Molecular Biology: Prokaryotic And Eukaryotic Transcription

Front 1

experimental method for studying prokaryotic RNA polymerase (133)

Back 1

1. isolate protein
2. separate subunits with SDS-PAGE

Front 2

list the prokaryotic RNA polymerase subunits (133)

Back 2

CORE POLYMERASE
1. 2 alphas
2. beta
3. beta prime
4. omega (not easily isolated)

5. sigma

Front 3

experimental method for separating sigma from other prok. RNA polymerase subunits (133)

Back 3

subject the RNA pol holoenzyme to ion exchange chromatography with neg-charged phosphocellulose resin.

Front 4

experiment to determine if sigma subunit of RNA polymerase is responsible for binding promotors (137)

Back 4

FILTER BINDING ASSAY
1. isolate core and holoenzyme
2. bind to labeled promoter DNA (lambda genomic DNA)
3. add excess unlabeled DNA (so that any DNA that dissociated would bind to this instead of labeled DNA)
4. measure dissociation rate of DNA-polymerase

RESULTS
found that the core enzyme dissociated from DNA at a much higher rate than the holoenzyme, meaning the latter binds DNA much tighter

Front 5

experiment to determine if prokaryotic RNA polymerase binds better at higher temperatures (137)

Back 5

METHOD
1. formed complexes between RNA pol holoenzyme and labeled DNA at three different temperatures.
2. added unlabeled DNA
3. removed samples at various times and passed them through nitrocellulose

RESULTS
complex was much more stable at higher temperatures, implying that local DNA melting occurs when pol is bound tightly to promoter

Front 6

experimental method for studying prokaryotic RNA polymerase (133)

Back 6

1. isolate protein
2. separate subunits with SDS-PAGE

Front 7

list the prokaryotic RNA polymerase subunits (133)

Back 7

CORE POLYMERASE
1. 2 alphas
2. beta
3. beta prime
4. omega (not easily isolated)

5. sigma

Front 8

experiment to determine how much local melting occurs at the prokaryotic transcription bubble

Back 8

METHOD: methylation S1-assay
1. use end-labeled promoter DNA
2. allow RNA pol to bind
3. treat with DMS which methylates at ss adenines
4. remove polymerase
5. treat with SDS under mild conditions
6. denature fragments and electrophorese

RESULTS
found that a region of ~10-12 bp was denatured

Front 9

open promoter complex (gloss)

Back 9

complex formed by tight binding between pol and promoter. Open in the sense that approximately 10 bp separate

Front 10

closed promoter complex (gloss)

Back 10

complex formed by relatively loose binding between pol and promoter. Closed in the sense that the DNA duplex does not open up.

Front 11

core promoter elements

Back 11

-10 box: TAtAaT

-35 box: TTGACa

Front 12

consensus sequences (138)

Back 12

average of several similar sequences. When perfect matches to consensus sequences are found, they tend to occur in very strong promoters that initiate transcription unusually actively. (up and down mutations look more and less like consensus sequences, respectively)

Front 13

UP element (139)

Back 13

Found 40 to 60 positions upstream of promoter

not always present

recognized by alpha subunits

Front 14

alpha subunit of prokaryotic RNA polymerase (152)

Back 14

to study, used alpha-subunit mutations (truncations, deletions, site-directed mutagenesis)
confirmed binding with DNase footprinting
found that it is the CTD that contacts the UP element

Front 15

CTD of prokaryotic RNA polymerase (154)

Back 15

is an independently folded CTD that recognizes and binds to a promoter’s UP element.

since RNA pol will bind to a promoter with an UP element with both its sigma and alpha subunits, UP elements allow very strong interaction between the two and thus high rates of transcription.

