Transcript Nessun titolo diapositiva

Overview of Transcription
The general
process is similar
in prokaryotes
and eukaryotes.
Three phases:
- Initiation
- Elongation
The word gene was coined in 1909 (W.
Johannsen).
The central dogma (1950s).
In prokaryotes a singe RNA polymerase
transcribes genes encoding mRNA, rRNA and
tRNA.
- Termination
Transcription in E. coli and in Eucaryotes
Procaryotes
Eucaryotes
Genes are grouped into operons
Genes are not grouped in operons
mRNA may contain transcript of
several genes (poly-cistronic)
each mRNA contains only
transcript of a single gene
(mono-cistronic)
Transcription and translation are coupled.
Transcript is translated already during
transcription.
Transcription and translation are
NOT coupled.
Transcription takes place
in nucleus, translation in cytosol.
Gene regulation takes place by
modification of transcription rate
Gene regulation via transcription
rate and by RNA-processing,
RNA stability etc.
mRNAs are not processed in prokaryotes
mRNAs are processed in in
eukaryotes (splicing , CAP, poly-A tail)
rRNA and tRNA are processed both in eukaryotes and prokaryotes
Transcription and translation is
coupled in prokaryotes
Initiation of transcription
Sense strand  nontemplate strand = mRNA
Antisense strand  template strand
As soon the growing
mRNA chain separates
from DNA, ribosomes
attach to it and begin
translation on the 5’ end of
the molecule following
right behind the RNA
polymerase while it is
transcribing the mRNA.
3
1
2
Conserved sequences in prokaryotic promoters
Pribnow box
(TATA box)
Startpoint  in most E. coli genes is an A
(the distance of the startpoint from the
TATA box may vary from 5 to 9
nucleotides).
-10 sequence  double-stranded DNA
separation.
-35 sequence  initial binding of RNA
polymerase.
spacer region (16-19 bp)  it is important
in maintaining the appropriate positions of
the -10 and -35 elements.
D. Pribnow (1975) P.N.A.S.
Promoter quality has a strong influence on the level of
expression. Some examples of s70 promoters are shown
(red indicates positions which are conserved). The
moderately matched lacZ promoter has about 1%
TTGACA
TATAAT
initiation efficiency compared with ideal, and effective
82 84 78 65 54 45
80 95 45 60 50 96
Promoter consensus sequences
expression is strongly dependent on activation by CAP.
The poorly matched lacI promoter is even less efficient
(LacI repressor is present only at few copy per cell).
Conserved sequences in eukaryotic promoters
The effects of mutations in the promoters region on transcription for the mouse b-globin gene.
 not recovered mutations (the relative
transcription level was not determined)
* more than one mutation was recovered for
the corresponding nucleotide
Upstream of the CAAT box most eukaryotic promoters (genes encoding mRNA) have additional conserved
sequences: CG box (GGGCGG) and CACCC box (GCCACACCC). Their role is still unclear.
DNAse I Footprinting
1. Prepare end-labeled DNA.
2. Bind protein.
3. Mild digestion with DNAse I (randomly
cleaves ds DNA on each strand)
4. Separate DNA fragments on denaturing
acrylamide gels.
DNase I footprint performed
on an end-labeled DNA
fragment
FIS
Footprint
Samples in lanes 2-4
had increasing amounts
of the DNA-binding
protein (lambda protein
cII); lane 1 had no
protein.
Partially DNase I digested DNA is subjected to linear PCR
DNA-protein complex
DNase I
Products of DNase I digestion
are primer extended by linear
PCR using a 5’ end-labeled
oligonucleotide
Sequencing gel
Gel retardation assay
Gel Shift
Electro Mobility Shift Assay (EMSA)
Band Shift
No protein
add protein
Non-denaturing PAGE
Incubating a purified protein, or a complex mixture of
proteins e.g. nuclear or cell extract, with a 32P endlabelled DNA fragment containing the putative
protein binding site (from promoter region).
*
*
Reaction products are then analysed on a nondenaturing polyacrylamide gel.
The specificity of the DNA-binding protein for the
putative binding site is established by competition
experiments using DNA fragments or
oligonucleotides containing a binding site for the
protein of interest, or other unrelated DNA
sequences.
