and Its Regulation January 21 Mechanism of Transcription Initiation January 23 Regulation of of Transcription Initiation January 27Mechanism and regulation of Transcription Elongation ID: 158887
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Slide1
Transcription
and Its Regulation
January
21
–Mechanism of Transcription Initiation
January
23– Regulation of of Transcription Initiation
January
27–Mechanism and regulation of Transcription Elongation
January
30– In class discussion of problem set
Mechanism of Transcription Initiation
References
I.
General
Chapter 12 of Molecular Biology of the Gene 6
th
Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M,
Losick
, R. 377-414
.
2.
Reviews
Murakami KS,
Darst
SA. (2003) Bacterial RNA polymerases: the
wholo
story.
Curr
Opin
Struct
Biol
13:31-9.
Campbell, E,
Westblade
, L,
Darst
, S., (2008) Regulation of bacterial RNA polymerase
factor activity: a structural perspective.
Current Opinion in Micro.
11
:121-127
Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along.
Annu
Rev
Biochem
.
77
:149-76.
Werner, Finn and Dina
Grohmann
(201). Evolution of
multisubunit
RNA polymerases in the three domains of life. Nature Rev. Microbiology
9
: 85-
98
Grunberg
, S. and Steven Hahn (2013) Structural Insights into transcription initiation by RNA polymerase II. TIBS
38:
603-11.
3.
Studies of Transcription Initiation
Roy S, Lim HM, Liu M,
Adhya
S. (2004) Asynchronous
basepair
openings in transcription initiation: CRP enhances the rate-limiting step.
EMBO J
.
23
:869-75.
Sorenson MK,
Darst
SA. (2006).Disulfide cross-linking indicates that
FlgM
-bound and free sigma28 adopt similar conformations. Proc
Natl
Acad
Sci
U S A.
103
:16722-7. Slide2
Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase
holoenzyme
sufficient for promoter melting.
Science.
303
:1382-1384
*
Kapanidis
, AN,
Margeat
, E, Ho,
SO,.Ebright
, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism.
Science.
314
:1144-1147.
Revyakin
A, Liu C,
Ebright
RH,
Strick
TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science.
314
: 1139-43.
Murakami KS, Masuda S, Campbell EA,
Muzzin
O,
Darst
SA (2002). Structural basis of transcription initiation: an RNA polymerase
holoenzyme
-DNA complex. Science.
296
:1285-90.
Kostrewa
D, Zeller ME,
Armache
KJ,
Seizl
M,
Leike
K,
Thomm
M, Cramer P.(2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature.
462
:323-30.
Discussion Paper
**
Feklistov
A and
Darst
, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase
s
Subunit.
Cell
147:
1257 – 1269
Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription:
s
–DNA Interaction.
Cell
:
147:
1218-1219Slide3
Reviews
Articles:
Chromosome conformation capture (CCC) technologies
de Wit, E. and de
Laat
, W. (2012) A decade of 3C technologies: insights into nuclear organization.
Genes Dev.
26: 11-24.
Elongation
BBA2013-- Issue 1874 devoted to reviews of transcription elongation
General Transcription Factors
Matsui, T.,
Segall
, J., Weil, P.A., and Roeder, R.G. (1980) Multiple factors required for accurate initiation of transcription by purified RNA polymerase II.
J
Biol
Chem
255: 11992-11996.
Thomas, M.C., & Chiang, C.M. (2006). The general transcription machinery and general cofactors.
Critical reviews in Biochemistry & Molecular Biology
, 41(3), 105-78.
Muller, F,
Demeny
, MA, &
Tora
, L. (2007). New problems in RNA polymerase II transcription initiation: matching the diversity of core promoters with a variety of promoter recognition factors.
The Journal of Biological Chemistry
, 282(20), 14685-9.
Mediator and Other Components
*Kornberg, R.D. (2005) Mediator and the mechanism of transcriptional activation.
Trends in Biochemical Sciences
30:235-239.
Fan, X, Chou, DM, &
Struhl
, K. (2006). Activator-specific recruitment of Mediator in vivo.
Nature Structural & Molecular Biology,
13(2), 117-20.
Sikorski
TW and
Buratowski
. (2009). The basal initiation machinery: Beyond the general transcription factors.
Current Opinion in Cell Biology.
21 344-351.Slide4
Key Points
1. Multisubunit RNA polymerases are conserved among all organisms
2. RNA polymerases cannot initiate transcription on their own. In bacteria
s
70
is required to initiate transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences.