Front 16

Fis sites (139)

Back 16

enhancers that are found at strong promoters between positions -60 and -150

binding site for the transcriptional activator Fis

does not bind RNA pol itself

Front 17

sigma subunit of prokaryotic RNA polymerase (140)

Back 17

stimulates initiation of transcription and can be reused

Front 18

experiment to determine how transcription initiation occurred (is there any elongation?) (140)

Back 18

METHOD
1. allowed RNA pol to synthesize 32P-labeled RNA in vitro using prokaryotic promoter + heparin + other nucleotides
2. electrophorese

RESULTS
found many small bands at bottom of gel: indicated that the polymerase made many small (9-10nt), abortive transcripts without ever leaving the promoter

Front 19

heparin (140)

Back 19

prevents reassociation between DNA and polymerase released at the end of a cycle of transcription

Front 20

steps in transcription initiation (L 1)

Back 20

1. closed promoter complex: RNA pol binding to DNA
2. open promoter complex: sigma stimulates pol to convert to open complex
3. polymerizing first few nucleotides while polymerase remains at the promoter
4. promoter clearance: transcript forms stable hybrid with template (pol clears promoter, loses sigma, begins elongation)

Front 21

experiment to determine what sigma subunit does (141)

Back 21

METHOD
1. run two reactions: one with RNA with 14C-ATP label (incorporates through all RNA), one with RNA with gamma-32P-GTP end label (incorporates only in 1st position of RNA)

RESULTS
appears that sigma contributes to both initiation and elongation, but initiation is rate limiting

Front 22

experiment to determine if sigma subunit activates initiation (141)

Back 22

METHOD
1. allow initiation to occur and then add rifampicin
2. ultracentrifugate
3. measure RNA chain length in presence and absence of sigma

RESULTS
found sigma made no difference in length of RNAs, thus it does not contribue to elongation, just initiation

Front 23

experiment to determine that sigma subunit is reused (142)

Back 23

METHOD
1. allowed pol to initiate and elongate RNA chains at low ionic strength, so that pol couldn't dissociate from template to start new RNA chains.(chains measured by gamma-32P-NTP labels)
2. then added new rif-resistant core pol in presence and absence of rifampicin.

RESULTS
immediate rise of both curves showed addition of core polymerase can restart RNA synthesis implying that sigma was reused. The fact that both curves increased means rif-resistance comes from core, not from sigma

Front 24

rifampicin (141)

Back 24

blocks prokaryotic transcription initiation, but not elongation

Front 25

experiment to determine if sigma subunit is bound to core polymerase (143)

Back 25

METHOD: FRET
trailing edge: fluorescence acceptor attached to 5' end of DNA. FRET efficiency decreases as txn occurs because pol gets farther from 5' end.

leading edge: fluorescence donor attached to 3' end. FRET efficiency is low at first, but will increase if sigma stays attached to core enzyme. Will not increase if sigma dissociates

RESULTS
found that FRET increases, so sigma does not dissociate. It is loosely bound to the core enzyme

Front 26

FRET (fluorescence resonance energy transfer)(143)

Back 26

used as a measure of the proximity of 2 fluorescent proteins relative to each other

if the donor and acceptor proteins are within 10nm, an excited donor will transfer E to the acceptor

Front 27

Determinants of UP element strength (Ross paper)

Back 27

1. relative affinity of the sequence for the alpha subunit
2. exact positioning of sites with respect to other promoter elements
3. extent to which a core promoter mechanism is rate-limited at a step affected by alpha-subunit—DNA interaction (e.g., lac UV5)

Front 28

UP element as defined by Ross et. al

Back 28

a sequence that increases transcription 2-fold or more in vivo.

Front 29

further experiments performed to evalutate sigma's associations with the polymerase core enzyme (Bar-Nahum) (144)

Back 29

METHOD
1. formed complexes between holoenzyme and promoter + 3/4 nucleotides to allow pol to move to position +32.
2. purify complex with oligos complementary to RNA transcript (beads)
3. release complex with nuclease, SDS-PAGE, immunoblot to identify proteins

RESULTS
shows sigma is less tightly bound during elongation

Front 30

elongation of transcription (L2)

Back 30

core extends ribonucleotide chain one ribonucleotide at a time

transcription bubble moves with RNA pol, maintains same approx. size (~17bp)

9bp transcript in RNA-DNA hybrid

Front 31

example of an intrinsic terminator (prokaryotes) (172)

Back 31

E. coli trp operon

Front 32

2 features of intrinsic terminators (172)

Back 32

1. string of T's in non-template strand: causes RNA pol to pause, allowing hairpin to form
2. inverted repeat just upstream of this: destabilizes the weak rU-dA pairs so that pol is ripped away from the RNA/DNA hybrid

Front 33

experiment showing if transcription termination worked or not (172)

Back 33

METHOD: in vitro run-off assay
1. digest trp with HpaII and transcribe frag with trp attenuator in vitro
2. electrophorese products

RESULTS
If attenuation works, should see a shorter (140nt) transcript. If it doesn't, should see longer (260nt) transcript.

