Transcription

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Transcription Part 2
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Hexons and pentons form capsid
(TP) Covalently linked to DNA
36 kbp
50 nm
Transcribed by RNA pol II
Transcribed by RNA pol III
Figure A-1 Adenovirus
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Fig. 1. Recent examples of the various levels of
regulation of eukaryotic gene expression and cell
biology by ncRNAs. dsRNA, double-stranded RNA
HMT, histone methyltransferases HP1,
heterochromatin protein 1 PARs,
promoter-associated RNAs PcG, Polycomb group
proteins RISC, RNA-induced silencing complex
RITS, RNA-induced initiation of transcriptional
gene silencing siRNA, small interfering RNA
TFIIB, transcription factor IIB and UCE,
ultraconserved element
Published by AAAS
P. P. Amaral et al., Science 319, 1787 -1789
(2008)
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Actually, lots of DNA codes for RNA on both
strands

• Interesting evidence
• Numbers of protein-coding genes do not change
  across metazoa as a whole!!!!!!!!!!
• C. elegans 1000 cells, 20,000 protein-coding
  genes
• Human 100 trillion cells, 25,000 protein-coding
  genes
• Regulatory genes scale quadratically with genome
  size
• Almost the entire genome of all eukaryotes is
  transcribed in one context or another
• Most loci are marked by the presence of
  transcripts with sense or antisense overlaps,
  intronic RNAs and bidirectional
  transcription!!!!!!
• More than 70 of known transcripts have evidence
  of overlap with RNA in the opposite orientation
  in mouse including 87 of the protein coding
  genes!!!!!
• These are lifted from Amaral and Mattick
  http//www.springerlink.com/content/t25m385772v56u
  4w/fulltext.pdf

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Figure 31-12a The sequence of a fork-junction
promoter DNA fragment. Numbers are relative to
the transcription start site, 1.
Page 1225
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Figure 31-10 The sense (nontemplate) strand
sequences of selected E. coli promoters.
Page 1223
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Figure 8.1 Pol II promoter
Figure 8.4 Pol II promoter architecture
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RNA polymerase

• http//www.pingrysmartteam.com/RPo/RPo.htm

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Figure 31-22 The proposed transcription cycle and
translocation mechanism of RNAP. (a) Nucleotide
addition cycle. (b) RNA DNA complex in RNAP II.
Page 1235
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Figure 31-21b X-Ray structure of an RNAP II
elongation complex.
Page 1234
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Fig. 2 Structure of pol II elongation complex in
the backtracked state Structure of pol II
elongation complex in the backtracked state. (A)
Complex with one mismatched residue at the 3'-end
of the RNA (12-nt oligomer RNA). The view is a
standard one, from the "Rpb2 side," as in the
past (2225). Difference electron density map
(Fobs Fcalc omit map, contoured at 3.0 sigma)
D. Wang et al., Science 324, 1203 -1206
(2009)
Published by AAAS
12

• RNA pol oscillates between forward and backward
  movement at every step in the process

Three states of a transcribing complex a
pretranslocation state, in which the nucleotide
just added to the growing RNA chain is still in
the nucleotide addition site a
posttranslocation state, in which the enzyme has
moved forward on the template, which makes the
nucleotide addition site available for entry of
the next nucleoside triphosphate (NTP) a
backtracked state, in which the enzyme has
retreated on the template, extruding the 3'-end
of the RNA Forward movement favored by NTP
binding traps the complex in the
posttranslocation state. Backtracking
predominates when forward movement is
impeded damage in the template or by nucleotide
misincorporation in the RNA Backtracking by one
or a few residues is reversible backtracking a
greater distance leads to arrest, recovery is
only possible by cleavage of the transcript in
the polymerase active center, transcription
factor SII (TFIIS) in eukaryotes and GreA and/or
Gre B in bacteria Backtracking and cleavage
enable proofreading of the transcript, through
the excision of misincorporated nucleotides and
resynthesis
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Fluorescence resonance energy transfer (FRET)

• Example of FRET between CFP and YFP (Wavelength
  vs. Absorption) a fusion protein containing CFP
  and YFP excited at 440nm wavelength. The
  fluorescent emission peak of CFP overlaps the
  excitation peak of YFP. Because the two proteins
  are adjacent to each other, the energy transfer
  is significanta large proportion of the energy
  from CFP is transferred to YFP and creates a much
  larger YFP emission peak.

