BIOL 405 Final Exam

Transcription factors, RNAP II. experiment to know order of binding

*EMSA* look at how much each migrates = binding order CONCLUSION - *TFIID binds first to promoter* - TFIIA is not crucial in vitro

assaying binding between the lac operator and the lac repressor

*FILTER BINDING ASSAY* - only labeled DNA bound to repressor = attaches to nitrocellulose - IPTG = gratuitous inducer RESULTS: - no IPTG = more DNA bound to repressor - IPTG present = low lacI bound to DNA

proximal/upstream promoter elements and enhancers

*GC box* is *orientation-independent* but *position-dependent* - if GC box is moved > a few dozen bp away from TATA, it can't stimulate transcription

how to identify a coactivator experiment

AFFINITY CHROMATOGRAPHY - bead-DNA 1. add Sp1 (TF) to column 2. load other proteins 3. see what is retained RESULTS: - only *TAF4* was retained CONCLUSION: - *TAF4* physically interacts w/ Sp1 = likely a coactivator of Sp1

1st model of positive regulation of the lac operon: CAP and RNAP bind to promoter cooperatively evidence

CAP and RNAP bind cooperatively @ RNAP's *a-CTD*, hence promote formation of closed promoter complex six lines of evidence: 1. *cosedimentation* of the two protein complexes in *ultracentrifugation* 2. *chemical cross-linking* when bound to cis-acting elements (operator + activator-binding site) 3. genetic study: footprinting + genetic data - *footprinting*: identified *CAP binding site AR1* - lac doesn't have *-35* or *UP* thus it needs CAP for high levels of transcription 4. CAP mutations decrease RNAP activation 5. deleting aCTD of RNAP = prevents activation - *run-off transcription* - a-CTD necessary for CAP-cAMP stimulation 6. XR crystallography - *AR1* of CAP (binding site) and *aCTD* do NOT touch each other

TFIID

CONTAINS TBP subunit in EMSA experiment: - no complex formed in absence of A or B - shows D can only bind in presence of A or B

experiment: how we know TFIIH phosphorylates the CTD of RNAPII

Mobility shift assay SDS PAGE experiment: - (b) chymotrypsin: cleaves peptides @ C side of Tyr, trp, phe - pol IIB lacks CTD - pol IIA has CTD - pol IIO is already pi'd but other regions can be pi'd - add complex w/ TFIIH to pi stuff RESULTS: - polIIB not pi'd bc no CTD CONCLUSION: - TFIIH pi's the CTD of RNAPII

Genetic analysis of Oc lac operator vs WT operator vs no operator

RESULTS: - Oc lac operator binds repressor w/ lower affinity than the WT operator (b/c its a mutant) - WT = full repressor binding - no operator = (-) ctrl, basically no repressor binding

eukaryotic RNA polymerases: location + what they synthesize

RNAP I - nucleolus - *pre-rRNAs* --> 28S, 5.8S, 18S rRNAs RNAP II - nucleoplasm - *mRNAs*, *snRNAs* (intron splicing), *miRNA* RNAP III - nucleoplasm - *tRNAs*, *5S rRNA*, + small stale RNAs (e.g. RNA of signal recognition particle for protein export to ER)

footprinting of the DABPolF complex

RNAP II is a very big complex predict: should cover a larger area than complex w/o enzyme result: - complex has larger footprint than DAB complex alone

nucleolus

RNAPs (RNAP I) in nucleolus make *rRNA* - higher CG content, repetitive

which region in the promoter determines the transcription start site? experiment

*S1 Mapping* Cell transfection w/ *SV40 DNA virus* 1. transfect human cells various things - SV40: lane 1 - plasmid w/ SV40 early gene: lane 2 - derivatives of pSV1: lane 3 and 4-9 2. isolated RNA after transfection and incubation 3. S1 mapping RESULTS: - derivates = mutants that had deletions downstream of TATA - more deletions just shifted transcription start down - H2 = deletion in TATA box = transcription starts wherever CONCLUSION: - TATA box is important for locating start of transcription - deleting TATA box made improper transcription start sites

high lactose, no glucose

*negative control* of lac operon - lactose high, glucose close to 0 - operon needs to be turned on quick via *removing lacI repressor* - need to transcribe lac genes @ high level

high lactose, high glucose

*positive control* of the lac operon *cAMP-dependent* - cAMP works w/ *CAP* - CAP is encoded by *crp* glucose inhibits *adenylate cyclase* which converts ATP --> cAMP