Retarded
mobility due
to protein
binding
Free DNA probe
virB
virF
virG
Bound DNA
EMSA
Free DNA
Evaluating the
Binding Affinity
Primer extension
mRNA
5’
3’
annealing

G A T C
primer-32P
Early-log Mid-log Late-log
37°C 10°C
37°C 10°C
37°C 10°C
mRNA
5’
primer -32P
3’
-10
+1
reverse transcriptase
+24
mRNA
5’
cDNA
primer -32P
run on denaturating gel
3’
+42
+77
cspA mRNA
S1 Mapping
[#]
La nucleasi S1
digerisce DNA o RNA a
singolo filamento
[*]
Il sito d’inizio della
trascrizione si trova a
300 basi dall’estremità
5’ marcata del
frammento di DNA
usato come sonda
[#]
[*]
Bacterial RNA polymerase
The overall reaction rate is ~40 nucleotides/second
at 37°C (for the bacterial RNA polymerase); this is
about the same as the rate of translation (15 amino
acids/sec). One mistake occurs every 10000
nucleotides added.
About 7000 RNA polymerase molecules are
present in an E. coli cell. Many of them are
engaged in transcription; probably 2000–5000
enzymes are synthesizing RNA at any one time.
The typical bacterial RNA polymerase consists of an essential four-subunit core enzyme
organized as aabb’ (449 kd, about ½ size of DNA Pol III). A fifth subunit w (rpoZ) interacts with
and stabilizes b‘
The a subunit (36.5 kDa, rpoA gene) is organized in two domains,
with the N-terminal (1-235) in contact with b or b' subunits. A flexible
linker connects to the C-terminal domain (249-329, a-CTD) which
lies outside the core polymerase and is the target for interaction both
exit
with activating factors such as Catabolite Activator Protein (CAP) and
entry
site
cis up-elements.
site
The b subunit (150 kDa, rpoB gene) and the b' subunit (155 kDa,
rpoC gene) form the catalytic site.
The rudder is a projecting loop of the b' subunit which is proposed to
act to separate the nascent RNA from the DNA template.
RNA polymerase passes through
several steps prior elongation.
1) Core enzyme + Sigma factor (σ)→ Holoenzyme
2) The enzyme remains at the promoter while it
synthesizes the first ~10 nucleotide bonds. The initiation
phase is protracted by the occurrence of abortive events,
in which the enzyme makes short transcripts (less than ~
10 nucleotides), releases them, and then starts synthesis of
RNA again.
15 bp
3) The initiation phase ends when the enzyme succeeds in
extending the chain and escapes from the promoter.
Transition to the elongation complex involves partial
dissociation of the holoenzyme. The sigma factor is left at
the promoter complex, and the core RNA polymerase
proceeds downstream. Conformational changes in the
core enzyme result in β subunits clamping around the
DNA, so that the polymerase never leaves the template.
This is critical for processive transcription, since RNA
polymerase can't resume synthesis of an incompletely
transcribed gene, and must be assured of remaining bound
for 104-105 reaction cycles.
Initiation requires tight binding only to particular sequences
(promoters), while elongation requires close association with all
sequences that the enzyme encounters during transcription.
1) The core enzyme has a general affinity for DNA because
of the electrostatic attraction between the basic protein and
the nucleic acid. The complex core enzyme-DNA is stable,
with a half-life for dissociation of the enzyme from DNA
~60 minutes. Core enzyme does not distinguish between
promoters and other sequences of DNA.
2) The affinity of RNA polymerase for DNA in general is
reduced by a factor of ~104, and the half-life of the
complex is less than 1 second when sigma factor is bound
to core enzyme.
When sigma
unattached,
hand is closed
3) In the presence of sigma factor, the holoenzyme binds to
promoters very tightly, with an association constant
increased from that of core enzyme by on average 1000
times and with a half-life of several hours.
4) There is wide variation in the rate at which the
holoenzyme binds to different promoter sequences. The
binding constants extend from ~1012 to ~106 M-1, reflecting
promoter strengths that support initiation frequencies of
~1/sec (rRNA genes) to ~1/30 min (the lacI promoter).
Transition in shape and size
identifies three forms of complex
The RNA
polymerase
directly recognizes
the promoter
The most likely
model is to suppose
that the bound
sequence is directly
displaced by another
sequence. The
enzyme continues to
exchange sequences
until a promoter is
found.
The RNA
polymerase moves
along DNA
RNA polymerase approaches DNA from one side and recognizes that face of the DNA
Prior modification experiments identify all those sites that the enzyme must recognize in order to
bind.
Protection experiments recognize all those sites that actually make contact in the binary complex
The regions at –35 and –10 contain most of the contact points for the enzyme. Within these regions,
the same sets of positions tend both to prevent binding if previously modified, and to show increased
or decreased susceptibility to modification after RNA polymerase binding.
The sigma factor, σ70 (MW = 72000)
Fragments 2.1 and 2.2 of s70 bind strongly to b'.
Adjacent helical segments located in fragments 2.3 and
2.4 are involved in recognition of the -10 region of the
promoter. The 2.3 region is required for melting.