Front 34

transcription termination: what happens when the string of T's at attenuator is mutated?

Back 34

see more of the longer transcript because non rU-dA bonds are harder to break apart

Front 35

transcription termination: what happens when iodo-CTP is substituted for CTP?

Back 35

since iodo-CTP forms a stronger bond with G, the hairpin is stabilized and thus, you see more short product.

Front 36

transcription termination: what happens with inosine is substitued for G?

Back 36

since inosine only forms 2 H-bonds with C, it weakens the hairpin and thus, you see more of the longer product.

Front 37

NusA protein

Back 37

the core polymerase has an upstream binding site that binds to part of the hairpin, slowing down hairpin formation.

NusA loosens the association between upstream binding site and hairpin, thus promoting termination.

Front 38

experiment to see how rho affects transcription

Back 38

METHOD
1. performed in vitro transcription with gamma-32P-GTP (just initiation) and 3H-UTP (total RNA)
2. add increasing amounts of rho

RESULTS
rho has little affect on initiation, but decreases total amount of RNA. Thus, probably terminates transcription.

Front 39

experiment to determine if rho acts as a nuclease (176)

Back 39

METHOD
1. allowed RNA pol to transcribe in absence of rho. Labeled RNA with 3H-UTP and ultracentrifugated
2. repeated this except using 14C-labeled RNA in the presence of rho + 3H-labeled RNA, then ultracentrifugated

RESULTS
14C labeled transcripts were found near the top of the gradient, indicating they were relatively small, but the 3H transcripts were bulky and the same size as they were originally. Thus, rho has no effect on the size of previously-made transcripts; only affects those made in its presence.

Front 40

experiment to determine if rho releases RNA product from DNA template (176)

Back 40

METHOD
1. transcribed DNA in the same method of other expt (fig. 6.54) in the absence and presence of rho.
2. subjected 3H labeled product to centrifugation

RESULTS
found that the RNA made in absence of rho cosedimented with DNA whereas that made in the presence of rho sedimented independently of DNA. Thus, transcription with rho releases transcripts from DNA template.

Front 41

properties of rho (177)

Back 41

an essential protein

a hexamer of identical subunits, each of which has an RNA-binding domain and an ATP-hydrolysis domain.

is an ATP-dependent helicase (propels rho down RNA 5'-->3')

C-rich, G-poor

can move faster than RNA pol, allowing it to catch up

Front 42

position of rho loading site (177)

Back 42

binds upstream of termination site at a sequence 60-100 nt long rich in C residues

the longer this region, the more efficient rho is

Front 43

antitermination (Yarnell paper)

Back 43

when the transcript is not released as usual (read through transcription occurs)

Front 44

Q82

Back 44

a protein that binds to the transcription complex and prevents termination and pausing.

It works by speeding RNA to the termination release site before mechanics of release are completed. Alternatively, it could stabilize the complex against the hairpin by inhibiting pausing in region of release.

Front 45

experiment used to assay binding between lac operator and repressor (189)

Back 45

METHOD
1. label lac operon DNA with 32P and add increasing amounts of lac repressor in presence or absence of IPTG (inducer).
2. assay binding via binding with nitrocellulose. Only labeled DNA bound to repressor will attach to filter.

RESULTS
found that repressor-operator interaction worked normally

Front 46

experiment that shows the lac operator binds to the lac repressor (189)

Back 46

METHOD
1. performed lac operator-repressor binding assay (figure 7.6) using 3 different DNAs: wt, one with an operator that doesn't bind repressor well, one with ns DNA

RESULTS
showed that a higher concentration of repressor was needed to achieve full binding with the mutated operator than with wild type, showing that the repressor binds to the operator.