exceptions to the universality of TAFs and TBP

- TAFs are not universally required for transcription of *class II* genes - Even *TBP* is not universally required, some promoters have alternate proteins in diff spp. such as TRF1 (TBP-related factor 1) - TBP-free TAFII-containing complex (TFTC) can help form pre-initiation complex w/ TFIID or TBP - general TF *NC2* = stims DPE-containing promoters, represses TATA-containing promoters

structure of the TBP-TATA box complex

- TBP (of TFIID) binds to MINOR groove + bends DNA 80 degrees - complex = saddle shaped - forces minor groove open

coactivators

- a protein that increases gene expression by binding to an *activator (transcription factor)* which contains a DNA binding domain - coactivator by itself cannot bind DNA - TAFs can be coactivators

characteristics of plasmids

- abx resistance = promotes uptake - MCS = digestion site for use in cloning i.e. MCS often w/i lacZ gene

regulation of the formation of open promoter complexes

- high initiating *[NTP]* = STABILIZES OPC - cells starved = uncharged tRNA @ A site - destabilizes OPC > *RelA* = senses alarm + makes *alarmone* = *pppGpp* + *ppGpp*

anion exchange chromatography

- positive bead: *DEAE* 1. + and - charged proteins put thru column 2. (-) charged proteins bind DEAE 3. can elute (-) w/ increasing amts of buffer (SO4-) - buffer competes w/ (-) protein for DEAE binding fraction 1: will be (+) proteins fraction 2: will be (-) proteins

generic class II promoter

1. *core promoter* aka minimal promoter - basal transcription 2. *UPE* aka proximal promoter element - up to 250 bp upstream of start

which TAFIIs are responsible for recognizing the initiator and DPE? experiment

1. *photocrosslinking experiment* - problems bc this experiment works well only for proteins that bind MAJOR groove - TBP binds MINOR groove 2. *footprinting experiment* - TBP/TAF1/TAF2 and TBP alone had a footprint over TATA CONCLUSION - TBP/TAF1/TAF2 complex = recognize initiator and DPE the best, and also recognize TATA

binding order of transcription factors to RNAP II

1. TFIID, TFIIA, TFIIB bind together = DAB complex 2. TFIIF binds RNAP II 3. RNAP II-TFIIF complex binds DAB complex 4. TFIIE and TFIIH join - DAB complex and RNAP II-F must be formed before DABPol-F complex can be formed - TFIID = essential for rest of protein binding

role of TAFIIs

1. facilitate binding of TBP to promoter 2. transcription stimulation provided by *activators* - in this case, TAFIIs are called *COACTIVATORS*

summary of glucose vs lactose in the lac operon

1. glucose, no lactose - "*gas off, brake on*" - low cAMP - repressor bound - no lac mRNA 2. glucose, lactose - "*gas off, brake off*" - low cAMP - repressor not bound - small amt of lac mRNA 3. no glucose, lactose - "*gas on, brake off*" - high cAMP - repressor not bound - a lot of lac mRNA 4. no glucose, no lactose - high cAMP - repressor bound - no lac mRNA

effect of a-amanitin on *small RNA* synthesis

1. label RNA w/ H3 + treat w/ diff [a-amanitin] 2. centrifugation = small RNAs in supernatant 3. PAGE 4. slice gel 5. count H3 signal on scintillation counter RESULTS: - inhibition of small RNAs by a-amanitin mirrors inhibition of *RNAP III* CONCLUSION - RNAP III likely synthesizes small RNAs (5S rRNA + 4S tRNA)

immunoprecipitation

1. label proteins by growing cells w/ labeled amino acids e.g. S35-methionine 2. precipitate a certain protein using *antibodies* coated on beads 3. electrophoresis 4. autoradiography more label = more protein accumulation

2 proposed mechanisms for the repression of the lac operon by lacI

1. lacI prevents *RNAP binding* to promoter - deems RNAP vs lacI binding as mutually exclusive - i.e. LacI prevent formation of OPC 2. lacI inhibits *transition from abortive transcription* to *processive transcription* - RNAP and lacI can bind at the same time - i.e. lacI prevents promoter clearance

how we know TFIID binds to MINOR groove of TATA box

1. methylation of *adenine* interferes w/ TFIID binding to promoter (b/c methylation is @ minor groove side) 2. *EMSA*: yeast TBP is smaller than human TBP, but does not change its ability to bind to human TATA vs yeast CICI

