Event Promoter Contracts - PowerPoint by lxv11378

VIEWS: 97 PAGES: 60

More Info
									   Chapter 9


Transcription
9.1 Introduction
9.2 Transcription is catalyzed by RNA polymerase
9.3 The transcription reaction has three stages
9.4 A stalled RNA polymerase can restart
9.5 RNA polymerase consists of multiple subunits
9.6 RNA Polymerase consists of the core enzyme and sigma factor
9.7 Sigma factor is released at initiation
9.8 Sigma factor controls binding to DNA
9.9 Promoter recognition depends on consensus sequences
9.10 Promoter efficiencies can be increased or decreased by mutation
9.11 RNA polymerase binds to one face of DNA
9.12 Supercoiling is an important feature of transcription
9.13 Substitution of sigma factors may control initiation
9.14 Sigma factors directly contact DNA
9.15 Sigma factors may be organized into cascades
9.16 Sporulation is controlled by sigma factors
9.17 Bacterial RNA polymerase has two modes of termination
9.18 There are two types of terminator in E. coli.
9.19 How does rho factor work?
9.20 Antitermination is a regulatory event
9.21 Antitermination requires sites that are independent of the terminators
9.22 More subunits for RNA polymerase
                        9.1 Introduction
Coding strand of DNA has the same sequence as mRNA.
Downstream identifies sequences proceeding farther in the direction of
expression; for example, the coding region is downstream of the initiation codon.
Primary transcript is the original unmodified RNA product corresponding to a
transcription unit.
Promoter is a region of DNA involved in binding of RNA polymerase to initiate
transcription.
RNA polymerases are enzymes that synthesize RNA using a DNA template
(formally described as DNA-dependent RNA polymerases).
Terminator is a sequence of DNA, represented at the end of the transcript, that
causes RNA polymerase to terminate transcription.
Transcription unit is the distance between sites of initiation and termination by
RNA polymerase; may include more than one gene.
Upstream identifies sequences proceeding in the opposite direction from
expression; for example, the bacterial promoter is upstream from the
transcription unit, the initiation codon is upstream of the coding region.
                9.1 Introduction




Figure 9.1 The function of RNA polymerase is to copy one strand
of duplex DNA into RNA.
9.1
Introduction



Figure 9.2
A transcription unit
is a sequence of
DNA transcribed
into a single RNA,
starting at the
promoter and ending
at the terminator.
9.2 Transcription
is catalyzed by
RNA polymerase


Figure 9.3 Transcription
takes place in a bubble, in
which RNA is synthesized
by base pairing with one
strand of DNA in the
transiently unwound region.
As the bubble progresses,
the DNA duplex reforms
behind it, displacing the
RNA in the form of a single
polynucleotide chain.
9.2 Transcription
is catalyzed by
RNA polymerase



Figure 9.4 During
transcription, the bubble is
maintained within
bacterial RNA polymerase,
which unwinds and
rewinds DNA, maintains
the conditions of the
partner and template DNA
strands, and synthesizes
RNA.
9.2 Transcription
is catalyzed by
RNA polymerase

Figure 9.5 Yeast RNA
polymerase has grooves
that could be binding
sites for nucleic acids.
The pink beads show a
possible path for DNA
that is ~25 Å wide and
5-10 Å deep. The green
beads show a narrower
channel.
9.2 Transcription
is catalyzed by
RNA polymerase




Figure 9.6 Phosphodiester
bond formation involves a
hydrophilic attack by the
3’-OH group of the last
nucleotide of the chain on
the 5’ triphosphate of the
incoming nucleotide, with
release of pyrophosphate.
9.2 Transcription
is catalyzed by
RNA polymerase


Figure 9.6 RNA
polymerase moves
like an inchworm
during elongation,
when it compresses
from the back end
and release from the
front end.
9.2 Transcription
is catalyzed by
RNA polymerase




Figure 9.6 A stalled
RNA polymerase
can be released by
cleaving the 3¢ end
of the transcript.
9.3 RNA polymerase consists of multiple
              subunits

Promoter is a region of DNA involved in
binding of RNA polymerase to initiate
transcription.
Terminator is a sequence of DNA, represented
at the end of the transcript, that causes RNA
polymerase to terminate transcription.
9.3 RNA polymerase
consists of multiple
subunits

Figure 9.7 Transcription has
four stages, which involve
different types of interaction
between RNA polymerase and
DNA. The enzyme binds to the
promoter and melts DNA,
remains stationary during
initiation, moves along the
template durign elongation, and
dissociates at termination.
9.3 RNA polymerase
consists of multiple
subunits




Figure 9.8 Eubacterial
RNA polymerases have
four types of subunit; a, b,
and b have rather constant
sizes in different bacterial
species, but s varies more
widely.
9.4 Sigma factor controls binding to DNA



Sigma factor is the subunit of bacterial
RNA polymerase needed for initiation;
is the major influence on selection of
binding sites (promoters).
9.4 Sigma factor controls
binding to DNA