2. E. coli
RNA polymerase holoenzyme, (core +
s)
finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins.
3. Transcription initiation proceeds through a series of structural changes in RNA polymerase,
s
70
and DNA.
4. A key intermediate in
E. coli
transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques.
5. Recognition of the -10 element of the promoter DNA is coupled with strand separation
6. When the open complex is given NTPs, it begins the
‘
abortive initiation
’
phase, in which RNA chains of
5-10 nucleotides are continually synthesized and released.
7. Through a
“
DNA scrunching
”
mechanism the energy captured during synthesis of one of these short
transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins
the elongation phase.
7. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase Slide5
rRNAs
snRNAs
miRNAs
Other non-coding RNAs
(e.g. telomerase RNA)
mRNAs
translation
proteins
transcription
(RNA processing)
Transcription is ImportantSlide6
Transcription/Splicing/Translation Provide
A Large Range of Protein ConcentrationsSlide7
I. RNA polymerasesSlide8
Cellular RNA polymerases in
all living organisms
are evolutionary related
A
common structural and functional frame work of transcription in the three domains of life
LUCA-Last universal common ancestor
Subunits
of
RNAPSlide9
Structure of RNAP in t
he
three domains
Werner and Grohmann
(2011),
Nature Rev Micro
9:85-98
Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities
Universally conserved
Archaeal/eukaryotic
Bacteria
Archaea
Eukarya
TranscriptionSlide10
Eukaryotic Cells have three RNA polymerases
TYPE OF POLYMERASE GENES TRANSCRIBED
RNA polymerase I 5.85, 18S, and 28S
rRNA
genes
RNA polymerase II all protein-coding genes, plus
snoRNA
genes,
miRNA
genes,
siRNA
genes, and some
snRNA
genes
RNA polymerase III
tRNA
genes, 5S
rRNA
genes, some
snRNA
genes
and genes for other small
RNAs
The
rRNAs
are named according to their
“
S
”
values, which refer to their rate of sedimentation in an ultra-centrifuge. The larger the S value, the larger the
rRNA
.Slide11
Evolutionary
relat
ionships
of general
transcription factors
s
Initiation
s
Gre
Transcript cleavage
Elongation
LUCA may have had elongating, not initiating RNA polymeraseSlide12
II. Challenges in initiating transcription
RNAP is specialized to ELONGATE, not INITIATE
2. Initiating RNAP must open DNA to permit transcription
3. RNAP must leave promoter—abortive initiationSlide13
The Initiating Form of RNA PolymeraseSlide14
‘
holoenzyme
’
'
K
D
~ 10
-9
M
+
‘
core
’
}
Can begin transcription on promoters and can elongate
}
Can elongate but cannot begin transcription at promoters
factor is required for bacterial RNA polymerase to initiate transcription on promoters
'
(1) The discovery of initiation factorsSlide15
How
was discovered (Burgess, 1969)
A.
Assay for RNA polymerase
:
E.coli lysate
buffer
*ATP
CTP
GTP
UTP
Calf thymus DNA
Look for incorporation of *ATP into RNA chains
B.
Initial purification
Lysate
various fractionation steps
(DEAE column, glycerol gradient etc)
Active fractions identified by assaySlide16
Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had
no activity on
DNA
Peak 1 restored activity
C.
Improved purification of RNA polymerase:
Improved fractionation
lysate
phosphocellulose column
salt
OD 280
1
2
Activity (*ATP)
CT DNA
Fraction #
SDS gel analysis
Peak 1 Peak 2
'
increases rate of initiation
g
Transcription
DNA
Assay:
incorporation
P
ATP
Slide17
(3)
s
undergoes a large conformational change upon binding
to RNA polymerase
Free
doesn
’
t bind DNA
in holoenzyme positioned for DNA recognition
Sorenson; 2006Slide18
-
10 logo
-
35 logo
Recognition of the prokaryotic promoterSlide19
s
is positioned for DNA recognitionSlide20
Initiating RNAP must open DNA to permit transcription:
Formation of the open complexSlide21
Is the -10 promoter element recognized as Duplex or SS DNA?
