Genetics 304
Lecture 8
Transcription Activation at Type I Promoters
-determinants within CAP and RNAP that are essential for
transcription activation specifically at Type I promoters have been
identified
1) CAP determinants for transcription activation
-mutants were sought which were specifically defective in
transcription from Class I promoters, but not in DNA binding or
DNA bending POSITIVE CONTROL MUTANTS
-random mutagenesis of gene encoding CAP, in vivo screen for
loss of ability to activate transcription at Class I promoter, but
retention of ability to bind DNA (still represses)
-mutants are designated crppc (positive control)
-all localize to a region that is surface exposed in the crystal
structure aa 156-162 (ACTIVATING REGION I, ARI)
-purified mutant proteins all bind and bend DNA but fail to
activate transcription of Class I promoters
-therefore, an additional step after DNA binding necessary to
activate transcription that requires AR1
2) RNAP determinants for transcription activation
-deletion of or point mutations in the C-terminal domain of the α
subunit preclude CAP-mediated activation at Class I promoters,
although the RNAP molecules are still able to bind DNA and
transcribe from an un-regulated promoter
-anti-α monoclonal antibodies inhibit CAP-mediated activation at
Class I promoters, but only partially inhibit CAP-independent
transcription
-aa 261, 265 and 270 essential for CAP-mediated transcription
-mutations alter a tightly folded C-terminal domain that is joined to
an N-terminal domain by a flexible linker
-hypothesize this region plays a role in AR1 activation
i)
Mechanism of transcription activation
-transcription activation at Class I CAP-dependent promoters
requires a protein-protein contact between AR1 and the C-terminus
of the alpha subunit of RNAP
-since CAP functions as a symmetrical dimer, which AR1 region is
required? Both?
-test using ORIENTED HETERODIMER EXPERIMENT
-CAP heterodimers are constructed that carry one subunit that has
altered DNA binding specificity V181 (but functional in
transcription activation); and one subunit defective in AR1 A158
(but having wild-type DNA binding specificity)
-the subunits are oriented on the promoter by using CAP binding
sites that are specific for the altered DNA binding mutant in one
half of the palindrome L29 or L8
-by determining transcription levels mediated by different
combinations of dimers and binding sites can determine which
AR1 region is important
-this approach determined that only the promoter proximal AR1
region is important for transcription activation at Class I CAPdependent promoters
Transcription Activation at Type II Promoters
-transcription activation requires 2 distinct protein-protein
interactions between different areas of CAP and RNAP that affect
different stages of transcription initiation
i)
A second activating region in CAP
-a screen was performed to determine if CAP contains a second
AR required for activation at Class II CAP promoters
-random mutagenesis of CAP screen for mutants specifically
defective in activation of Class II genes
-isolated mutations in His-19, His-21, and Lys-101
-all are next to each other in crystal structure, all are positively
charged AR2
-positive charge of AR2 is required for transcription activation of
Class II promoters
-these mutations affect transcription activation at Class II, but not
Class I CAP-dependent promoters
-they have no effect on DNA binding or bending
ii) Identification of residues in RNAP that interact with AR2
-mutations at amino acids 162-165 of rpoA (N-terminal domain of
α subunit) yield RNAP that is defective in transcription activation
at Class II promoters, but not Class I, or unregulated promoters
-these amino acids are all negatively charged, suggesting they
might interact with the positively charged AR2
-in complex of CAP, RNAP, and DNA, AR2 and aa 162-165 of α
are located next to one another
iii) Mechanism of transcription activation at Class II promoters
-transcription activation at Class II promoters involves an
interaction between AR2 and the NTD of the α subunit of RNAP
-which AR2 in the dimer is required ORIENTED
HETERODIMERS
-AR2 in the downstream (i.e., 3’) CAP subunit of the dimer is
required for transcription activation at Class II promoters
-NB AR1 is also required at Class II promoters
-because Class II promoters don’t contain an optimal –35,
formation of the closed complex is inhibited
-binding between AR1 and the CTD of alpha overcomes this
inhibition
Class II promoters require 2 separate interactions between
CAP and RNAP
-AR1 enhances formation of closed complex, AR2 stimulates open
complex formation
Other Characterized Activator-RNAP Contacts
-protein-protein contact between activators and RNAP is a
common requirement for transcription activation
α
-transcription factors which bind upstream of the promoter require
α CTD to activate transcription
-RNAP carrying an α subunit with a deletion of the CTD no longer