Figure 9.9 RNA
polymerase passes through
several steps prior to
elongation. A closed binary
complex is converted to an
open form and then into a
ternary complex.
9.4 Sigma factor
controls binding
to DNA


Figure 9.10 RNA polymerase
initially contacts the region
from -55 to +20. When sigma
dissociates,the core enzyme
contracts to -30; when the
enzyme moves a few base pairs,
it becomes more compactly
organized into the general
elongation complex.
9.4 Sigma factor
controls binding
to DNA




Figure 9.11 Core
enzyme and
holoenzyme are
distributed on DNA,
and very little RNA
polymerase is free.
 9.4 Sigma factor
 controls binding
 to DNA




Figure 9.12 How does
RNA polymerase find
target promoters so
rapidly on DNA?
9.4 Sigma factor
controls binding
to DNA




Figure 9.13 Sigma
factor and core enzyme
recycle at different
points in transcription.
       9.4 Sigma factor controls binding to DNA




Figure 9.13
Sigma factor
and core
enzyme recycle
at different
points in
transcription.
       9.4 Sigma factor controls binding to DNA




Figure 9.13
Sigma factor
and core
enzyme recycle
at different
points in
transcription.
 9.5 Promoter recognition depends on
         consensus sequences


Consensus sequence is an idealized
sequence in which each position
represents the base most often found
when many actual sequences are
compared.
9.5 Promoter recognition depends on consensus sequences




Figure 1.33
Control sites in
DNA provide
binding sites for
proteins; coding
regions are
expressed via the
synthesis of RNA.
9.5 Promoter
recognition depends
on consensus
sequences



Figure 1.34
A cis-acting site
controls the
adjacent DNA but
does not influence
the other allele.
9.5 Promoter recognition depends on consensus sequences




Figure 9.14 A typical promoter has three components,
consisting of consensus sequences at -35 and -10, and
the startpoint.
9.5 Promoter
recognition depends
on consensus
sequences

Figure 9.9 RNA
polymerase passes
through several steps
prior to elongation. A
closed binary complex
is converted to an open
form and then into a
ternary complex.
   9.6 RNA polymerase binds to one
            face of DNA


Footprinting is a technique for
identifying the site on DNA bound
by some protein by virtue of the
protection of bonds in this region
against attack by nucleases.
9.6 RNA
polymerase binds to
one face of DNA



Figure 9.15
Footprinting
identifies DNA-
binding sites for
proteins by their
protection against
nicking.
9.6 RNA
polymerase binds to
one face of DNA


Figure 9.10
RNA polymerase initially
contacts the region from -55 to
+20. When sigma
dissociates,the core enzyme
contracts to -30; when the
enzyme moves a few base pairs,
it becomes more compactly
organized into the general
elongation complex.
        9.6 RNA polymerase binds to one face of DNA




Figure 9.16 One face of the promoter contains the contact points for RNA.
9.6 RNA
polymerase binds
to one face of
DNA

Figure 9.3 Transcription takes
place in a bubble, in which
RNA is synthesized by base
pairing with one strand of
DNA in the transiently
unwound region. As the bubble
progresses, the DNA duplex
reforms behind it, displacing
the RNA in the form of a
single polynucleotide chain.
        9.6 RNA polymerase binds to one face of DNA

Figure 9.17
Transcription may
generate more
tightly wound
(positively
supercoiled) DNA
ahead of RNA
polymerase, while
the DNA behind
becomes less
tightly wound
(negatively
supercoiled).
9.6 RNA polymerase
binds to one face of DNA




Figure 14.15
Separation of the
strands of a DNA
double helix could be
achieved by several
means.
 9.7 Substitution of sigma factors may control initiation




Figure 9.18 E. coli sigma factors recognize promoters
with different consensus sequences. (Numbers in the
name of a factor indicate its mass.)
9.7 Substitution of
sigma factors may
control initiation



Figure 9.19 A map of the
E. coli s70 factor identifies
conserved regions.
Regions 2.1 and 2.2
contact core polymerase,
2.3 is required for melting,
and 2.4 and 4.2 contact the
-10 and -35 promoter
elements.
  9.7 Substitution of sigma factors may control initiation

Figure 9.20
Amino acids
in the 2.4 a-
helix of s70
contact
specific bases
in the
nontemplate
strand of the -
10 promoter
sequence.
   9.8 Sigma factors may be organized
              into cascades



Sporulation is the generation of a
spore by a bacterium (by
morphological conversion) or by a
yeast (as the product of meiosis).
9.8 Sigma factors may
be organized into
cascades




Figure 9.21
Transcription of phage
SPO1 genes is controlled
by two successive
substitutions of the
sigma factor that change
the initiation specificity.
9.8 Sigma factors may
be organized into
cascades




Figure 9.22 Sporulation
involves the
differentiation of a
vegetative bacterium into
a mother cell that is
lysed and a spore that is
released.
        9.8 Sigma factors may be organized into cascades