-
10 logo
-
35 logo
Helix-turn-helix in Domain 4
Recognizes -35 as duplex DNA
The Strand Separation/Melting StepSlide22
Approach
1. Determine a high resolution structure of
s
2
bound to non-template strand of the -10 element
2. Determine whether this structure represents the
“
initial binding state
”
or endpoint state
SchematicSlide23
Identifying eukaryotic “initiation factors”Slide24
Transcription Initiation by PolII requires many General Transcription Factors
RNA Pol II
+ NTPs
+ DNA containing a real promoter
NO TRANSCRIPTION
promoter
RNA Pol II
+ NTPs
+ DNA with real promoter
TRANSCRIPTION INITIATION and ELONGATION
nuclear extractSlide25
Purification scheme for partially purified general transcription factors. Fractionation of
HeLa
nuclear extract (Panel A) and nuclear pellet (Panel B) by column chromatography and the molar concentrations of
KCl
used for
elutions
are indicated in the flow chart, except for the Phenyl
Superose
column where the molar concentrations of ammonium sulfate are shown. A thick horizontal (Panel A) or vertical (Panel B) line indicates that step
elutions
are used for protein fractionation, whereas a slant line represents a linear gradient used for fractionation. The purification scheme for
pol II, starting from sonication of the nuclear pellet, followed by ammonium sulfate (AS) precipitation is shown in Panel B. (Figures are adapted from Flores et al., 1992 and from
Ge et al., 1996)
NAME # OF SUBUNITS FUNCTION
TFIIA 3 Antirepressor; stabilizes TBP-TATA complex; coactivator
TFIIB 1 Recognizes BRE;Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment
TFIID
TBP 1 Binds TATA box; higher eukaryotes have multiple TBPs
TAFs ~10 Recognizes additional DNA sequences; Regulates TBP binding; Coactivator;
Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs
TFIIF 2 Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH;
enhances efficiency of pol II elongation
TFIIE 2 Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance
TFIIH 9 ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase
phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoterSlide26
Transcription Initiation by RNA Pol II
The stepwise assembly of the
Pol
II
preinitiation
complex is shown here. Once assembled at the promoter,
Pol
II leaves the
preinitiation
complex upon addition of the nucleotide precursors required for RNA synthesis and after
phosphorylation
of serine resides within the
enzyme’
s “tail”
.
PIC =
preinitiation complexSlide27
The first two steps of Eukaryotic transcription
Many archae have a proliferation of TBPs and TFBs, suggesting that
they provide choice in promoters, akin to alternative
s.
In archae, TBP and TFB are sufficient for formation of the pre-initiation complex (PIC), suggesting that they are key to the mechanism of transcription initiation in eukaryotes
Promoter
TFB
TBPSlide28
The Pol II promoter has many recognition regions
Positions of various DNA elements relative to the transcription start site (indicated by the arrow above the DNA). These elements are:
BRE (TFIIB recognition element); there is also a second BRE site downstream of TATA
TATA (TATA Box);
Inr (initiator element);
DPE (downstream promoter element);
DCE (downstream core element).
MTE (motif ten element; not shown) is located just upstream of the DPE. Slide29
Steps in transcription initiation
K
B
K
f
initial
binding
“
isomerization
”
Abortive
Initiation
Elongating
Complex
RP
o
RP
c
R+P
NTPsSlide30
K
B
K
f
initial
binding
“
isomerization
”
Abortive
Initiation
Elongating
Complex
RP
o
RP
c
R+P
NTPs
Abortive Initiation and Promoter escape
D
uring abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly.
How can the active site of RNAP move forward along the DNA while maintaining
c
ontact with the promoter?
Slide31
Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution
Experimental set-up for single molecule FRET
: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope
Using single molecule FRET to monitor movement of RNAP and DNASlide32
Three models for Abortive initiation
#1
Predicts expansion and contraction of RNAP
Predicts expansion and contraction of
DNA
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
#3 Slide33
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
Initial transcription involves DNA scrunching
Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP induces DNA bending
Open complexSlide34
Initial transcription involves DNA scrunching
Higher E* in Abortive initiation complex than open complex results from DNA scrunching
Open complex
Abortive initiation complexSlide35
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complexSlide36
At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].
The energy accumulated in the DNA scrunched
“
stressed intermediate could disrupt interactions between RNAP,
and the promoter, thereby driving the transition from initiation to elongationSlide37
s
is positioned to block elongating transcriptsSlide38
Validation of the prediction that
occlusion of the RNA exit channel promotes
“
abortive initiation
”
#1: transcription by holoenzyme with full-length
#2: transcription by holoenzyme with
truncated at Region 3.2: lacks
in
the RNA exit channel
Murakami, Darst 2002