facilitates transcription from promoters of genes requiring some
activator proteins
-eg. PhoB, OmpR, OxyR
β’
-ssDNA binding protein of bacteriophage N4 stimulates
transcription of late phage genes by interacting with a conserved
domain of β’
σ
-cI encodes dual function activator/repressor responsible for the
lytic/lysogenic growth decision in λ
-mutants in an N-terminal acidic patch are specifically defective in
transcription activation, however still bind DNA normally
-suppressor mutations in σ restore activation of transcription to
these mutants
DEBATE OVER THE MECHANISM
-2 possible mechanisms of transcription activation through proteinprotein contact with RNAP:
1) recruitment of RNAP/stimulation of closed complex formation
2) stimulation of conformational changes in RNAP that facilitate
transcription initiation (AR2 of CAP)
-several experiments suggest that ANY protein-protein contact
between a DNA binding protein located upstream of a promoter
and RNAP will facilitate transcription, arguing that (I) is correct
-eg. if the C-terminal dimerization domain of lambda cI is fused to
the NTD of α in place of the native CTD, lambda cI can function
as an activator of transcription if a binding site is placed upstream
of a promoter
COMPLEX ACTIVATION
-transcription activation involving more than one activator protein
-often, one will be a GLOBAL REGULATOR that controls
expression of large numbers of genes in response to a global
metabolic signal, while the other will be a SPECIFIC
REGULATOR, triggering expression of a small number of genes
in response to a specific inducer molecule
-several mechanisms of complex regulation:
I.
nucleoprotein structures
-binding of multiple activators forms a nucleoprotein complex that
triggers transcription activation
II. repositioning
-binding of a second activator repositions an activator that is
already bound to DNA, such that it is now in position to activate
transcription
III. simultaneous touching
-multiple activators interact with distinct regions of RNAP to
facilitate transcription activation
-common with regulators that interact with σ70
IV. recruitment of bystanders/DNA looping
-IHF or other DNA bending protein facilitates contact of RNAP
with transcription factors bound far upstream of the promoter
-eg. σ54 promoters
TRANSCRIPTION TERMINATION
Mechanisms of Transcription Termination
# intrinsic (Rho/Factor-independent)
# Rho-dependent
& Rho as a transcription/translation coupling
factor
& Accessory factors
Controlling Transcription Termination to Regulate Gene
Expression
# Attenuation
& E. coli trp operon
& B. subtilis trp operon
# Anti-termination
& λN
# Other mechanisms
MECHANISMS OF TRANSCRIPTION TERMINATION
-transcription termination determines end of transcription
cycle
RPo RPc RPi RPe termination
-proper termination VERY important in bacteria since regions
between transcription units small
-RPe is highly processive & stable, special mechanisms
required to stop transcription
-specific termination systems act to release transcript +
RNAP from template DNA
-gene expression can be precisely regulated by controlling
transcription termination:
i)
site
ii) efficiency
Intrinsic Termination
-Rho-independent, factor-independent
-comparison of many sites of spontaneous termination
identifies consensus elements:
L region of dyad symmetry 30 bp upstream of
termination point; folds into GC rich stem-loop
L 4-8 A’s 10 nt downstream from center of dyad
symmetry (template strand)
-do sequence elements act at the level of DNA or RNA?
1) only base changes in template strand of DNA affect
termination
2) transcription with modified nucleotides that decrease
or increase RNA secondary structure decrease or
increase termination efficiency
Mechanism of Intrinsic Transcription Termination
Event
Description
RNAP pausing -RNA hairpin formation causes
RNAP to pause
Destabilization -hairpin formation energetically
of RPe
favored over RNA:DNA hybrid
formation
-displaces existing RNA:DNA
hybrid
Dissociation of -RNAP & mRNA dissociate from
RPe
template
-pausing & terminator strength determined by stability of
hairpin
-terminator site differentiated from pause site by string of U’s
following hairpinrole?
-rU:dA hybrids very weak, facilitate destabilization of
RNA:DNA hybrid by hairpin formation
Sequences Separate From Terminator Affect
Termination
-sequences downstream from the termination site affect
termination efficiency, likely due to effects on:
2 unwinding
2 RNAP binding
-no consensus
-sequences upstream from termination site (near promoter!)
also affect termination frequency
-transcription complex may undergo conformational
transitions upon passing through early transcribed region
(+1-+30) that convert it to a terminationR RNAP
-formation of terminationR RNAP requires:
i)
ii)
iii)
iv)
specific early transcribed region present only in
some promoters
transcription through early transcribed region
spacing of early transcribed region with respect to
promoter must be conserved
only works with intrinsic terminators