Lac operon: two constituitive "on" mutants

1. mutations in repressor gene *lacI* - constitutively on b/c lacI will not be made - will not bind to operator 2. mutations in *operator* - lacI cannot bind to operator - will make products in the absence of lactose

Coactivator experiment

1. quaternary complex w/ TAF4 can help activation by Sp1 2. TAF2 and TAF6 = coactivators of *GAL4-NTF1* = synthetic transcriptional activator - w/o correct coactivator, TBP-TAF1 cannot activate transcription RESULTS: - *TAF1* always present = serves as assembly factor to attract other molecules

evidence for 1st mechanism of lacI repression

1st mechanism: RNAP vs lacI binding = mutually exclusive *FOOTPRINTING* - RNAP = -48 to +5 - lacI = -3 to +5 CONCLUSION: - b/c the sites they bind to overlap, binding should be mutually exclusive

evidence for contradicting 1st model of lacI repression

1st model: mutually exclusive model EXPERIMENT: 1. incubate RNAP + DNA w/ lac operator in presence of lacI 2. add *IPTG* and *rifampicin* - rifampicin will block formation of first phosphodiester bond in RNA synthesis - i.e. will not inhibit transcription if OPC formed RESULTS: - observed *Rifampicin-resistant transcription* CONCLUSION: - RNAP + LacI can bind at the same time - OPC forms even w/ RNAP + lacI = *contradicts 1st model* - hypothesis: LacI *blocks promoter clearance*

lac repressor does not appear to block formation of the OPC experiment

RUN OFF TRANSCRIPTION ASSAY 1. cut cloned DNA w/ REs 2. add lacI + incubate 10 mins 3. add RNAP + incubate 20 mins = *allow OPC* to form 4. add *heparin* = blocks new initiation - add ATP+UTP+GTP + buffer 5. add *32-P CTP* with or without *IPTG* 6. allow 10 mins for elongation 7. run a gel + expose to XR CONCLUSION: - OPC was formed even in the presence of lacI - thus lacI does NOT prevent formation of OPC (contrary to 1st model) - *lacI* blocks transition from *initial transcribing complex* to *elongation state* = PREVENTS PROMOTER CLEARANCE - supports 2nd model

S1 mapping

S1 mapping to determine *start site* of transcription if you *know the sequence* already EXPERIMENT: 1. cut w/ 2 RE's (BamHI and SalI) 2. treat w/ *alkaline phosphatase* (removes Pi) and *polynucleotide kinase* --> Pi added to both side 3. cut with SalI again (site is towards end) = only one end 5' labeled 4. denature = one DNA strand 5. hybridize to transcript 6. *S1 nuclease* 7. denature + electrophorese AUTORADIOGRAPH - will have have DNA fragments of known length for comparison - length of transcript will indicate how far from the probe the transcription start site is e.g. if the marker is 350nt long, start site is 350 bp upstream of BAMHI site

TAFIIs experiment

TAFIIs = TBP-associated factors experiment: 1. immunoprecipitation w/ abs against TBP 2. urea to strip TAFs from TBP ppt 3. SDS-PAGE of TAFs results: found 8 TAFs associated w/ TBP

archaea TBP-like protein

TATA boxes in promoter regions of archaeal genes - TFIIB-like protein found in archaea

difference of TFIID and TBP in supporting transcription

TBP = part of TFIID - TFIID better than TBP alone for certain types of promoters - some promoters = TAFs are dispensable for in vitro transcription

TATA-less promoter with GC boxes

TFIID CANNOT bind to TATA alone - *BINDS* other components - *Sp1* bind to *GC* box and will facilitate binding of TFIID - *TAF1,2,4* will anchor TBP to *Sp1*

TATA-less promoter w/ Inr and DPE

TFIID CANNOT bind to TATA alone - it will *BIND* other components - then will bind to TATA box - facilitated by *TAF1 and TAF2* helping bind TBP to *Inr* and *DPE*

