Transcript PowerPoint 簡報 - Tunghai University

Mechanisms of Transcription
• RNA polymerase does not need a primer to
initiate transcription.
• The RNA product does not remain basepaired to the template DNA strand.
• Transcription is less accurate than
replication.
RNA polymerases and the
transcription cycle
• RNA polymerase performs essentially the
same reaction in all cells.
• From bacteria to mammals, the cellular
RNA polymerases are made up of multiple
subunits.
Transcription is catalyzed by RNA polymerase, which catalyzes the
synthesis of RNA using DNA as a template.
RNA polymerase from E.coli contains two  subunits, ’, , ω and .
The E. coli holoenzyme is composed of a core, which is competent to
carry out RNA synthesis, and a σ factor, which directs the core to
transcribe specific genes.
Comparison of the crystal structures of prokaryotic and eukaryotic
RNA polymerases
Transcription in Prokaryotic cells
Transcription involves three stages
•Initiation: A promoter is the DNA
sequence that initially binds the RNA
polymerase. Only one of the DNA
strands acts as a template. The choice
of promoter determines which stretch
of DNA is transcribed and is the main
step at which regulation is imposed.
•Elongation: Once the RNA polymerase
has synthesized a short stretch of RNA
approximately ten bases), it shifts into
the elongation phase.
•Termination: Once the polymerase has
transcribed the length of the gene, it must
stop and release the RNA product.
The transcription cycle in
bacteria
• Bacterial promoters vary in strength and
sequence, but have certain defining features
• 3 regions of conservation: -35, -10 and the
length of spacer 17 - 19 bp, called
consensus sequence.
• Promoters with sequences closer to the
consensus sequence are generally stronger
than those that match less well.
• An additional DNA element, UP-element,
that binds RNA polymerase is found in some
strong promoters and increase polymerase
binding by providing an additional specific
interaction between the enzyme and the
DNA.
• Another class of promoters lacks a -35
region and instead has a so-called
“ extended -10” element.
The sigma factors mediates binding of
polymerase to the promoter
The best evidence for the functions of these regions shows that subregions 2.4 and 4.2 are involved in promoter –10 box and –35 box
recognition.
The extended –10 element is recognized by an αhelix in σregion 3.
σ70 family: There are four conserved regions in sigma 70 family
proteins.
The RNA polymerase α subunit has an independently folded Cterminal domain that can recognize and bind to a promoter’s UP
element. This allows very tight binding between polymerase and
promoter.
The α -CTD is connected to the αNTD by a flexible linker.
α subunit response to activator, repressor, elongation factor and
transcription factors
Transcription Initiation involves
three defined steps
• The first step in RNA synthesis is the
binding of RNA polymerase to a DNA
promoter- to form what is called a closed
complex.
• In the second step, the closed complex
undergoes a transition to the open complex
in which the DNA strands separate over a
distance of some 14 bp around the start site.
• Once an RNA polymerase molecule has
bound to a promoter site and locally
unwound the DNA double helix, initiation
of RNA synthesis can take place.
• Once the enzyme gets further than 10 bp, it
is said to have escaped the promoter. A
stable ternary complex contains enzyme,
DNA and RNA. This is a transition to
elongation phase.
Transition to the open complex involves
structural changes in RNA polymerase
and in the promoter DNA
The transition from closed to open complex involves structural changes
in the enzyme and the opening of the DNA double helix to reveal the
template and nontemplate strands.
In bacterial enzyme with σ70, this transition called isomerization, does
not require energy from ATP hydrolysis.
The active site of the enzyme, which is made up of regions from both
the β and β’ subunits, is at the base of the pincers within the active
center cleft.
Transcription is initiated by RNA
polymerase without the need for a primer
• This requires that the initiating ribonucleotide be
brought into the active site and held stably on the
template while the next NTP is presented with
correct geometry for the polymerization to occur.
• The enzyme has to make specific interactions with
the initiating NTP.
• The interactions are specific for the nucleotide
on A, and only chains initiated with A are held in a
manner suitable for efficient initiation.
RNA polymerase synthesizes several short
RNAs before entering the elongation phase
• Abortive initiation: the enzyme synthesizes short
RNA molecules of less than ten nucleotides and
then released from the polymerase. And the
enzyme begins RNA synthesis again.
• Once a polymerase manages to make an RNA
longer than 10 bp, a stable ternary complex is
formed. This is the start of the elongation phase.
• Region 3.2 of σ factor may be involved, and it
mimics RNA.
The elongation polymerase is a processive
machine that synthesizes and proofreading
RNA
• Double-stranded DNA enters the front of
the enzyme between the pincers.