Front 47

Pastan's experiment with lac repressor (189)

Back 47

METHOD
1. added dsDNA + RNA pol + repressor + NTPs
2. add IPTG + rifampicin

RESULTS
found that transcription still occurred, implying that the repressor did not block access by RNA pol to promoter

Front 48

2 models for repression (189)

Back 48

1. repressor blocks RNA pol's access to the promoter (competition between repressor and pol)

2. repressor blocks transition from transcriptional initiation state to elongation state (open promoter complex forms, but pol never clears the promoter because of repressor binding)

Front 49

Lee and Goldfarb's experiment with lac repressor (190)

Back 49

METHOD: run-off assay
1. incubated a DNA frag containing control region + lacZ with repressor for 10 min
2. add RNA pol. Wait 20 min for open promoter complexes to form
3. add heparin + 3/4 NTPs. Then, add labeled NTP in the presence or absence of IPTG.
4. electrophorese

RESULTS
transcription occurred even when repressor bound to DNA before polymerase could. Thus, repressor does not prevent polymerase from binding and forming an open promoter complex

when repressor was bound, shorter abortive transcripts were made (6 nt instead of 9)

Front 50

Record's experiment with lac repressor (190)

Back 50

METHOD: in vitro assay
1. form RNA pol-lac promoter complex
2. measure the rate of transcript synthesis in presence of gamma-fluor-labeled UTP: alone, with heparin, or with lac repressor (transcription rate correlates with amount of tagged PP released)

RESULTS
found repressor mimics heparin, inhibiting reinitiation of abortive transcription (blocking dissociated RNA polymerase from reassociating with the promoter)

Front 51

3 lac operators (191)

Back 51

O3: -82
O1: +11
O2: +412

removing O1 causes 18-fold decrease in transcription, removing O2 and O3 causes 50-fold decrease, removing all 3 causes 1300-fold decrease

operators function cooperatively

Front 52

how does the lac repressor bind? (192)

Back 52

as a tetramer (2 dimers)

each dimer contains 2 DNA-binding regions

the 2 dimers can bind independently to the operator

Front 53

catabolite repression (192)

Back 53

repression of a gene or operon by glucose, or more likely, by a catabolite (breakdown product, cAMP) of glucose

Front 54

CAP (catabolite activator protein) (193)

Back 54

a protein necessary for lac operon stimulation by cAMP (the two together cause positive control)

Front 55

experiment that shows beta-galactosidase production is stimulated by cAMP-CAP (193)

Back 55

METHOD
1. combined bacterial extracts with increasing concentrations of cAMP and either wt or mutant CAP.

RESULTS
mutant CAP made much less beta-galactosidase, which makes sense since the mutant CAP had less affinity for cAMP. Too much cAMP interfered with beta-galactosidase production.

Front 56

where does CAP-cAMP bind? (193)

Back 56

on the activator site, just upstream of the promoter

all activator sites contain TGTGA sequence

everywhere where activators are needed have very weak promoters, scarcely recognizable -35 boxes.

Front 57

experiment that shows that CAP-cAMP allows formation of an open promoter complex (194)

Back 57

METHOD
incubate lac promoter + RNA pol + rifampicin + NTPs in presence and absence of CAP-cAMP

RESULTS
found transcription can occur only in the presence of CAP-cAMP, meaning this complex helps RNA pol form an open promoter complex (since rifampicin won't allow txn unless an open promoter complex is formed)

Front 58

2 steps in recruitment of RNA pol by CAP-cAMP (195)

Back 58

1. formation of the closed promoter complex
2. conversion of the closed promoter complex to the open promoter complex

Front 59

2 promoters in lac operon (195)

Back 59

P1: strong
P2: inefficient (22 bases upstream of P1)

CAP-cAMP reduced initiation at P2 while stimulating initiation at P1, thus freeing up more RNA pol to bind at P1.

Front 60

how does CAP-cAMP facilitate binding of RNA pol to the promoter? (195)

Back 60

CAP-cAMP interacts with pol via the pol's alpha-CTD and bind cooperatively.

evidence: CAP-cAMP and pol cosediment during centrifugation and x-link during DNA-footprinting

X-ray crystallography shows 1 subunit of alpha binds DNA alone and the other binds DNA and CAP.

