Lecture 27

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FCH 532 Lecture 16
Chapter 31
Pili/Fimbriae
•
•
•
•
Exclusively in Gram - bacteria
Short, fine, hair-like appendages (hollow core)
Composed of helical protein subunits
Functions
–
–
–
Not involved in motility.
Important for attachment of some bacterial species, e.g. Neisseria gonorrhoeae => pathogenesis
Sex Pilus: conjugation - transfer of genetic material between bacterial cells using the F-plasmid
Page 1218
Figure 31-3 Bacterial
conjugation.
Bacteria can transmit genes via conjugation
•
Bacterial conjugation is a process through which bacteria
transfer genetic material.
•
Ability is conferred by a plasmid (F factor) for fertility factor.
•
Designated F+, has many hairlike projections called F-pilli
that bind to cell-surface receptors on bacteria that lack the F
factor (F- cells).
•
The F factor passes through a cytoplasmic bridge into the Fcell.
Page 1218
Figure 31-4 Diagram
showing how an F– cell
acquires an F factor from an
F+ cell.
Figure 31-5 Transfer of the bacterial
chromosome from an Hfr cell to an F– cell
and its subsequent recombination with the
F– chromosome.
Sometimes the F factor will
spontaneously integrate into the
chromosome of an F+ cell.
Page 1219
Hfr (high frequency of
recombination) cells.
F factor behaves like in the
plasmid state.
Part of the bacterial
chromosome is transferred over
in a fixed order.
Page 1224
Figure 31-11a
X-Ray structure of Taq RNAP core
enzyme. a subunits are yellow and green, b subunit is
cyan, b¢ subunit is pink, w subunit is gray.
Figure 31-11b
X-Ray structure of Taq RNAP. (b)
The holoenzyme viewed as in Part a.
Page 1224

Page 1225
Figure 31-12a
The sequence of a fork-junction
promoter DNA fragment. Numbers are relative to the
transcription start site, +1.
Page 1225
Figure 31-12b
X-Ray structure of Taq holoenzyme
in complex with a fork-junction promoter DNA fragment.
Page 1225
Figure 31-13a
Model of the closed (RPc) complex
of Taq RNAP with promoter-containing DNA extending
between positions –60 and +25.
Page 1225
Figure 31-13b
Model of the open (Rpo) complex of
Taq RNAP with promoter-containing DNA showing the
transcription bubble and the active site.
RNA polymerase
•
RNAP holoenzyme (459 kD) abbw.
•
Crystal structure for Taq RNAP solved by Seth Darst.
•
Active site has a Mg2+ ion.
•
DNA template lies across one face of the enzyme outside
the active site.
•
Open and closed complexes.
•
Closed complex has UP element contacts.
•
Open complex, template strand of transcription bubble is in
a tunnel formed by the bbsubunits lined with basic
amino acids.
•
This tunnel leads to the active site.
Rifamycins inhibit prokaryotic RNAP
•
•
Two related antibiotics:
rifamycin B and rifampicin
•
2 X 10-8 M rifampicin
inhibits 50% RNAP
Binds to the b subunit and prevents chain elongation.
Page 1226
Figure 31-14 The two possible modes of RNA chain
growth. Growth may occur (a) by the addition of
nucleotides to the 3¢ end and (b) by the addition of
nucleotides to the 5¢ end.
Chain elongation proceeds in the 5’  3’ direction with
RNAP
• Experimentally proven with radiactively labelled [32P]GTP.
•
For 5’  3’ elongation, the 5’ -P is permanently labeled
so that the chain’s level of radioactivity does not change
upon replacement of labeled GTP with unlabeled GTP.
•
For 3’  5’ elongation, the 5’  -P would be added with
each nucleotide, so that on replacement of labeled GTP
by unlabeled GTP, the RNA chains lose their
radioactivity.
•
The 5’  3’ elongation is observed experimentally,
therefore, chain elongation proceeds 5’  3’.
Page 1227
Figure 31-15 RNA chain elongation by RNA
polymerase.