Figure 9.23
Sporulation involves
successive changes in
the sigma factors that
control the initiation
specificity of RNA
polymerase. The
cascades in the
forespore (left) and
the mother cell (right)
are related by signals
passed across the
septum (indicated by
horizontal arrows).
     9.8 Sigma factors may be organized into cascades




Figure 9.24 sF
triggers synthesis
of the next sigma
factor in the
forespore (sG)
and turns on
SpoIIR which
causes SpoIIGA
to cleave pro-sE.
    9.8 Sigma factors may be organized into cascades




Figure 9.25 The
criss-cross
regulation of
sporulation
coordinates timing
of events in the
mother cell and
forespore.
   9.9 Bacterial RNA polymerase has
        two modes of termination


Terminator is a sequence of DNA,
   represented at the end of the
   transcript, that causes RNA
     polymerase to terminate
          transcription.
 9.9 Bacterial RNA
polymerase has two
modes of termination


Figure 9.26 Intrinsic
terminators include
palindromic regions that
form hairpins varying in
length from 7-20 bp. The
stem-loop structure
includes a G-C-rich
region and is followed by
a run of U residues.
    9.10 How does rho factor work?


               Polarity
refers to the effect of a mutation in
    one gene in influencing the
   expression (at transcription or
translation) of subsequent genes in
    the same transcription unit.
            9.10 How does rho factor work?




Figure 9.27 A rho-dependent terminator has a sequence rich in
C and poor in G preceding the actual site(s) of termination.
 9.10 How does rho
 factor work?




Figure 9.28 Rho factor
pursues RNA polymerase
along the RNA and can
cause termination when it
catches the enzyme
pausing at a rho-
dependent terminator.
                    9.10 How does rho factor work?


Figure 9.29 The
action of rho
factor may
create a link
between
transcription and
translation when
a rho-dependent
terminator lies
soon after a
nonsense
mutation.
  9.11 Antitermination depends on
            specific sites



Antitermination proteins allow
RNA polymerase to transcribe
through certain terminator sites.
9.11
Antitermination
depends on
specific sites

Figure 9.30
Antitermination can
be used to control
transcription by
determining
whether RNA
polymerase
terminates or reads
through a particular
terminator into the
following region.
9.11
Antitermination
depends on
specific sites




Figure 9.31 Switches in
transcriptional
specificity can be
controlled at initiation or
termination.
9.11
Antitermination
depends on
specific sites




Figure 9.22 Sporulation
involves the differentiation
of a vegetative bacterium
into a mother cell that is
lysed and a spore that is
released.
9.11
Antitermination
depends on
specific sites
Figure 9.32 Host RNA
polymerase transcribes
lambda genes and
terminates at t sites. pN
allows it to read through
terminators in the L and
R1 units; pQ allows it to
read through the R¢
terminator. The sites at
which pN acts (nut) and
at which pQ acts (qut) are
located at different
relative positions in the
transcription units.
9.11
Antitermination
depends on
specific sites


Figure 9.33 Ancillary factors bind to
RNA polymerase as it passes certain
sites. The nut site consists of two
sequences. NusB-S10 join core
enzyme as it passes boxA. Then
NusA and pN protein bind as
polymerase passes boxB. The
presence of pN allows the enzyme to
read through the terminator,
producing a joint mRNA that
contains immediate early sequences
joined to delayed early sequences.
9.12 More subunits
for RNA
polymerase




Figure 9.34 RNA
polymerase may alternate
between initiation-
competent and termination-
competent forms as sigma
and Nus factors
alternatively replace one
another on the core enzyme.
9.12 More subunits
for RNA
polymerase




Figure 9.34 RNA
polymerase may alternate
between initiation-
competent and termination-
competent forms as sigma
and Nus factors
alternatively replace one
another on the core enzyme.
9.12 More subunits
for RNA
polymerase




Figure 9.34 RNA
polymerase may alternate
between initiation-
competent and termination-
competent forms as sigma
and Nus factors
alternatively replace one
another on the core enzyme.
                     Summary
1. A transcription unit comprises the DNA between a
promoter, where transcription initiates, and a terminator,
where it ends.
2. Core enzyme has a general affinity for DNA.
3. Bacterial promoters are identified by two short
conserved sequences centered at 35 and 10 relative
to the startpoint.
4. The binary complex is converted to a ternary complex
by the incorporation of ribonucleotide precursors.
5. The "strength" of a promoter describes the frequency at
which RNA polymerase initiates transcription.
                        Summary
6. The core enzyme can be directed to recognize promoters with
different consensus sequences by alternative sigma factors.
7. The geometry of RNA polymerase-promoter recognition is
similar for holoenzymes containing all sigma factors.
8. Bacterial RNA polymerase terminates transcription at two
types of sites. Intrinsic terminators contain a G C-rich hairpin
followed by a run of U residues.
9. The Nus factors increase the efficiency of rho-dependent
termination, and provide the means by which antitermination
factors act.
10. Antitermination is used by some phages to regulate
progression from one stage of gene expression to the next and
(less often) in bacteria.

								
To top