Front 61

epitope tagging (276)

Back 61

genetically alter the sequence of proteins of interest in eukaryotes to include a 9 aa epitope tag. Then subject translated proteins to a monoclonal antibody (specific to the epitope tage) and purify the protein of interest from the rest of the proteins

used in Kolodzie paper to purify RNA pol II

Front 62

3 forms of RPB1 (279)

Back 62

IIa: "parent"
IIo: phosphorylated version of IIa
IIb: missing the CTD

Front 63

how CAP-cAMP binds DNA (195)

Back 63

CAP-cAMP bends DNA at 100 degree angle, confirmed with DNA mobility assay evidence (bent DNA does not travel as fast as straight DNA--lac DNA digested with RE and bound to CAP-cAMP migrated on gel at different rates, showing it is bent.

confirmed by crystal structure

Front 64

araO1 (198)

Back 64

one of the ara operators, responsible for transcription of araC gene (makes a control protein)

Front 65

araO2 (198)

Back 65

one of the ara operators, found far upstream of the promoter (-265 --> -294).

controls PBAD

Front 66

how does araO2 regulate transcription if it is so far upstream of the promoter? (198)

Back 66

DNA looping

need integral number of loops (multiples of 10.4 bp) because DNA cannot interact with araC binding site if it is twisted 180 degrees.

Front 67

araC, general (198)

Back 67

ara control protein

acts as both a positive and negative regulator

Front 68

how is araC a negative regulator for the ara operon? (198)

Back 68

in the absence of arabinose, monomers of araC bind to O2 and I1, bending the DNA and blocking access to the promoter by RNA pol

Front 69

how is araC a positive regulator for the ara operon? (198)

Back 69

in the presence of arabinose, arabinose binds to araC, causing a conformational change. This causes araC to bind as a dimer to I1 and I2 and not to O2, which opens up the promoter to access by RNA pol. Glucose must be absent/depleted for the operon to work because CAP-cAMP must bind to the CAP binding site to activate transcription.

Front 70

experiment that gives evidence for the looping model of ara operon repression (200)

Back 70

METHOD
1. prepared minicircles containing either wt or mutant araC binding sites
2. added araC to form a complex with labeled DNA, then excess (competitive) DNA containing an araI site as a competitor.
3. electrophoresed to see whether in looped or unlooped form (looped travels faster)

RESULTS
found that looping was inhibited by both the araO2 and araI mutant, demonstrating that both sites are important in binding to araC.

with mutant O2: loop stable for less than 60 seconds
with wt O2: loop stable for more than 90 minutes

Front 71

experiment that shows that arabinose can break the araC repression loop (200)

Back 71

METHOD
1. added arabinose to preformed loops before electrophoresis in the presence and absence of arabinose

RESULTS
found that the loop was broken in the presence of arabinose

Front 72

methylation interference assay in ara operon (201)

Back 72

a means of detecting the sites on a DNA that are important for interacting with a particular protein. These are the sites where methylation interferes with binding to the protein.

used to show that after the ara loop opens, the araC monomer bound to araO2 binds to araI2. (araC and araI2 don't interact in looped state)

in assay, the wt araI2 DNA bound to araC in linear state, but not the mutant araI2.

Front 73

eukaryotic RNA polymerase, 2 types: early notions (272)

Back 73

early studies revealed there were at least 2 different RNA polymerases
1) one made rRNA genes
2) the other made protein-coding genes

Front 74

evidence for the eukaryotic RNA pol that makes rRNA genes (272)

Back 74

ribosomal genes are different than nuclear:
1) they have 60% GC comp, vs. 40%
2) they are repetitive
3) they are found in the nucleolus, mRNA in the nucleoplasm

Front 75

experiment that showed where eukaryotic RNA polymerases were found (272)

Back 75

METHOD
purify with DEAE-Sephadex ion-exchange chromatography

RESULTS
1. II and III found in the nucleoplasm fractions
2. I found in the nucleolus fraction

Front 76

RNA polymerase I (272)

Back 76

found in the nucleolus

(large) rRNA-synthesizing enzyme

Front 77

RNA polymerase II (273)

Back 77

found in nucleoplasm

makes heterogeneous nuclear RNA (hnRNA) and small nuclear RNA (snRNA)--> leading to mRNA

Front 78

RNA polymerase III (273)

Back 78

found in nucleoplasm

makes tRNA and 5S rRNA genes

Front 79

sensitivity of RNA polymerases to alpha-amanitin (274)

Back 79

RNA pol II sensitive to a lower concentration of it

RNA pol III sensitive to higher concentrations of it

RNA pol I not sensitive even at high concentrations

Front 80

effect of alpha-aminitin on small RNA synthesis (274)

Back 80

METHOD
synthesized labeled RNA in presence of increasing amounts of alpha-amanitin. Small RNAs leaked out of nucleus and were found in supernatant. PAGE