TATA-containing promoter

TFIID can bind alone to the TATA box then *RECRUIT* the rest of the components

DNA footprinting

Technique for determining the DNA sequence to which a DNA-binding protein binds if the more negative number is on top, that indicates that the 5' end TEMPLATE STRAND was labeled indicating that is a TEMPLATE STRAND if the more negative number is on the bottom, indicates that the 5' end NONTEMPLATE was labeled, indicating this is a NON-TEMPLATE STRAND

mechanism of cAMP stimulation of the lac operon

experiment: - mix RNAP + lac promoter + w/ or w/o CAP-cAMP - add nts along w/ *rifampicin* > rifampicin blocks initiation > also blocks weak promoter/RNAP interaction results: - no CAP-cAMP: rifampicin can act before pol can initiate - CAP-cAMP present: *OPC forms* immediately, nts reached pol site before rifampicin = resistant to rifampicin until it reinitiates CONCLUSION - *CAP-cAMP appears to allow formation of OPC* - however this result could be like how we saw with the sigma factor seemingly stimulating both initiation and elongation - CAP-cAMP could promote CPC and thus stimulate more OPC?

experiment: which operator is most important to lac system?

experiment: 1. portion of lac operon w/ different combinations of operators deleted (1,2, or all 3) 2. ligate that to *phage lambda* 3. lysogenize E. coli + integrate 4. measure *B-galactosidase* levels *with IPTG and w/o) - ratio of w/ vs w/o = fold change RESULTS: - WT: 1300 - no O2: 440 - no O3: 700 - *no O1: 1.9* CONCLUSION: - *Operator 1* = most important to lac system

2nd model of positive regulation of the lac operon: CAP-cAMP inhibits RNAP binding to the less efficient promoter evidence

footprinting experiment - AR1 (binding site for CAP-cAMP) = -87 to -49 - RNAP = -48 to +5 RESULTS: do not overlap = *Cooperative binding*

Footprinting of the DA and DAB complexes

footprinting with D, DA, and DAB components: OP-Cu+2 (gives a finer footprint) DNase RESULTS: - areas w/ DA, DAB more protected from DNase and OP-Cu+2 (makes OH radicals) CONCLUSION: DAB complex binds to TATA box

RNAP sensitivity to alpha-amanitin

graph: [a-amanitin] vs % maximal activity RESULTS: - RNAP I = not sensitive - RNAP III = sensitive - RNAP II = most sensitive > needs least amount to get 50% inhibition

structure and function of TFIID

has several subunits - *TBP* = TATA-box binding protein > highly conserved > binds to MINOR groove of TATA - TBP bends DNA 80 degrees

TAF1 importance + activities

important bc of its activities 1. *histone acetyltransferase (HAT)*: attaches to acetyl to lysine in histones - chromatin remodeling - *activates* transcription 2. *protein kinase*: Pi's itself, TFIIF,A,E - modulates *efficiency of assembly* of preinitiation complex

run off transcription assay: lac promoter

in vitro, demonstrates that RNAP forms lac OPC even in presence of lacI 1. cut cloned gene with REs e.g. *SmaI* 2. transcription w/ labeled NTPs 3. RNAP runs off template = no termination 4. electrophorese run off RNA to determine size experiment: done w/ lac promoter in presence of lacI CONCLUSION: - RNAP formed OPC even in presence of lacI

silencers

inhibit transcription by interacting w/ *transcription repressors* - cause chromatin to *condense* - inaccessible for transcription sometimes same DNA element can act as either an enhancer or silencer: - *TRE*: silencer when bound by thyroid receptor protein - functions as enhancer when bound by thyroid receptor bound to thyroid hormone (ligand)

creating a fusion protein

insertions of DNA need to be *IN FRAME* e.g. GST-sigma4.2 GST DNA + sigma DNA need to be ligated in frame (sigma in frame relative to GST not the lac ORF) NOTE: if DNA fragment is *small* <450 bp is inserted IN FRAME, then the colony can still be BLUE b/c B-galactosidase can still be in frame

CAP-cAMP binding to DNA

it bends DNA see figure 7.18 - hypothetical: bent DNA has least electrophoretic mobility - actual: CAP-cAMP-DNA has least mobility CONCLUSION: - CAP-cAMP bends DNA

how does a promoter know which pol to recruit?

