Transcript FCH 532 Lecture 16 Extra credit assignment for Friday March 2Seminar Speaker Matt DeLisa, 3PM-4:30PM 148 Baker New Homework/Study Guide posted Chapter 31

FCH 532 Lecture 16
Extra credit assignment for Friday March 2Seminar Speaker Matt DeLisa, 3PM-4:30PM
148 Baker
New Homework/Study Guide posted
Chapter 31
Major classes of RNA
•
Ribosomal RNA (rRNA)
•
Transfer RNA (tRNA)
•
Messenger RNA (mRNA)
•
The role that proteins are specified by mRNA and
synthesized on ribosomes was defined by experiments in
enzyme induction.
•
Enzyme induction is a consequence of mRNA synthesis
by proteins that specfically bind to the mRNA’s DNA
template.
Enzymes
• Constitutive enzymes - enzymes that are involved in
basic cellular housekeeping functions and are
synthesized at a constant rate.
•
Adaptive/inducible enzymes-enzymes that are
synthesized at rates that vary with the cell’s
circumstances.
•
Example: lactose metabolizing enzymes conversion of
lactose to galactose and D-glucose.
•
Two main proteins: -galactosidase-catalyzes the
hydrolysis of lactose to galactose and glucose
•
Galactosidde permease (lactose permease)transports lactose into the cell.
Enzymes
•
•
In the absence of lactose only a
few molecules of galactosidase or lactose
permease.
Minutes after lactose
introduced increased by ~1000fold.
•
Lactose unavailable - returns
to the basal level.
•
Metabolic product triggers the
synthesis of proteins-inducer.
H
H
H
H
H
H
H
H
H
H
H
+
H
H
H
H
H
H
H
H
H
Page 1217
Figure 31-1 The induction kinetics of -galactosidase
in E. coli.
Inducers of the lac operon
•
Physiological inducer-lactose
isomer 1,6-allolactose.
H
H H
H
H
H
H
•
•
•
H H
Results from transglycosylation
of lactoase by -galactosidase
H
Most studies of lactose system
use isopropylthiogalactoside
(IPTG).
H
H
H
Potent inducer that is not
degraded.
H
H
H
Page 1218
Figure 31-2 Genetic map of
the E. coli lac operon.
lac repressor inhibits synthesis of lac operon proteins
•
PaJaMo experiment-Hfr bacteria of genotype I+Z+ were
mated to F- strain with a genotype I-Z- in the absence of
inducer while -galactosidase activity was monitored.
•
At first, no -galactosidase activity because the Hfr donors
lacked inducer and F- recipients were unable to to produce
active enzyme.
•
After 1 h conjugation when the I+Z+ enter the F- cells galactosidase began and ceased after another hour.
•
The donated Z+ gene entering the cytoplasm of the I- cell
causes constitutive expression of the -galactosidase gene.
•
After the I+ gene is expressed (after 1 h) it represses galactosidase synthesis.
•
I must be a diffusible product.
Page 1219
Figure 31-6 The PaJaMo experiment.
F- strain resistant to T6 phage and streptomycin.
Page 95
Figure 5-25 Control of transcription of the lac operon.
Other constitutive mutations
•
Second type of constitutive mutation in the lactose system is
OC - operator constitutive.
•
Independent of I gene-maps to space between I and Z
genes.
•
In a partially diploid F’ strain OC Z-/F O+ Z+, -galactosidase
activity is inducible by IPTG.
•
In the strain OC Z+/F O+ Z-, -galactosidase is constitutively
produced.
Other constitutive mutations
•
An O sequence can only control expression of the Z gene
on the same chromosome.
1. The structural genes on DNA are transcribed onto
complementary strands of mRNA.
2. The mRNAs transiently associate with ribosomes, which
they direct in polypeptide synthesis.
The lac repressor binds to the O sequence to prevent the
transcription of mRNA. In an OC mutant, the repressor
cannot bind to the sequence.
Other constitutive mutations
•
The simultaneous or coordinate expression of the lac
enzymes under the control of a single operator indicates
that the lac operon is transcribed as a single polycistronic
mRNA
•
Cistron-gene
•
Control sequence that act only on the same DNA molecules
as the genes they control are called cis-acting elements.
•
Agents of diffusible products can be on different DNA
molecules. Trans-acting factors.
RNA polymerase
•
RNAP is the enzyme responsible for the DNA-directed
synthesis of RNA.
•
Enzyme couples together the ribonucleoside triphosphates
ATP, CTP, GTP, and UTP on DNA templates in a reaction
driven by the release and hydrolysis of pyrophosphate.
(RNA)n residues + NTP
(RNA)n + 1 residues + PPi
•
RNAP holoenzyme (459 kD) .
•
Once RNA synthesis is initiated the subunit dissociates
from the core enzyme which carries out the
polymerization.
Page 1221
Table 31-1
Components of E. coli RNA Polymerase
Holoenzyme.
Page 1222
Figure 31-9 An electron micrograph of E. coli RNA
polymerase (RNAP) holoenzyme attached to various
promoter sites on bacteriophage T7 DNA.
RNA polymerase
•
(+) strand (sense strand) or coding strand-has the same
sequence as the transcribed RNA.
•
(-) strand (antisense strand) acts as the template.
•
By convention, the sequence template DNA is repesented
by its template (nontemplate strand) so that it will have the
same directionality as the transcribed RNA.
•
RNAP holoenzyme binds to its initiation sites called
promoters that are recognized by the corresponding 
factor.
•
Promoters consist of ~40-bp sequence located 5’ to the
transcription start site.
•
Holoenzyme forms tight complexes with promoters (K ~10-14
M)
Promoter sequences
•
In E. coli, most conserved sequence is a hexamer at about
the -10 region (Pribnow box) of TATAAT.
•
Upstream sequence at -35 is also highly conserved,
TTGACA.
•
Sequence unimportant between -35 and -10 but length is
(16-19 bp).
•
Initiating nucleotide (+1) is always A or G.
•
Additional A+T rich sequence between -40 and -60
upstream promoter (UP) element binds to the C-terminal
domain of RNAP’s  subunits.
•
Promoter mutations that increase or decrease the rate at
which the associated gene is transcribed are called up
mutations and down mutations.
Page 1223
Figure 31-10 The sense (nontemplate) strand
sequences of selected E. coli promoters.
Page 1224
Figure 31-11a
X-Ray structure of Taq RNAP core
enzyme.  subunits are yellow and green,  subunit is
cyan, ¢ subunit is pink,  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) .
•
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 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  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.