levels CumI gh if The 54quot233ru39m394 ill Curr 211 i a Int Warn 53 till Itqui39eIJ Ir lELllEdUL IlUquot disp39 y Activator binding site Promcmew Polymerase lac CAP binding site binding site Dperatnr lac l l i L1UBN3 UDI I quot l LOW Glucose gt High CAMP gt activation of lac operon thru CAP binding site CyclicAMP Figs 716 amp713 CAPCAMP binding creates DNA bending and activates lac operon transcription Copyright The McGrawHlli Companies Inc Permlssion required for reproduction or dispiay CAP CAMP dimer Figs 717 amp 719 The trp operon attenuator Cantrqu rrra artarmtrauma tut permrwr quotrm tarrwruuuctmr at War Low lryplopltan transcription at trp structural genes trpor P IrpL Header Attenuator mRNA f AUG start Leader peptrde Met rm rrr UM 39r trr Sar Hrgn tryptoptran attenuatron premature ermtnallon trpo P leLHeader Attenuator trpE mRNA w 4 AUG UGA Leader termtnatton start stop Leader peptide Ma r Rr r tr Met Lys Ata Ite Phe quotvat L eu39i y 39 t39y Trp Trp Arg Thr Set Sta p pppA llllltlr lAr Hr A t m H 39t39t7rH391t P IIHIY HF FlgS 728 Alternative We structures of the trp operon M AC attenuator RNA Termination uuuuuuu N CsuAAA G U c s c G u c A H A C HA G A n C 7 N0 termlnatlon 53 cu AA AGEL HAH A UUUUUUU i A Fig 730 Translation of the trp operon uORF regulates transcription termination awauam mame 5 Tryplcphan slarvahon m Tryptophan abundance Hibosome siaiis UUUUUUUU No iermmaion polymerase Conlmues UUUUUUUUgt V A Terminator hairpin R39gfgfgzgggfquot paymemesops Fig 732 MCDB 3500 Lecture 9 2802 Figures 621 537 530 630 642 643 645 650 151 Ed 526 531 532 620 621 27 647 648 649 654 Transcription Elongation By Core polymerase i subunit Nucleotide binding Phosphodiester bond formation subunit Binds to DNA Movement of the bubbleuses topoisomerases to relieve the strain Transcription Termination Occurs at the terminators RNA polymerase falls off Rho 9 Independent Terminators Inverted repeat A stretch of A on the template Exp Make mutations what kinds Rho m Dependent Terminators Rho 9 Factor D0es not affect transcription initiation Affects e10ngati0nresults in shorter transcripts Releases RNA transcripts from the DNA template Binds RNA Has an RNA helicase activity ATPase Alternate Splicing Determining factors 1 The intrinsic strength of the splice site sequence 2 Formation of more elaborate secondary RNA structures 3 Changes in the concentration of general splicing factors 4 Tissue stagespeci c transacting regulators Alternate 5 ends Alternate 3 ends Skip exon Alternate exon T0 splice or Not to splice 1 U15 splice site recognition example MER 2 required for meiotic recombination in yeast 2 U1 repression example DrosophilaP elements in somatic ce11the third intron is not removedgtTransposition Repressor protein is made in germ cellsthe third intron is removedgtTransposase is made The repressor for splicing is made in somatic cells 3 Alternate seconda structure example ribosomal protein L32 of yeast High amount of L32 in the cell blocks splicing of L32 transcripts by forming alternate secondary structure Alternate Splicing Choosing between two 1 5 Splice choice example S V40 virus large T antigen vs small I antigen 2 3 splice site repression example Drosophila Ira gene 3 3 splice site enhancer example Drosophila dsx double sex Chapter 11 General Transcription Factors in Eukaryotes Page 303311 Two important assays to examine proteinDNA interaction DNase I footprint analysis Gel mobility shift assay or Gel retardation assay D N A fragment Prutein incubate with protein Protein beam to spewi c DNA