• At the opening of the catalytic cleft, the
strands separate to follow different paths
through the enzyme before exiting via their
respective channels and reforming a double
helix behind the elongation polymerase.
RNA polymerase carries out two
proofreading functions
• Pyrophosphorolytic editing: the enzyme uses its
active site to catalyze the removal of an
incorrectly inserted NTP.
• Hydrolytic editing: the polymerase backtracks by
one or more nucleotides and cleaves the RNA
product, removing the error-containing sequence.
• Hydrolytic editing is stimulated by Gre factors,
which also serves as elongation stimulating factors.
Transcription is terminated by signals
within the RNA sequences
• In bacteria, terminators come in two types:
rho-independent and rho-dependent.
•Rho-independent Terminators, also called intrinsic terminators, a
short inverted repeats (about 20 nucleotides) followed by a string of
about eight A:T bp.
Rho-dependent terminators
• Have less well-characterized RNA elements
and requires the action of the rho factor.
• Rho is an RNA helicase, composed of 6
identical subunits, each subunit has an RNA
binding domain and ATPase domain,
requires ATP to function.
• Rho releases the RNA product from the
DNA template.
How is Rho directed to a
particular RNA molecule?
• There is some specificity in the sites it binds
(the rut sites, Rho utilization). Optimally
these sites consist of stretches of about 40
nucleotides that do not fold into a
secondary structure; they are also rich in
C.
• Rho fails to bind any transcript that is being
translated.
Transcription in Eukaryotes
• Eukaryotic cells have three different
polymerase (Pol I, II and III)
• Whereas bacteria require only one initiation
factor, several initiation factors are required
for efficient and promoter-specific initiation
in eukaryotes. These are called the general
transcription factors (GTFs).
Three different RNA polymerases
• RNA polymerase I resides in the nucleolus and is
responsible for synthesizing three of the four types of
rRNA found in eukaryotic ribosomes (28S, 18S,and
5.8 S rRNA).
• RNA polymerase II is found in the nucleoplasm and
synthesizes precursors to mRNA, the class of RNA
molecules that code for proteins.
• RNA polymerase III is also a nucleoplasmic enzyme,
but it synthesizes a variety of small RNAs, including
tRNA precursors and the smallest type of ribosomal
RNA, 5S rRNA.
Core promoter: defined as the minimal set of DNA sequences
sufficient to direct the accurate initiation of transcription by RNA
polymerase
Four types of DNA sequences are involved in core promoter
function in RNA polymerase II (1) a short initiator (Inr) (2) the
TATA box (3) the TFIIB recognition element (BRE) (4) the down
stream promoter element (DPE)
RNA polymerase II core promoters are made up of combinations of
four different sequence elements
Regulatory sequences
• Beyond the core promoter, there are other
sequence elements required for efficient
transcription in vivo.
• Together these elements constitute the
regulatory sequences: promoter proximal
elements, upstream activator sequences,
enhancers, silencers, boundary elements and
insulators. All these elements bind
regulatory proteins.
RNA polymerase II forms a pre-initiation
complex with general transcription factors
at the promoter
• The complete set of general transcription
factors and polymerase, bound together at
the promoter and poised for initiation, is
called the pre-initiation complex.
In vitro, TFIIA, TFIIB, TFIIF
together with polymerase, and
then TFIIE and TFIIH form the
pre-initiation complex.
Promoter melting in eukaryotes
requires hydrolysis of ATP and is
mediated by TFIIH.
In eukaryotes, promoter escape
involves phosphorylation of the
polymerase.
• The largest subunit of Pol II has a C-terminal
domain (CTD), which extends as a tail. The
CTD contains a series of repeats of the
heptapeptide sequence: Tyr-Ser-Pro-Thr-SerPro-Ser. They are 27 of these repeats in the
yeast and 52 in the human case.
• Each repeat contains sites for phosphorylation
by specific kinases including that is a subunit
of TFIIH.
TBP binds to and distorts DNA using a beta sheet inserted into the
minor groove
TBP uses an extensive region of  sheet to recognize the minor
groove of the TATA element.
It also bends the DNA by an angle of approximately 80o
The interaction between TBP and DNA involves only a limited
number of hydrogen bonds between the protein and the edges of the
base pairs in the minor groove.
TBP-DNA complex
The other General Transcription factors also
have specific roles in initiation
• TAFs: TBP is associated with
about ten TAFs.
• TFIIB: a single polypeptide
chain, enters the pre-initiation
complex after TBP. This protein
appears to bridge the TATAbound TBP and polymerase.
• TFIIF: This two-subunit factor
associated with PolII and is
recruited to the promoter
together with the enzyme.
TFIIB-TBP-Promoter complex
• TFIIE and TFIIH:TFIIE
recruits and regulates TFIIH.