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Transcription

• RNAP bind to promoter ?closed complex
• unwinds 14 bp DNA ? open complex
• Upstream boundary protected by footprinting is
  unchanged but the open complex can synthesize
  9-11 nt RNA
• HUH? 3 models

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Fig. 1. Background and experimental approach. (A)
Background. Three models have been proposed for
RNAP active-center translocation during initial
transcription (48) see also (915) transient
excursions, inchworming, and scrunching. White
circles, RNAP active center red dashed lines,
RNA black rectangles promoter 10 and 35
elements. (B) Experimental approach. (Top) Use of
confocal microscopy with ALEX (1921) to monitor
fluorescence of single transcription complexes.
Single transcription complexes labeled with a
fluorescent donor (D, green) and a fluorescent
acceptor (A, red) diffuse through a
femtoliter-scale observation volume (green oval
transit time 1 ms) each molecule is illuminated
with light that rapidly alternates between a
wavelength that excites the donor and a
wavelength that excites the acceptor. For each
single molecule, and for each excitation
wavelength, fluorescence emission is detected at
both donor and acceptor emission wavelengths.
This configuration permits calculation of two
parameters the donor-acceptor stoichiometry
parameter, S, and the observed efficiency of the
donor-acceptor energy transfer, E (1921). The
parameter S permits identification of molecules
containing both donor and acceptor (S 0.4 to
0.9 desired species boxed in blue), molecules
containing only a donor (S gt 0.9 undesired
species, arising from the presence of free 70
molecules and buffer impurities), and molecules
containing only an acceptor (S lt 0.4 undesired
species, arising from the dissociation of
nonspecific complexes after heparin challenge).
Subsequent analysis is performed only on
molecules containing both donor and acceptor.
(Bottom) Nucleoside triphosphate (NTP) subsets
and corresponding RNA products and complexes.
A. N. Kapanidis et al., Science 314, 1144
-1147 (2006)
Published by AAAS
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3 Models

• 1. Transient excursions multiple cycles of
  forward and backward movement
• 2. Inchworming a flexible element in RNAP
  translocates downstream and retracts upon
  abortive RNA production
• 3. SCRUNCHING flexible element in DNARNAP
  pulls downstream DNA into itslf and DNA
  accumulates as a ss bulge within the unwound
  region

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Fig. 2. Initial transcription does not involve
transient excursions
Panels display negative results. For POSITIVE
results, see next slide.
A. N. Kapanidis et al., Science 314, 1144
-1147 (2006)
Published by AAAS
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Fig. 4. Initial transcription involves scrunching
Fig. 4. Initial transcription involves
scrunching. (A) Experiment documenting
contraction of DNA between positions 15 and 15
Cy3B as donor at DNA position 15 Alexa647 as
acceptor at DNA position 15. Subpanels as in
Fig. 2A. The two donor-acceptor species in the
E histograms comprise free DNA (lower-E
species) and RPo or RPitc,  7 (higher-E
species higher FRET attributable to RNAP-induced
DNA bending). Free DNA is present in all
experiments, arising from dissociation of
nonspecific complexes after heparin challenge
during preparation of RPo, but is detected only
in this experiment, because DNA contains both
donor and acceptor only in this experiment. (B)
Summary of results. Structural model of RPo (28)
showing all donor-acceptor distances monitored in
this work (Figs. 2 to 4A and figs. S2 to S8).
Distances that remain unchanged on transition
from RPo to RPitc,  7 are indicated with thin
blue lines. Distances that decrease on transition
from RPo to RPitc,  7 are indicated with thick
blue lines. The red and pink arrows show the
proposed positions at which scrunched
templatestrand DNA and scrunched
nontemplate-strand DNA, respectively, emerge from
RNAP (i.e., near template-strand positions 9 to
10 and near nontemplate-strand positions 5 to
6).
Published by AAAS
A. N. Kapanidis et al., Science 314, 1144
-1147 (2006)
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(No Transcript)
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•  Generalized scheme for the events occurring
  during initiation of transcription
• The core promoter is shown in blue and the
  transcription initiation site is indicated by a
  green dot. After RNA polymerase attachment, the
  closed complex is converted into the open complex
  by breakage of base pairs within a short region
  of the DNA double helix. RNA synthesis begins but
  successful initiation is not achieved until the
  polymerase moves away from the promoter region.

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MVA Fig. 26.6
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Figure 31-15 RNA chain elongation by RNA
polymerase.
Page 1227
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Figure 31-16 An electron micrograph of three
contiguous ribosomal genes from oocytes of the
salamander Pleurodeles waltl undergoing
transcription.
Page 1228
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MVA Fig. 26.8
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RNA Backtracking
MVA Fig. 26.10
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MVA Fig. 26.15
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MVA Fig. 26.16
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Figure 31-18 A hypothetical strong (efficient) E.
coli terminator.
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Fig. 1 The three states of a pol II transcription
elongation complex
The three states of a pol II transcription
elongation complex. RNA transcript is red, DNA
template is blue. The nucleotide base just added
to the 3'-end of RNA and the complementary base
in the DNA template are represented by cyan and
green bars, respectively. The dashed oval
represents the empty nucleotide addition site in
the posttranslocation state. The green circle
represents the pol II bridge helix.
D. Wang et al., Science 324, 1203 -1206
(2009) (including R. Kornberg)
Published by AAAS
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• Simple animation
• Very nice animation from the PBS production DNA
  The Secret of Life
• look for the baby chick at the beginning?
• also the purple ribosome?
• A pretty animation showing eukaryotic export of
  RNA into the cytoplasm