quttence lDigcst wnh DNase ill chnatum II UM tit Radioartive label l Divide sample Incubate without protein DigPsl with DNase itiiiijiit EIECU OIJI39IUI ESiS Aumrnciiugraphy Regier of DNA plolL Cli d by protein binding Wi i i1iiiiky D i i DNA mm xixmmme Divide sample Protein Incubate with Incubate without protein protein Protein lmunrl 0 DNA we mmmmm Electrophoresis Amoracl lugrnphy DNAeproiei n complex i Free DNA Class II preinitiation complex Six class II factors TFIIA TFIIB TFIID TFIIE TFIIF and TFIIH Preinitiation complex is assembled in a precise order D gt DA gt DAB gtDABPolF gt DABPolFEH CoyvyhmV 39IwUccmwuHCQ LL u 9 E 5 as ea 2 lt5 o Ip lt5 9 lt lt D k a lt mmew mmamoxu D I D lt o o I I I 0 DABPOIF gt M DBPoIF gt quot t H DAB nun u WW I up n 123456789101j1213141516 H aw m McGIawHIImethe Irv um w qu m n WWW w W DBPolF EH w 8 m m o I I l l papaquot gt u u 1 DBPoIFEH 39 DBPOIFE TFIID TFIID binds to TATA box TFIID is composed of TATAboxbinding protein TBP and 810 TBPasscociated factors TAFs TBP is a highly conserved protein from yeast to humans The Cterminal 180 amino acid of TBP is the TATA binding domain TBP not only functions With Pol II promoters With TATA box but also With TATAless pol II promoters Surprisingly TBP also works with Pol I and Pol III w NGmwnm Comm m m mm m mmamu WW DNase DNas e V V 5 s 7 a 5 s 7 B Nomanplate DNA swam Templaze DNA grand Vlsda ed complexes Isolated oomp exesw TBP is a universal eukaryotic transcription factor that operates at all promoters regardless of their TATA content or the nature of polymerase P65 8 1143 gtN Figure 1111 1 D 1 40 123456 Poll Pol II F Pol III Pol III 24 37D 24037D 24 13 2 WT I143N P65 3 Schuhzelm u an m y The TBPAssociated Proteins TAFs a I M we Flgure 23L 3 JTAFHZSO 11 2 TAF 150 116 TAFHHO 957 wlt TAFHBO 6 TAF 60 TBP 43 TAFH4O 31 N mason TAFHSDu 1 2 cm a al Assemb y av recombmam mm reveals umevenua mommy veqwemenls my mum Iranscnphonal munstars Del79 7 om tee p mo 4 7a Rammed by permwss cn m asewev Smence TAFs from three different species Figure 1113 09 3132 20 TAFHS 30 What are the roles of TAFs AdML H 70 5 l 51539 mm L p TATA lnr D E1B E4 AdML HSp7O Q Q Q Q 3 4 873 4 r and M 12 34 56 7 8 E1B E4Igt TATA l F AdML Hsp70 I TATA lnr D TAFIIs help TBP with transcirption from promoters with initiators and downstream elemens Chapter 11 General Transcription Factors in Eukaryotes Class I factors u mm Cce Upstream control element Cm element Upstream promoter element Upstream bind ing factor I39UBF SL1 SL1 Direct transcription from the rRNA promoter in combination with Poll SL1 does not bind promoter DNA by itself SL1 is speciespeci c Mouse extract Figure 1139 Mouse rRNA promoter UCE Cars Human rRNA promoter UCE Core Human SL1 Learned e a Ma mam 5 1935 13551 e Amencan Soc ety my Microbiology Upstreambinding factor UBF UBF is composed of 97kD and 94 kD proteins 97kD protein is suf cient for binding to Core element and Upstream control element UCE UBF stimulates transcription of the rRNA gene in the presence or absence of W drtypa u UCE Core What is SL1 HOW does TBP t in Structure and Function of SL1 SL1 is composed of TBP and three TAFs TAF1110 TAFI63 and TAFI48 Heparinagarose Elysero gradient sm UBF PoH was m m r m gt m TBP prolem arbllrary uan Prolein concentration mgmL PM so 25 2b 15 IO 5 u so Fm 35 32 25 24 20 1s 2 a 4 I lt SL1achny Smac vi y Fr 1 W q t 33163 6 gt9 r 4 TBP 1 2 3 4 5 6 7 E TEPprolem F 123456789101112 Igure 1145 ComaxelaThe CEHGEKG Mar 99219 was 2m Immunodepletion of SL1 prevent rRNA transcription TBP alone cannot restore SL1 