Page 1228
Figure 31-16 An electron micrograph of three
contiguous ribosomal genes from oocytes of the
salamander Pleurodeles waltl undergoing transcription.
RNA polymerase cannot proofread
•
Cannot rebind polynucleotide it has released.
•
Enzyme is processive.
•
No exonuclease activity.
•
Error rate is one wrong base for every ~104 transcribed.
•
DNA Pol I is one nt incorrect for every 107
•
RNAP error rate is tolerable because most genes are
repeatedly transcribed.
•
The genetic code has synonyms (redundancy).
•
Amino acid substitutions can be functionally innocuous.
•
Large portions of many eukaryotic transcripts are
excised when forming mature mRNAs.
Chain termination
•
Transcriptional terminators share two common features:
1. A series of 4 - 10 consecutive A-Ts with the A’s on the
template strand. The transcribed RNA is terminated in
or just past this sequence.
2. A G-C rich region with a palindromic (2-fold) symmetric
sequence that is immediately upstream of the series of
A-Ts.
This sequence forms a self-complementary “hairpin” that is
very stable.
Page 1229
Figure 31-18 A hypothetical
strong (efficient) E. coli
terminator.
Rho factor aids in termination
•
Rho factor is a helicase that unwinds RNA-DNA and
RNA-RNA double helices dependent on the hydrolysis
of NTPs.
•
Require a specific recognition sequence (80 -100 nt that
lack a stable secondary structure and have multiple C
rich regions, G poor regions) on the newly transcribed
RNA upstream of the termination site.
•
Attaches to nascent RNA at recognition site and
migrates in the 5’ 3’ direction until it encounters RNAP
paused at termination site and unwinds the RNA-DNA
duplex that forms the transcription bubble.
•
This releases the RNA transcript.
Page 1231
Figure 31-19a
X-Ray
structure of Rho factor in
complex with RNA. (a) The Rho
protomer with its N-terminal
domain cyan, its C-terminal
domain red, and their
connecting linker yellow.
Figure 31-19b
XRay structure of Rho
factor in complex with
RNA. (b) The Rho
hexamer. Its six subunits,
each of which are drawn
in a different color, form
an open lock washershaped hexagonal ring.
Page 1231
Figure 31-19c
X-Ray structure of Rho factor in
complex with RNA. (c) The solvent-accessible surface
of the Rho hexamer as viewed from the top of Part b.
Control of transcription in prokaryotes
•
Prokaryotes need to respond to sudden environmental
changes such as the influx of nutrients, by inducing the
synthesis of proteins.
•
Transcription and translation are tightly coupled.
•
Ribosomes commence translation near the 5’ end of the
nascent mRNA soon after it is made by RNAP.
•
Most prokaryotic transcripts are degraded within 1 - 3
min after their synthesis.
•
In contrast, eukaryotic induction takes hours or days to
respond because the transcription takes place in the
nucleus and has to be exported to the cytoplasm for
translation.
Page 1237
Figure 31-24 An electron micrograph and its interpretive
drawing showing the simultaneous transcription and
translation of an E. coli gene.
Promoters
•
The more the promoter resembles the consensus
sequence, the stronger the promoter.
lac repressor binding
•
lac repressor is a tetramer of 360 residue subunits
which are each capable of binding one IPTG with a K =
10-6 M.
•
In the absence of inducer, binds to duplex DNA
nonspecifically (K = 10-4)
•
Binds to the lac operator tightly (K = 10-13 M).
•
Binds faster than diffusion rate constant in solution, so
lac repressor slides along DNA quickly until it finds the
lac operator sequence.
•
lac operator sequence is nearly palindromic.
•
lac repressor prevents RNAP from forming a productive
initiation complex.
Page 1239
Figure 31-25 The base
sequence of the lac operator.
Figure 31-26 The nucleotide sequence of the E. coli lac
promoter–operator region.
Page 1239
C-terminus LacI
N-terminus LacZ
Catabolite repression
•
Glucose is the carbon source of choice for E. coli, so if it
is present in large amounts, the bacterium will suppress
the expression of genes encoding proteins involved in
other catabolites’ metabolism.
