Assay for RNA polymerase

Report
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 6th 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
perspective. Current Opinion in Micro. 11:121-127
factor activity: a structural
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: 60311.
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.
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 DNAscrunching 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: 1218-1219
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.
Key Points
1. Multisubunit RNA polymerases are conserved among all organisms
2. RNA polymerases cannot initiate transcription on their own. In bacteria s70 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, s70 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
Transcription is Important
transcription
(RNA processing)
rRNAs
mRNAs
translation
proteins
snRNAs
Other non-coding RNAs
(e.g. telomerase RNA)
miRNAs
Transcription/Splicing/Translation Provide
A Large Range of Protein Concentrations
I. RNA polymerases
Cellular RNA polymerases in all living organisms are evolutionary
related
LUCA-Last universal common
ancestor
A common structural and functional frame work of transcription in the
three domains of life
Structure of RNAP in the three domains
Universally conserved
Archaeal/eukaryotic
Bacteria
Archaea
Eukarya
Transcription
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
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.
Evolutionary relationships of general transcription factors
s
Initiation
Transcript cleavage
s
Gre
Elongation
LUCA may have had elongating, not initiating RNA polymerase
II. Challenges in initiating transcription
1. RNAP is specialized to ELONGATE, not INITIATE
2. Initiating RNAP must open DNA to permit transcription
3. RNAP must leave promoter—abortive initiation
The Initiating Form of RNA Polymerase
(1) The discovery of initiation factors
factor is required for bacterial RNA polymerase to
initiate transcription on promoters
'
'
+
KD ~ 10-9 M
}
‘core’
}
‘holoenzyme’
Can begin transcription
on promoters and can
elongate
Can elongate but cannot
begin transcription at
promoters
How
was discovered (Burgess, 1969)
A. Assay for RNA polymerase:
E.coli lysate
*ATP
CTP
GTP
UTP
Calf thymus DNA
buffer
Look for incorporation of *ATP into RNA chains
B. Initial purification
Lysate
various fractionation steps
(DEAE column, glycerol gradient etc)
Active fractions identified by assay
C. Improved purification of RNA polymerase:
lysate
Improved fractionation
OD 280
salt
2
1
Activity (*ATP)
CT DNA
phosphocellulose column
Fraction #
Peak 1
Peak 2
'
Peak 1 restored activity
increases rate of initiation
Transcription
DNA
SDS gel analysis
Labmate Jeff Roberts
reported that the new,
improved preparation of
RNAP (peak 2) had no
activity on DNA
Assay:
incorporation
ATP
g
P
(3) s undergoes a large conformational change upon binding
to RNA polymerase
Free
doesn’t bind DNA
Sorenson; 2006
in holoenzyme positioned for DNA recognition
Recognition of the prokaryotic promoter
-35 logo
-10 logo
s is positioned for DNA recognition
Initiating RNAP must open DNA to permit transcription:
Formation of the open complex
The Strand Separation/Melting Step
-35 logo
-10 logo
Helix-turn-helix in Domain 4
Recognizes -35 as duplex DNA
Is the -10 promoter element recognized as Duplex or SS DNA?
Approach
1. Determine a high resolution structure of s2 bound to non-template strand of the
-10 element
Schematic
2. Determine whether this structure represents the “initial binding state” or
endpoint state
Identifying eukaryotic “initiation factors”
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
nuclear
extract
+ DNA with real promoter
TRANSCRIPTION INITIATION and ELONGATION
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
TAFs
1
~10
Binds TATA box; higher eukaryotes have multiple TBPs
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 promoter
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 complex
The first two steps of Eukaryotic transcription
In archae, TBP and TFB are sufficient for formation of the preinitiation complex (PIC), suggesting that they are key to the mechanism
of transcription initiation in eukaryotes
TFB
TBP
Promoter
Many archae have a proliferation of TBPs and TFBs, suggesting that
they provide choice in promoters, akin to alternative s.
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.
Steps in transcription initiation
NTPs
KB
R+P
RPc
Kf
RPo
initial “isomerization”
binding
Abortive
Initiation
Elongating
Complex
Abortive Initiation and Promoter escape
NTPs
KB
R+P
RPc
Kf
RPo
initial “isomerization”
binding
Abortive
Initiation
Elongating
Complex
During abortive initiation, RNAP synthesizes many short transcripts, but
reinitiates rapidly.
How can the active site of RNAP move forward along the DNA while maintaining
contact with the promoter?
Using single molecule FRET to monitor movement of RNAP and DNA
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
Three models for Abortive initiation
#1
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
Predicts expansion and contraction of RNAP
#3
Predicts expansion and contraction of DNA
Initial transcription involves DNA scrunching
Open complex
Lower E* peak is free DNA; higher E* peak is DNA in
open complex; distance is shorter because RNAP
induces DNA bending
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complex
Higher E* in Abortive initiation complex than open
complex results from DNA scrunching
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complex
The energy accumulated in the DNA scrunched “stressed
intermediate could disrupt interactions between RNAP, and
the promoter, thereby driving the transition from initiation to
elongation
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)].
s is positioned to block elongating transcripts
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

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