it is encoded in the promoter via - core promoter element - UPE other elements: - *TFBII recognition element: (G/C)(G/C)(G/A)CGCC* - DCE: downstream core element, three parts (CTTC, CTGT, AGC) overlaps w/ MTE - MTE: motif 10 element - DPE - TATA box - Inr not every promoter has TATA not every promoter has Inr some have both, some have one - Inr can couple w/ TATA, GC or DCE

lac operon summary

lac operon is controlled by: *2 sugar sensors* - lacI - glucose (indirectly via regulating adenylate cyclase activity) *two TFs* - lacI - CAP *three cis-acting elements* - AR1 - RNAP binding site - operator *two small molecules* - allolactose - cAMP

lacI operator interaction

lacI is a *Tetramer* - binds to *MAJOR GROOVE* of DNA at four half sites - makes DNA *loop* of about 400 bp - lacI binds at lower affinity sites (other DNA than operator) when inducer is present = less lac operators bound - repressor never leaves DNA: just binds tight vs loose sites

allolactose

lactose (B-1,4 linkage) cleaved by B-galactosidase to make *Allolactose* (B-1,6 linkage)

TFIIH

last TF to join pre-initiation complex 2 functions: - *kinase*: *pi's CTD* of RNAPII - *helicase*: unwind DNA @ start site to make transcription bubble

ampicillin resistance, lac, and DNA insertion into vector

media will have ampicillin - selects for transformed cells (that have plasmid w/ Ampr) media has IPTG - gratuitous inducer of lac operon RESULTS - non-successful transfections killed by Amp - successful transfections = more likely to be white (b/c more out of frame insertions) - some colonies = blue despite successful transfection due to *in-frame insertions* = will make functional B-galactosidase

fluorography

modified version of autoradiography - done w/ *32P Beta electrons* bc they have high energy - electrons emitted by 32P are stronger than H3 ones 1. soak gel in solution w/ FLUOR: compound that fluoresces when hit by 32P B e- 2. light exposes XR film

discovery of the lac operon using Western blot

monitor protein level of *B-galactosidase* Results: - high lactose = high B-galactosidase - correlation btw lvl of B-galactosidase and bacterial density

experiment: deleting TATA box

overall: abolishes transcription from both the OG start site and aberrant start site techniques: linker scanning + primer extension - *Tk* (thymidine kinase) gene 1. make DNA w/ diff deletions using linker scanning 2. inject DNA into frog oocytes + purify mRNA 3. primer extension RESULTS: - changing TATA box abolished transcription from the mutated promoter

subcellular localization of the three RNA polymerases

rat liver ANION EXCHANGE CHROMATOGRAPHY 1. extract protein from nuclei 2. load on DEAE column 3. wash + elute (-) proteins w/ buffer 4. in vivo transcription RESULTS: - graph of fraction # vs UMP incorporation (activity = RNA produced) > blue line = ammonium sulfate (buffer) > green = protein [ ] > red = amt of RNA from in vitro transcription, labeled w/ H3 - nucleoplasmic = one RNAP II peak - nucleolar = one RNAP I peak CONCLUSION - RNAP I = nucleolar - RNAP II and III = nucleoplasmic

separation of eukaryotic RNA polymerases

sea urchin embryo extracts + *ANION EXCHANGE CHROMATOGRAPHY* w/ DEAE 1. extract protein from nuclei 2. load on DEAE column 3. wash + elute (-) proteins w/ buffer 4. in vivo transcription RESULTS: - graph of fraction # vs UMP incorporation (activity = RNA produced) > blue line = ammonium sulfate (buffer) > green = protein [ ] > red = amt of RNA from in vitro transcription, labeled w/ H3 - 3 distinct red peaks - second peak = most activity (RNAP II) CONCLUSION: - where the names for the RNAPs come from

TFIIA

stabilizes binding of *TBP* (TATA-box binding protein) to *TFIIB* - not crucial in vitro TBP + TFIIB = increase binding affinity of TBP to promoter has 9 Zinc finger domains

Primer extension

technique to determine transcription start site 1. do transcription (make RNA) 2. hybridize RNA to a labeled primer 3. extend primer w/ *REVERSE TRANSCRIPTION* 4. denature hybrid + electrophorese can also do *dideoxy chain termination sequencing method* w/ same primer but template for DNA sequencing is the *non-template strand* for transcription

linker scanning

technique to systematically remove portions of a promoter then add a linker to see what deletions have what effect 1. cut + digest ends 2. ligate linker 3. repeat for different areas