• TFIIH controls the ATPdependent transition of the
pre-initiation complex to the
open complex. It has nine
subunits. Within TFIIH are
two subunits that function as
ATPases, and another that is
a protein kinase, with roles
in promoter melting and
escape
In vivo, transcription Initiation requires
additional Proteins, including the mediator
complex
• The high, regulated levels of transcription in vivo require the Mediator
Complex, transcriptional regulatory proteins and in many cases,
nucleosome-modifying enzymes.
• Mediator is associated with the CTD “tail” of the large polymerase
subunit through one surface, while presenting other surfaces for
interaction with DNA-bound activators.
• Different Mediator subunits to bring polymerase to
different genes.
• Mediator aids initiation by regulating the CTD
kinase in TFIIH.
• The need for nucleosome modifiers and remodellers
also differs at different promoters.
Mediator consists of many subunits, some
conserved from yeast to human
• There are various forms of Mediator,
each containing subsets of Mediator
subunits.
• A complex consisting of Pol II,
Mediator, and a some of the general
transcription factors can be isolated
from cells as a single complex in
the absence of DNA---RNA Pol II
holoenzyme.
A new set of factors stimulate Pol II
elongation and RNA proofreading
• Elongation factors (such as TFIIS and hSPT5) are
factors stimulate elongation.
• Phosphorylation of the CTD leads to an exchange of
initiation factors for those factors required for
elongation and RNA processing.
• Various proteins are thought to stimulate elongation
by Pol II. One of them is the kinase P-TEFb.
TFIIS
• TFIIS stimulates the overall rate of
elongation by limiting the length of time
polymerase pauses.
• TFIIS stimulates an inherent RNAse
activity in polymerase and contributes to
proofreading by polymerase.
Kinase P-TEFb
• Kinase P-TEFb, is recruited to polymerase by transcriptional
activators.
• Once bound to Pol II, it phosphorylates the serine residue at
position 2 of the CTD.
• P-TEFb phosphorylates and activates hSPT5, another
elongation factor. hSpT5 stimulates 5’ capping enzyme.
• P-TEFb also recruits TAT-SF1, an elongation factor, to
stimulate elongation. TAT-SF1 recruits components of the
splicing machinery.
Elongating Polymerase is associated with a new set
of protein factors required for various types of RNA
Processing
• There is an overlap in proteins involved in elongation, and
those required for RNA processing.
• hSPT5 recruits and stimulates the 5’ capping enzyme.
Elongation factor TAT-SF1 recruits components of the
splicing machinery.
Phosphorylation on ser 5: capping factors
Phosphorylation on ser 2: splicing factors
The structure and formation of the 5’ RNA cap
• The first RNA processing
event is capping.
• It is a methylated guanine,
and it is joined to the RNA
transcript by an unusual 5’ 5’ linkage.
• The RNA is capped when
it is still only 20-40
nucleotides long- when the
transcription cycles has
progressed only to the
transition between the
initiation and elongation
phases.
Polyadenylation and termination
• The final RNA processing
event, polyadenylation of
the 3’ end of the mRNA, is
linked with the termination
of trancription.
• The polymerase CTD tail is
involved in recruiting the
enzymes necessary for
polyadenylation.
• Once polymerase has
reached the end of a gene, it
encounters specific
sequences called poly-A
signals.
• CPSF (cleavage and
polyadenylation
specificity factor) and
CstF (cleavage
stimulation factor) are
carried by the CTD of
polymerase as it
approaches the end of the
gene.
• Once the CPSF and CstF
are bound to the RNA,
other proteins are
recruited as well, leading
initially to RNA cleavage
and then polyadenylation.
• Polyadenylation signal is
required for termination.
RNA polymerase I and III recognize distinct
promoters, using distinct sets of transcription
factors, but still require TBP
• Each of these enzymes also works with its own
unique set of general transcription factors. However,
TBP is universal for most of the cases.
• RNA polymerase I resides in the nucleolus and is
responsible for synthesizing three of the four types of
rRNA found in eukaryotic ribosomes (28S, 18S,and 5.8
S rRNA).
• The promoter for the rRNA genes comprise : the core
element and the UCE (upstream control element).
• Pol I promoter initiation requires Pol I, SL1 and UBF.
SL1 comprises TBP and three TAFs, and binds DNA
only in the presence of UBF.
Pol III transcription
• Pol III initiation requires Pol III, TFIIIB and TFIIIC (for the
tRNA genes), and those plus TFIIIA for the 5S rRNA gene.
• TFIIIC binds to the promoter region and recruits TFIIIB to the
DNA just upstream of the start site, where it in turn recruits
Pol III to the start site.