RESULTS
found that the inhibition of 5S rRNA and 4S tRNA precursor synthesis closely parallels the effect of the toxin on polymerase III (showing polymerase III transcribes these genes)

Front 81

core subunits of RNA polymerase II (276)

Back 81

Rpb1: homologous to beta prime (both bind DNA)

Rpb2: homologous to beta (both near active site)

Rpb3: homologous to alpha

Front 82

common subunits of RNA polymerase II (276)

Back 82

Rpb5, 6, 8, 10, 12

Front 83

nonessential subunits of RNA polymerase II (277)

Back 83

Rpb4, 9: mutants viable at 37 degrees but not at low or high temp. Rpb4 may anchor Rpb7.

deltaRpb4 can still transcribe DNA, but doesn't initiate at native promoter

Front 84

2 parts of RNA polymerase II promoter (287)

Back 84

1. core promoter
2. upstream promoter element

Front 85

components of the RNA polymerase II core promoter (287)

Back 85

1. TATA box centered at -25
2. TFIIB recognition element (BRE) upstream of the TATA box
3. Initiator centered at transcription start site
4. downstream promoter element (DPE)

Front 86

RNAi (Wright paper)

Back 86

interference RNA (dsRNA)

specific to the gene that encodes a protein of interest-->prevents translation

more efficient than knocking out genes genetically and allows one to look at proteins in vivo.

Front 87

TATA box (287)

Back 87

consensus sequence: TATAAA (non-template)

centered at -25

no TATA box found in housekeeping (constitutively "on") genes or developmentally-regulated genes (e.g., homeotic genes)

Front 88

what kind of genes have TATA boxes? (287)

Back 88

specialized genes encoding tissue-specific proteins

Front 89

2 functions of TATA boxes (287)

Back 89

1. positions RNA pol
2. allows transcription to occur

function depends on the gene

Front 90

experiment showing that TATA boxes are important for positioning RNA pol (288)

Back 90

METHOD
1. promoter deletion mutagenesis on SV40 early promoter
2. located initiation sites by primer extension, S1 mapping
3. made series of deletions

RESULTS
found that overall transcript size was proportional to the deletion. Distance between TATA box and transcription initiation sites remained constant (~30bp). Initiation moved to a downstream purine.

when TATA box deleted altogether, transcription started at random sites

Front 91

experiment showing that TATA boxes are important for transcription to occur (289)

Back 91

METHOD: linker scanning mutagenesis in herpes virus tk promoter
1. cut DNA with REs and digest the ends so that the DNA is missing about 10 bp
2. fill the gap with synthetic DNA linker
3. repeat with gaps farther and farther down the DNA-->allows one to scan through DNA of interest
4. injected mutated DNA into frog oocytes along with wt-DNA
5. assayed transcription via primer extension

RESULTS
found mutations in the TATA box destroyed promoter activity

Front 92

DPE (downstream promoter elements) (290)

Back 92

commonly coupled with initiators in the absence of TATA boxes

just like the TATA box, can bind TBP of TFIID

typically 30bp downstream of start site

Front 93

initiator of eukaryotic promoter (289)

Back 93

a site surrounding the transcription start site that is important in the efficiency of transcription from some class II promoters, especially those lacking TATA boxes.

along with TATA box is sufficient for basal transcription

Front 94

BRE (TFIIB recognition element) (290)

Back 94

TFIIB binds to promoter with RNA pol and other factors

BRE elements help TFIIB bind

Front 95

upstream element (2 types) (290)

Back 95

1. GC boxes: bound by Sp1, a specific transcription factor. Orientation independent, but position-dependent (must be upstream). Found in SV40 promoter.

2. CCAAT box: bound by the CCAAT binding transcription factor (CTF). Found in herpes TK promoter.

Front 96

first enhancer discovered (296)

Back 96

that in the SV40 5'-flanking region (72 bp repeats)

deletion of this region drastically affects transcription

orientation and position independent

bound by enhancer-binding proteins

Front 97

experiment showing effects of deletion in gamma2b H-chain enhancer (297)

Back 97

METHOD
1. made deletions in intron of gene where they suspected the enhancer was
2. transfected plasmacytoma cells with wt gene and the deletion mutant genes.
3. added radioactive aa to label newly made protein, immunoprecipitated and electrophoresed