function NXT aTBP SL1 1 2 3 4 5 6 QTBP bTBP MOCK i4 SUPPpL W 1 3 W Figure 1146 Three TAFs TBP associated factors are important for the SL1 activity Figure 1147 14 quots kD 4 0 A 97 56 45 TAF110 TAF 63 lgG TAF as TBP TBP uses completely different sets of TAFs for RNA pol I and RNA pol 11 Two sets of TAFs compete for binding to the TBP Class III Factors 39 Intermediate Flgure 1036 99mm SS rRNA Box A Box 0 tRNA or VA RNA Box A Box B Three factors TFIIIA TFIIIB and TFIIIC are important for transcription of Ss rRNA gene TFIIIA is not required for transcription of tRNA genes TFIIIB and TFIIIC are important for all Pol III genes M Nonspeci c Ab 9quot TFIIIA Ab Figureagt 1 1 1 48 SSIHNA 3 A anC SSrRNA RNAuv 1 VA RNA p preIRNA RNA N at ax PNAS7EMar19mp w am Frog Oocyte extract TFIIIA is a zinc nger DNA binding protein What are the roles of TFIIIB and TFIIIC 39N in reSASy i Y dm quot TFIIIC helps TFIIIB bind to the promoter 3 b C quot After that TFIIIB by itself binds to the promoter tightly without TFIIIC Factors C C C B Bheparin TFIIIC can bind internal elements Box A and Box B TFIIIB can not bind DNA by itself but can do so in the presence of TFIIIC binds to the upstream region right near the transcription start Once it binds to DNA TFIIIB can stay bound in the absence of TFIIIC Promoter bound TFIIIB can initiate multiple rounds of transcription The order of binding of TFIII factors to the Pol III gene is important for the transcription TFIIIB is composed of TBP and TAFs TAF 172 and TAF L Figure 1152 BOX A BOX 8 TFIIIC is a large protein complex with siX subunits It has a dumbbell shape with two globular domains connected by a stretchable linker tRNA or VA RNA Copyright e W wpiuuuwuu or display 80 n A A 20 itquot I i 10 H 39 50 nm 0 Source Schultz at a EMEO Journal 8 p 3817 07 l989 J Three unifying principle for preinitiation complex formation 1 It always starts With an assembly factor that recognizes a speci c binding site in the promoter TATAcontaining class II promoters TBP TATAless class II promoters TAFs or upstream element binding factors such as Spl Class I promoter UBF Class III promterTFIIIC TFIIIA 2 TBP is always involved in the assembly process playing an organizing role in preinitiation compleX formation 3 The speci city of TBP is governed by its associated factors TAFs Figure 115 E Q DAB PolF TATAcontaining promoters Class I rRNA Class II 6 General TATAless promoters factors GC boxes Initiator Class III VA 1 Lecture 20 31302 MCDB 3500 Figures 1810 18271835 1St Ed 1891827 1834 Polypeptides Made from Amino Carboxyl direction Exp Howard Dintzis 1961 mRNA Read from 5 9 3 Exp synthetic mRNA translation The triplet code Exp synthetic mRNA translation 64 codons Degenerate Wobble hypothesis Translational Elongation Ribosome P peptidyl siteoccupied by Vlet tRNA 1st A amonacyl site occupied by incoming aminoacyl tRNA 1 EFTu and GTPbrings aminacyltRNA onto the A site Proofreading Happens when incorrect codon anticodon base pair occurs 2 Peptidyl transferase ribosome transfers aa from P to A By 23S rRNA in SOS particle 3 Empty tRNA on the P site leaves Via E site 4 Prokaryotic EFG Eukaryotic EF2 and GTP Translocates mRNA so that A site is available GTP is used to release EFG from the ribosome Structures of EF Tu and EFG Molecular mimicry Translational Termination Termination codons are recognized by the Release Factors RFs RFl UAA UAG RFZ UAA UGA RF3 ribosomedependent GTPase binds GTP and helps the other RFs bind to the ribosome eRF recognize all three stop codons GTP dependent Molecular mimicry