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Fig. 3. Transport to the nucleus. In the absence of ligand, GR–Hsp90 preferentially contains the immunophilin FKBP51. After
hormone binding, it is replaced by FKBP52. FKBP52 associates with dynein and thereby links the GR–Hsp90 complex to
microtubules for transport to the nucleus. It is not known whether transport complexes still contain Hsp90 or whether FKBP52
bound to GR associates with dynein and microtubules without Hsp90. Alternatively, GR alone or in a complex with FKBP52 might
bind importins to travel to the nuclear pore. MT, microtubules; C, cytoplasm; N, nucleus.
I. Grad, D. Picard / Molecular and Cellular Endocrinology 275 (2007) 2–12
Figure 1 Whole-genome mapping of Pol II binding in Drosophila S2 cells. (a) Top, percentage of input DNA obtained by ChIP versus
chromosome position (in kilobase units), which represents the center point between primers used for quantitative PCR. Bottom, fold
enrichment over genomic DNA observed by ChIP-chip versus chromosome position of probes. Relative probe intensities from Pol II ChIP-chip
are shown in grayscale (black, higher intensity; white, lower intensity); below, arrows within genes denote the direction of transcription. (b)
Flowchart describing the strategy used to determine and validate global Pol II promoter occupancy. (c) A histogram showing the position of the
maximally bound probe within each bound gene (n ¼ 5,403) with respect to the transcription start site (TSS).
Figure 2 Pol II is enriched near the promoters of a subset of genes. ChIP-chip data for Pol II using antibodies that recognize the Rpb3 subunit
(black squares) and the serine-2-phosphorylated CTD of Rpb1 (gray circles) are shown for six bound genes, plotted as fold enrichment over input
versus chromosome position in kilobase units. The start site and direction of transcription are shown by arrows, with boxes depicting exons and
lines representing introns
5’P
3’P
Figure 4 Permanganate mapping of open transcription bubbles reveals
engaged Pol II within the promoter-proximal region of genes with PPEP.
In vivo permanganate footprinting demonstrates the presence and
locations of engaged Pol II in the promoter-proximal region of five genes
with PPEP
3’P
5’P
Se determinó la relación de veces de enriquecimiento en esa
región (zona promotor) . Un valor de 1 (eje x) representa un
promedio de dos veces mas de señal de Pol II cerca del promotor
que downstream.
(Log(2)=1)
(Log(4)=2)
(log2(8)=3)
Se analizaron 1500 genes que poseen Pol II pausada. Los CORE promotores de esos genes
estaban enriquecidos en una secuencia determinada PAUSE BOTTON un motivo rico en
GC-rich sequence motif, (KCGRWCG).
Aproximadamente ¼ de los 1500 promotores poseían una copia de PB motivo entre 1 y 60
pb del TSS
Se propone que esa secuencia atenúa la velocidad de Pol II
Alto contenido en GC podría requerir mas energía para depararear el DNA
Elongación Productiva
NELF factor
Figure 5 Depletion of NELF relieves promoter-proximal stalling, globally reducing PPEP. (a–c) Pol II (anti-Rpb3) ChIP-chip data from
NELF-depleted (open circles) and mock-treated ChIP samples (blue squares) for genes with PPEP,
FIGURE 1. Reconstitution of elongation control in vitro. A, kinetics of RNAPII
elongation in the absence or presence of DSIF and NELF. Isolated EECs were allowed
to elongate transcripts for indicated times in the absence or presence of purified
DSIF and NELF. The synthesized transcripts were extracted and analyzed on a 6%
denaturing RNA gel followed by autoradiography of the dried gel
FIGURE 2. Reconstitution of P-TEFb function through prephosphorylation
reactions. Isolated EECs and the indicated add-back components were either
left untreated () or subjected to prephosphorylation (P) by incubating with
recombinant P-TEFb and 500 M ATP for 5 min at room temperature
P-TEFb es necesario in vivo para la transición de Pol II del estado pausado al
estado de elongación productiva, no para completar transcripción.
FRAP: fluorescence
fluorescence recovery after photobleaching
THE JOURNAL OF BIOLOGICAL CHEMISTRY
Vol. 280, No. 6, Issue of February 11, pp. 4017–4020, 2005
FIG. 2. dEloA is redistributed
to heat shock puffs during
the heat shock response.
A, polytene chromosomes
from the salivary glands of
Oregon R third-instar larvae
were fixed as described and
immunostained with polyclonal
antisera against dEloA
FIG. 3. dEloA colocalizes with phosphorylated
Pol II at heat shock loci. Chromosomes were
prepared from heat-shocked third-instar larvae as
described in Fig. 2. Dual immunostaining was
performed using dEloA polyclonal antisera (A) and
monoclonal anti- Ser2 phospho-Pol II (B). Both
dEloA and phospho-Pol II are enriched at the heat
shock loci. A merge of these images is shown in C.