experiment: enhancer is orientation independent and position independent

techniques 1. transfection 2. immunoprecipitation 3. PAGE 4. fluorography GRAPH: - lane 1: ctrl, non-transfected cells - lane 2: wt fragment - lane 3-4: deleted enhancer - lane 5-12: changing position and orientation of enhancer - M: protein marker RESULTS: - changing position + orientation of enhancer had no effect on transcription CONCLUSION: enhancer = orientation and position INDEPENDENT

effects of mutations in TBP on transcription by all 3 RNAPs

techniques: - S1 mapping RESULTS: - mutations in TBP affected all 3 RNAPs ability for transcription

effect of TBP and TFIID on different promoters experiment

techniques: - in vitro transcription - primer extension to quantify RNA products experiment: - TBP or TFIID with 4 diff promoters 1. in vitro transcription --> RNA 2. convert to cDNA w/ RTase note: signal is DNA that reflects RNA products in sample CONCLUSION: - TFIID does better when promoter has other elements like *DPE and Inr*

experiment: deletion of enhancers *within* an intron

techniques: immunoprecipitation (protein) fluorography northern blot (RNA) 1. delete enhancers 2. immunoprecipitate proteins with antibodies 3. soak gel in fluor 4. expose to XR film 2. also do northern blot to analyze RNA RESULT: - deleting enhancers decreased expression

Gel Mobility Shift Assay (EMSA)

tells does protein bind to DNA? e.g. - DNA bound to two proteins = moves less - DNA not bound moves most

graph of lactose operon

time vs bacterial density *DIAUXIC* - "help" meaning there is a delay in the curve - first part of curve is glucose being used - pause = switching gene expression to metabolize lactose - second part of curve = lactose is metabolized

transcription and translation in eukaryotes

transcription - occurs in nucleus - capping enzyme - intron splicing - poly A addition translation - bigger ribosome complex - multiple RNAPs for diff things - NO f-Met-tRNAfMet (only proks)

experiment: how we know TFIIH Pi's RNAPII

two experiments: 1. EMSA: - DNA is radiolabeled - PolIIO = pi'd form - PolIIA = unpi'd form RESULT: complex w/ TFIIH made pi'd RNAP - this could be the result of the complex, not just TFIIH alone 2. *mobility shift assay* (not EMSA) - gamma 32P added directly, DNA not radiolabeled - checks *pi of proteins NOT DNA* RESULT: TFIIH alone is sufficient to pi conclusion: - TFIIH Pi's RNAPII - TFIIE stimulates pi activity of TFIIH

lac operon: mutant repressor gene (I-d)

use merodiploid (partial diploid) has one WT copy, one mutant copy RESULTS: - lac products in both copies in absence of lactose - lacI cannot bind to operator CONCLUSION: - *dominant negative* mutation - negative bc cannot bind - *NONREPRESSIBLE*

lac operon: mutant repressor gene (Is)

use merodiploid (partial diploid) has one WT copy, one mutant copy RESULTS: - one normal copy, one mut copy = tetramer of lacI that cannot bind allolactose - no lac products in either - lacI can still bind CONCLUSION - mutation is *Cis and trans dominant* b/c it affects both copies - this is because the lac repressor cannot be derepressed - *UNINDUCIBLE*

lac operon: mutant operator (Oc)

use merodiploid (partial diploid) has one WT copy, one mutant copy RESULTS: - only the mutant was nonrepressible - lac products formed from mutant CONCLUSION - mutation is *cis-dominant* = only affects same piece of DNA that its on

lac operon: mutant repressor gene (I-)

use merodiploid (partial diploid) has one WT copy, one non-functional mutant copy RESULTS: - mutation repressed BOTH lac operons - both = no lac products in absence of lactose CONCLUSION - mutation is *recessive* - b/c one lacI is functional = enough to repress both operons

models of positive regulation of the lac promoter

via *CAP-cAMP* 1. CAP-cAMP and RNAP bind cooperatively to the promoter 2. CAP-cAMP inhibits RNAP binding to the less efficient promoter = more RNAP available for transcription of the more efficient promoter

beta galactosidic bond

bond btw galactose and glucose = lactose beta galactosidase breaks that bond

number of operators in the lac system

3 - 1 classical - 2 auxiliary - classical + 1 auxiliary are near transcription start - high conserved seq across spp in all 3

structure of the lac operon

3 genes that each have their own *SHINE DALGARNO* = 3 SD seqs - 3 translation events, NOT one protein cut into 3 - B-galactosidase - permease - transacetylase induced by allolactose - binds to lacI = *NOT* part of lac operon b/c it is not under the control of the lac promoter

