BioReg2014_Transcription_1x - UCSF Tetrad Program

swedishstreakΜηχανική

22 Φεβ 2014 (πριν από 3 χρόνια και 8 μήνες)

108 εμφανίσεις

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.








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
-
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
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

rRNAs

snRNAs

miRNAs

Other non
-
coding RNAs


(e.g. telomerase RNA)

mRNAs

translation

proteins

transcription

(RNA processing)

Transcription is Important


Transcription/Splicing/Translation Provide

A Large Range of Protein Concentrations

I. RNA polymerases

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



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

Transcription

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
relat
ionships

of general
transcription factors

s



Initiation

s

Gre

Transcript cleavage

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




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 factors

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 assay

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




(3)
s

undergoes a large conformational change upon binding


to RNA polymerase

Free

doesn

t bind DNA

in holoenzyme positioned for DNA recognition

Sorenson; 2006

-
10 logo

-
35 logo

Recognition of the prokaryotic promoter

s

is positioned for DNA recognition



Initiating RNAP must open DNA to permit transcription:

Formation of the open complex

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 Step

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

Schematic

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

+ DNA with real promoter

TRANSCRIPTION INITIATION and ELONGATION

nuclear
extract

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 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

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

TBP

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

K
B

K
f

initial

binding


isomerization


Abortive

Initiation

Elongating

Complex


RP
o

RP
c

R+P

NTPs

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?


Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluoresc
ent

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 emis
sio
n of a
longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance
bet
ween
the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the inte
nsi
ty
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 DNA

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


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 complex









Initial transcription involves DNA scrunching

Higher E* in Abortive initiation complex than open
complex results from DNA scrunching

Open complex

Abortive initiation complex



Initial transcription involves DNA scrunching

Open complex

Abortive initiation complex



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 b
e
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 break
age 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 ene
rgy 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/mo
l
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
elongation

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