In addition, sequences near the N-terminal (1.1 and 1.2)
of s70 were found to be inhibitory to DNA binding.
The addition of s to the polymerase core gives the RNA polymerase holoenzyme recognizing a site at -10
to form the closed complex. In the holoenzyme form, an additional DNA binding domain of s, the region
4.2, become unmasked, and this recognizes a second site at -35, approximately 2 helical turns of DNA
away. If the -35 site is recognized, the holoenzyme melts the region -11 to +3 in the DNA, giving the open
complex, and the bubble is stabilized by the ssDNA binding domain of s at region 2.3.
The region 2.5 interacts with dsDNA from -11 to –17 (spacer region).
Melting of the transcription bubble admits the template strand to the catalytic site, allowing initiation to
proceed.
Cambiamenti strutturali che avvengono nella RNA polimerasi durante
l’ isomerizzazione (transizione complesso chiuso-complesso-aperto)
+3
-11
(non template)
(template)
- Le pinze (pincers) bloccano il DNA nel complesso aperto.
- Cambiamento di posizione della regione 1.1 del fattore sigma sigma. Quando l’oloenzima
non è legato ad un promotore la regione s 1.1 impegna il sito attivo bloccando l’accesso al
DNA. Quando si forma il complesso aperto la regione s 1.1 è spostata 50 A° fuori dall’enzima
permettendo l’ingresso del DNA nel sito attivo. La regione s 1.1 mina il DNA in quanto è
carica negativamente. Il sito attivo sull’enzima che interagisce alternativamente con il DNA o
con s 1.1, è carico positivamente.
Alternative sigma factors respond to general environmental changes
E. Coli sigma factors recognize promoters with different consensus sequences
•
•
Sigma70 (rpoD)
(-35)TTGACA (-10)TATAAT
Primary sigma factor, or housekeeping sigma factor.
Sigma54 (rpoN)
(-35)CTGGCAC
(-10)TTGCA
Alternative sigma factor involved in transcribing nitrogen-regulated genes (among others).
•
Sigma32 (rpoH)
(-35)TNNCNCCCTTGAA
(-10)CCCATNT
Heat shock factor involved in activation of genes after heat shock.
•
SigmaS (rpoS)
intrinsic curvature
(-10)TGNCCATA(C/A)T
Alternative sigma factor transcribing genes of stationary phase of growth.
Note the extended -10 element.
The use of different sigma factors gives E. coli flexibility in responding to different conditions.
a2bb’s43
The production of new
sigma factors occurs
during infection of B.
subtilis by
bacteriophage SPO1
gp28
gp28
gp33
gp34
gp33
gp34
Elongation
1) The antisense strand of DNA is used as template.
2) Transcription proceeds in 5’--3’ direction.
3) The double stranded RNA-DNA hybrid is very
transient. At any given time during transcription, the
number of nucleotides of RNA that remain paired
with the DNA template may vary between 8 and 10.
Several proteins can affect the rate of elongation.
NusA slows elongation when RNA polymerase
encounters certain sequences keeping the rate of
transcription similar to the rate of translation so that
the ribosomes are able to follow on RNA molecule
right behind the RNA polymerase.
Termination in prokaryotes
1) Core enzyme can terminate in vitro at certain sites in the absence of any other factor. These sites
are called intrinsic terminators.
2) Rho-dependent terminators are defined by the need for addition of rho factor in vitro
transcription assay.
1) Intrinsic terminator
loop
Stem
(CG rich)
The importance of the run of U bases is confirmed
by making deletions that shorten this stretch;
although the polymerase still pauses at the hairpin,
it no longer terminates.
2) Rho dependent termination.
-rho factor is an essential protein in
E. coli (~275 kD) hexamer of identical
subunits).
-Mutations in rpoB gene (b subunit of
RNA polymerase) can reduce
termination at a rho-dependent site.
A consensus sequence for rho-dependent terminators cannot
be defined (high C and low G content).
- E. coli has relatively few rhodependent terminators; most of the
known rho-dependent terminators are
found in phage genomes.
- rho has a 5’-3’ helicase action that
can cause an RNA-DNA hybrid to
separate; hydrolysis of ATP is used
to provide energy for the reaction.
- The idea that rho moves along RNA
leads to an important prediction about
the relationship between transcription
and translation. The RNA polymerase
pauses when it reaches a terminator,
and termination occurs if rho catches
it there.
Stop codon
In some cases, a nonsense mutation in one gene of a transcription unit prevents the expression of subsequent
genes in the unit. This effect is called polarity. Rho and NusA create a link between transcription and
translation.
UAA
UGA
UAG