TFIIH subunits

9 total kinase activity: four subunits, two of them are the active site helicase activity: five subunits, two have distinct helicase activities

effect of cryptic mutant (lacY-) on accumulation of galactoside

Y- = cryptic mutant b/c it behaves like WT in a lysed cell - Y = permease, makes sense bc dont need permease to get lactose in cell if cell is lysed EXPERIMENT: - Looked at WT (Z+Y+) and mutant (Z+Y-) - treated w/ *radioactive galactoside* to induce and looked @ accumulation of galactoside RESULTS: - WT not induced = no galactoside in cell - WT induced = galactoside - *MUT not induced or induced = no galactoside*

Immunoblotting

aka Western Blot 1. SDS-PAGE 2. transfer to polymer sheet 3. antibody exposure (can be direct or primary and secondary abs) 4. expose film 3 = direct or two abs - two abs = cheaper - secondary abs w/ alkaline phosphatase or horseradish peroxidase

DNA Constructs

aka molecular cloning used for many things: - transfection or agrobacterium-mediated transformation - making fusion proteins - sequencing DNA fragments - mutagenesis of DNA sequences - amplification of DNA fragments

lacI and allolactose interaction

allolactose binds lacI = conformational change in lacI result: DEREPRESSION

autoradiography vs fluorography

auto - *DIRECT* exposure of film by B particles or gamma rays fluoro - exposure of film by *SECONDARY* light that was generated by exciting a fluor or a screen by B particles or gamma rays

TFIIB

binds to TBP - increasing binding affinity of TBP to the promoter

experimental evidence for positive control of the lac operon (3)

cAMP-CAP mediated control (1) *KD* - WT CAP had a lower KD than mutant CAP - indicates that mutant CAP binds cAMP less tightly (2) *graph of [cAMP] vs B-galactosidase activity* - mutant CAP (cant bind cAMP) stimulates a lot less B-galactosidase than WT CAP - even w/ increasing [cAMP] (3) *rescue effect* - B-galactosidase synthesis decreased w/ mutant CAP shown to be rescued by an addition of WT CAP

photo-cross-linking

can cross link proteins and DNA to see what interacts 1. incorporate *Br-dU* into 32P labeled promoter 2. allow TFIID to bind to promoter 3. irradiate complex w/ UV light + wash 4. digest DNA w/ nuclease to release labeled protein-DNA complex 5. SDS-PAGE

blue-white screening

components: - cloning vector (plasmid) - synthetic inducer - substrate for color selection

new evidence to support 1st model of lacI repression

effect of lac repressor on dissociation of RNAP from lac promoter-operator region experiment: - time vs fluorescence of *pppU* - 4 conditions: > + repressor > + heparin > no DNA (- ctrl) > no additions to DNA (+ ctrl) results: - fluorescence intensity of heparin vs repressor were similar - no inhibitor = reinitiation - heparin = no re-initiation - repressor = no re-initiation conclusions: - repressor functions similar to heparin - i.e. prevents RNAP reassociation w/ promoter

enhancers vs promoters

enhancers - work anywhere in the gene - orientation-independent promoters - position dependent - orientation dependent

TFIIE and TFIIH

enter the complex at the end - creates a larger complex

TBP not universally required experiment

exceptions to the universality of TAFs and TBP *TRF1* = TBP related factor-1 FOOTPRINTING drosophila gene w/ 2 distinct promoters 1. one w/ TATA box recog by TBP-complex 2. one w/ *TC* box recog by *TRF1* RESULTS: - promoter 1 was protected by TBP - promoter 2 only protected by complex w/ *TRF1*, not by TBP

general transcription factor NC2

exceptions to the universality of TAFs and TBP - NC2 blocks *TFIIA and TFIIB* from joining TBP-TATA complex - acts to repress transcription - is a *negative cofactor*

microarray experiment and TAF's necessity for transcription of class II genes

exceptions to the universality of TAFs and TBP MICROARRAY tells what genes are expressed 1. cDNA labeled + hybridized to DNA on microarray results: - TFIID not 100% necessary for all genes

some promoters can be stimulated by TFTC rather than by TFIID

exceptions to the universality of TAFs and TBP TFTC = TBP-free-TAFII-containing complex 3D models of TFIID and TFTC show that they have similar grooves for DNA interaction = to hold DNA like a clamp