•
This happens even when metabolites such as lactose,
arabinose, or galactose are present in high
concentrations.
•
Catabolite repression-prevents the wasteful
duplication of energy-producing enzymes.
Page 1240
Figure 31-27 The kinetics of lac operon mRNA
synthesis following its induction with IPTG, and of its
degradation after glucose addition.
cAMP is the signal molecule for lack of glucose
•
cAMP is the signal molecule indicating a lack of
glucose.
•
In the presence of glucose, cAMP levels are diminished.
•
Addition of cAMP overcomes catabolite repression by
glucose.
•
cAMP binding protein responsible for the actioncatabolite activator protein (CAP); cAMP receptor
protein (CRP).
•
CAP is a homodimer of 210 residue subunits that
undergoes large conformational change upon binding to
cAMP.
•
CAP-cAMP complex binds to the lac operon and
stimulates transcription in the absence of lac repressor.
CAP-cAMP promotes high levels of expression for a weak
promoter
• CAP-cAMP complex binds to the lac operon and
stimulates transcription.
•
CAP is a positive regulator-turns on transcription
•
lac repressor is a negative regulator - turns off
transcription
•
lac operon has a weak (low-efficiency) promoter
because it differs significantly from the consensus
sequence.
•
CAP interacts directly with RNAP via the C-terminal
domain (aCTD).
 aCTD binds to dsDNA nonspecifically but with higher
affinity to A-T rich sites (UP elements).
Page 1241
Figure 31-28a
XRay structures of
CAP–cAMP
complexes. (a) CAP–
cAMP in complex with
a palindromic 30-bp
duplex DNA.
Page 1241
Figure 31-28b
X-Ray structures of CAP–cAMP
complexes. (b) CAP–cAMP in complex with a 44-bp
palindromic DNA and the aCTD oriented similarly to
Part a.
Page 1241
Figure 31-28c
X-Ray structures of CAP-cAMP
complexes. (c) CAP dimer’s two helix-turn-helix motifs
bind in successive major grooves of the DNA.
CAP-dependent promoters
•
Class I promoters (lac operon) require only CAP-cAMP
for transcriptional activation. CAP binding site can be
located at various distances on the DNA.
•
Class II promoters also only require CAP-cAMP for
transcriptional activation. CAP binding site only
occupies a fixed position that overlaps the RNAP
binding site.
•
Class III promoters require multiple activators to
maximally stimulate transcription. May be more than
one CAP-cAMP complexes or a CAP-cAMP complex in
concert with promoter specific activators.
DNA binding motifs
•
CAP proteins form a supersecondary structure called a
helix-turn-helix (HTH) motif that binds to DNA.
•
HTF motifs associate with target base pairs mainly via
side chains extending from the second helix of the HTH
motif (recognition helix).
•
HTH motifs are observed in the lac repressor, trp
repressor, cI repressors, and Cro proteins from
bacteriophages.
•
Another type of structural motif observed in DNA binding
proteins are b-ribbons or two stranded anti-parallel bsheets.
 b-ribbons are found in the met repressor (MetJ).
Page 1243
Figure 31-29
X-Ray structure of the N-terminal
domain of 434 phage repressor-target DNA complex.
(a) A skeletal model (b) HTH (a2, a3) interaction with
target DNA (c) A space-filling model.
Page 1243
Figure 31-30 X-Ray structure of the 434 Cro protein in
complex with DNA. (a) A skeletal model. (b) HTH (a2,
a3) interaction with target DNA (c) A space-filling model.
Page 1244
Figure 31-31 X-Ray structure of an E. coli trp
repressor– operator complex.
Page 1245
Figure 31-32a
X-Ray structure of the E. coli met
repressor- SAM-operator complex. (a) The overall
structure of the complex as viewed along its 2-fold axis
of symmetry.
Page 1245
Figure 31-32b
X-Ray structure of the E. coli met
repressor-SAM-operator complex. (b) The antiparallel b
ribbon (yellow) in the DNA’s major groove.
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