FIG. 4. dEloA is required for efficient
expression of HSP70. A. RNAi efficiently
reduces levels of dEloA protein on polytene
chromosomes. Chromosomes from thirdinstar larvae in which RNAi was activated
against dEloA (top panels) and control
larvae (bottom panels) were prepared and
stained with antibodies against dEloA and
serine 2-phosphorylated Pol II. C, larvae
with RNAi-mediated depletion of dEloA
express dramatically less HSP70 transcript
than their siblings that do not carry the
RNAi construct.
OBSTACULOS EN LA ELONGACION
Cambios en estado de Fosforilación de CTD
NTP’s diffuse through funnel and pore at a rate of ~200 NTPs per second.
The Number of binding events in the E site is estimated to be <20 per
second. The In vivo rate of transcription is estimated to 10 nt per second.
Batada et al. 2004. PNAS 101:17361-17364
Figure 3
Schematic representation of the extended model
for the nucleotideaddition cycle (NAC). The
vertical dashed line indicates register +1. The
steps where -amanitin interferes with the NAC are
indicated. For details, refer to the text. This figure
was adapted from Brueckner & Cramer (2008)
with modifications
Figure 1. Genome-Wide Distribution Pattern of
Histone Modifications from a Transcription
Perspective The distribution of histones and their
modifications are mapped on an arbitrary gene
relative to its promoter (5’ IGR), ORF, and 3’ IGR
(original references were reviewed in Shilatifard,
2006; Workman, 2006). The curves represent the
patterns that are determined via genome-wide
approaches. The squares indicate that the data are
based on only a few case studies. With the exception
of the data on K9 and K27 methylation, most of the
data are based on yeast genes.
Cell 128, 707–719, February 23, 2007
Reportero:
Lex Cyc P
Lex DBD
Set2
Lacz
Figure 2 Loss of histone–DNA contacts at the active GAL1 and GALVPS13 loci. The amount of histone H3 (grey bars) and H2B (white
bars) on the GAL1 promoter (A) or coding region (B) was determined
by ChIP assays with anti-FLAG and anti-H3 C-terminal antibodies,
respectively
Chaperonas
Asf1,Nap, Nucleoplasmina
Fig. 1. Possible histone octamer fates during intense and moderate transcription by Pol II in vivo. DNA is shown in white, the H3/H4 tetramer
in purple, and two H2A/H2B dimers in green and yellow. The histone displacement and exchange pathways are indicated by dashed and
solid arrows, respectively.
EC45
(D) FACTfacilitated transcription through the nucleosome results in the formation of hexasomes. Immobilized, DNA-end-labeled
nucleosomes were transcribed in the presence or absence of rFACT, rFACTC, or 300 mM KCl. The beads were pelleted, and the
supernatant was analyzed in a native gel. Supernatant contains fully transcribed and some nontranscribed templates (24).
Fig. 4. Gene transcription at low and high Pol II densities. During transcription at a moderate or low level the histone octamer is not likely
to be displaced from DNA even transiently. However transcription by Pol II is accompanied by frequent transient displacement of
H2A/H2B dimer(s); this results in extensive and fast transcription-dependent exchange of H2A/H2B histones and possibly in rare exchange
of H3/H4 histones. Transcription through nucleosomes and displacement of the dimers could be accomplished by Pol II itself; however the
rate of elongation is strongly increased by TFIIS and FACT. Re-binding of displaced H2A/H2B dimer to DNA occurs almost immediately after
Pol II passage and most likely is facilitated by FACT. At a higher density of Pol II molecules complete histone octamer can be displaced (by
one of the mechanisms described in Fig. 3). Since efficiency of this process depends only on the density of Pol II molecules, Pol II is the
primary player in the reaction. If the rate of transcription is decreased, the octamer rebinds to DNA almost immediately; the recovery of
chromatin structure depends upon activity of Spt2, Spt6 FACT and Hir proteins. Other designations are as in Fig. 1.
TERMINACION
Modelo Alostérico
Modelo Torpedo
Modelo Híbrido
Figure 1. Models for Termination by RNA Polymerase II
Cell, Vol. 119, 911–914, December 29, 2004
FIG. 1. MAZ element promotes poly(A)-dependent transcriptional
termination in vivo. (A) -Globin construct with exons shown as gray
boxes and CoTC element as hatched box. NRO probe positions are
underlined, and RT-PCR probe positions are dotted lines. The panels
below show NRO signals for CoTC, CoTC, MAZ4, mMAZ4, MAZ4pA, and
mMAZ4pA constructs. M (empty M13 vector) shows the background
signal
TRENDS in Biochemical Sciences Vol.28 No.4 April 2003
REINICIACION
Templado modificado por complejos
proteína_DNA intermediarios que
permanecen asociados durante los
subsecuentes ciclos
Fig. 1. Template-based reinitiation mechanisms.
Fig. 2. Protein-based reinitiation strategies. These strategies involve modification of transcription factors (TF) or RNA polymerase (Pol
1) Templado inmovilizado
incubado 40’ con NE para
formar PIC.
2) Lavado
3) Incubación 2’con NTPs
4) Lavado
Solo 1er NE
1er NE wt y 2o depletatado
p,t FISH RNA : 200-400 RNAs