RESULTS
found that the larger delta 2 mutation had profound effects on transcription

Front 98

silencers in eukaryotic promoters (298)

Back 98

cause chromatin to coil up into a condensed, inaccessible form, preventing transcription of neighboring genes

Front 99

2 types of eukaryotic transcription factors (L5)

Back 99

1. general TF
2. gene-specific TF

Front 100

general transcription factors in eukaryotes (L5)

Back 100

recruit RNA pol (weakly)

provide basal level of transcription

Front 101

class II TF pre-initiation complex (303)

Back 101

general TF and RNA pol II

factors: IIA-H

TFs assemble in a specific order

Front 102

experiment that shows what order eukaryotic class II TFs bind (303)

Back 102

METHOD: gel mobility shift assay
Performed assays with TFIID, A, B, F, RNA pol II, labeled DNA containing promoter.

RESULTS
shows TFIIF does not bind in absence of pol II (responsible for bringing it to the complex)

Nothing binds if TFIID is absent-->is the first to bind. A is next, then B. E, then H adds to F.

Front 103

TFIID (305)

Back 103

complex protein containing TBP and 8-12 TBP-associated factors (TAFs)

Front 104

TBP (305)

Back 104

highly conserved

CTD is most important (confirmed by trunctations in which the CTD was enough for transcription to occur)

saddle shaped: lines up with DNA along minor groove and bends TATA box to an 80-degree curve, opening it up.

required by all 3 RNA pols

Front 105

experiment showing footprinting of DABPolF complex (306)

Back 105

METHOD: DNase footprinting
DNase footprinting with TFIID, A, B and with TFIID, A, B, F, RNA pol II

RESULTS
when RNA pol and F joined the complex, the footprint was extended to +17, which correlates with large size of RNA pol II.

Front 106

experiment showing how TBP binds to TATA box (307)

Back 106

METHOD
1. Replaced T's with C's and A's with I's, yielding a CICI box instead of a TATA box (C and I look the same as T,A in the minor groove)
2. Bound TBP to CICI box and performed gel mobility shift assays using DNA frags containing: CICI boxes, TATA boxes, or non-specific DNA with no promoter elements.

RESULTS
found that substituting CICI for TATA had little effect on yield. Thus, TBP binds at minor groove of DNA.

Front 107

2 functions of TAFIIs (309)

Back 107

1. interaction with the promoter
2. interaction with gene-specific transcription factors

Front 108

experiment that shows the activity of TBP and TFIID on 4 different promoters (310)

Back 108

METHOD
1. tested Drosophila transcription system containing either TBP or TFIID on templates with 4 different promoters. 2 of the promoters had just a TATA box, 2 had a TATA box + Inr + DPE.
2. transcribed in vitro, assayed by primer extension

RESULTS
found TPB and TFIID fostered transcription equally well when just a TATA box was present, but TFIID did much better when the Inr and DPE were present. Thus, the interaction between TBP and other TAFs is important.

Front 109

experiment to determine which TAFs are bound to Inr, DPE of promoter (311)

Back 109

METHOD
1. photo-crosslinked TFIID to 32P-labeled template containing: TFIID found to labeled template (substituted with BrdU); TBP/250/150; TBP alone.
2. Irradiated complexes with UV light to crosslink
3. digested DNA with nuclease and did SDS-PAGE.

RESULTS
TAF250 and 150 became labeled, indicating they were in close contact with the DNA's major groove and bind to the Inr and DPE. TBP was not labeled (binds only to TATA box).

This binding was later confirmed with DNase I footprinting.

Front 110

coactivators (311)

Back 110

factors that have no transcription-activation ability of their own, but help other proteins to stimulate transcription (e.g., Sp1 with TAF110 binding to GC boxes).

Front 111

model for interaction between TBP and TATA-containing or TATA-less promoters (312)

Back 111

TATA containing promoter: TBP can bind by itself to TATA box and can also bind in company of all other TAFs of TFIID or a subset of them.

TATA-less promoter with Inr + DPE: TBP cannot bind by itself. It can bind with all TAFIIs through interactions with TAF250 and 150. These two are sufficient for TBP binding.

TATA-less promoter with GC boxes: TBP cannot bind by itself. It can bind with all TAFs along with coactivator Sp1. TAF250, 150 and 110 are sufficient to anchor TBP to Sp1 bound to GC boxes.