Transcript Lecture 27

FCH 532 Lecture 17
Extra Credit Assignment for Friday Mar. 2
DeLisa seminar, 148 Baker, 3:00PM
Study guide 8 posted
Exam scheduled for Friday, March 9-will cover
up to translation (Chapter 32). Amino acid
metabolism will be next exam
Chapter 31
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 (CTD).
 CTD 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 CTD 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 -ribbons or two stranded anti-parallel bsheets.
 -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 (2, 3) 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 (2,
3) 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 
ribbon (yellow) in the DNA’s major groove.
trp operon regulation.
• Encodes 5 polypeptides that make 3 enzymes
mediating the synthesis of tryptophan from chorismate.
•
Under control of trp repressor (homodimer, 107
residues)-binds L-tryptophan to form a complex that
binds to the trp operator to reduce the rate of
transcription.
•
Trp forms a hydrogen bond to DNA phosphate group
increasing the repressor-operator association
(corepressor-acts in conjunction with trp repressor).
•
Controls 2 other operons: trpR and aroH involved in
chorismate synthesis.
trp operon attenuation
• Transcriptional control through which bacteria regulate
the expression of certain operons involved in amino acid
biosynthesis.
•
Discovered with E. coli trp operon-before they thought it
was just the trp repressor responsible for regulating
operon.
•
trp deletion mutants downstream of trpO increased trp
operon expression 6-fold-additional transcriptional
control elements.
•
Sequence analysis revealed trpE is preceeded by a 162
nt leader sequence (trpL).
•
The new control element is located in trpL ~30-60 nt
upstream of trpE.
trp operon attenuation
• When W is scarce, the entire 6720-nt polycistronic trp
mRNA, including trpL is synthesized.
•
As W increases, rate of trp transcription decreases as a
result of the trp-repressor-corepressor complex.
•
Of the trp mRNA that is transcribed, an increasing
amount consists of only a 140-nt segment
corresponding to the 5’ end of trpL.
•
The availability of tryptophan results in the premature
temination of the trp operon transcription.
•
The control element responsible is an attenuator.
Page 1251
Figure 31-39 A genetic map of the E. coli trp operon
indicating the enzymes it specifies and the reactions
they catalyze.
Page 1251
Figure 31-40 The base sequence of the trp operator.
The nearly palindromic sequence is boxed and its –10
region is overscored.
trp operon attenuation: mechanism
• The attenuator transcript has 4 complementary
segments that form one of two sets of mutually
exclusive base paired hairpins.
•
Segments 3 and 4 together with the succeding residues
make a normal rho-independent transcription
terminator: G-C rich sequence that forms a hairpin
with several sequential U residues.
•
Transcription rarely proceeds beyond this termination
site when W is scarce.
•
A section of the leader sequence (segment 1) is
translated to form a 14-residue polypeptide with 2
consecutive Trp residues.
•
This provides a clue to the mechanism.
trp operon attenuation: mechanism
• When an RNAP that has escaped repression initiates
the trp operon transcription, a ribosome attaches the
ribosomal initiation site of trpL mRNA and begins
translation of the leader peptide.
•
When W is abundant, lots of tryptophanyl-tRNATrp, the
ribosome follows closely behind the transcribing RNA
polymerase to sterically block the formation of the 2-3
hairpin.
•
The prevention of the 2-3 hairpin allows the formation of
the 3-4 hairpin which results in the termination of
transcription.
•
If there are low levels of Trp, the ribosome stalls on the
1 position and the 2-3 antiterminator forms allowing
transcription of the trp operon.
Page 1252
Figure 31-41 The alternative secondary structures of
trpL mRNA.
Page 1253
Figure 31-42a
Attenuation in the trp operon. (a)
When tryptophanyl–tRNATrp is abundant, the ribosome
translates trpL mRNA.
Page 1253
Figure 31-42b
Attenuation in the trp operon. (b)
When tryptophanyl–tRNATrp is scarce, the ribosome
stalls on the tandem Trp codons of segment 1.
Page 1253
Table 31-3 Amino Acid Sequences of Some Leader
Peptides in Operons Subject to Attentuation.
Regulation of rRNA synthesis: Stringent Response
•
Under optimal conditions, E. coli divides every 20 min.
•
These cells contain up to 70,000 ribosomes so 35,000
ribosomes must be made per cell division.
•
RNAP can initiate transcription at 1 gene per sec.
•
In order to meet the needs of the cell for ribosomes, there
are multiple copies (7) or the rRNA operon in the E. coli
genome.
•
Rapidly growing cells contain multiple copies of their
replicating chromosomes.
•
The rate of rRNA synthesis is proportional to the rate of
protein synthesis.
•
Stringent response: a shortage of any species of amino
acid charged tRNA that limits the rate of protein synthesis
triggers a metabolic adjustment.
Stringent Response
• Stringent response: a shortage of any species of
amino acid charged tRNA that limits the rate of protein
synthesis triggers a metabolic adjustment.
•
Can cause a 10 to 20-fold reduction in the rate of rRNA
and tRNA synthesis.
•
Stringent control depresses numerous metabolic
processes (DNA replication, biosynthesis of
carbohydrates, lipids, nucleotides, proteoglycans,
glycolytic intermediates) while stimulating other
pathways (amino acid biosynthesis).
Stringent Response
• 2 nucleotides regulate the stringent response
ppGpp and pppGpp. Together known as (p)ppGpp.
•
The accumulation and decay of these regulates the
stringent response.
•
Relaxed control mutants designated relA-, do not
exhibit the stringent response-lack (p)ppGpp.
•
(p)ppGpp inhibits the transcription of rRNA genes but
stimulates transcription of the trp and lac operons.
•
Stringent factor (RelA) catalyzes the reaction:
ATP + GTP
AMP + pppGpp
ATP + GDP
AMP + ppGpp
Stringent Response
• Several ribosomal proteins convert pppGpp to ppGpp.
•
Stringent factor is only active in association with a
ribosome that is actively engaged in translation.
•
(p)ppGpp synthesis occurs when ribosome binds its
mRNA specified but uncharged tRNA.
•
The binding of a specified and charged tRNA greatly
reduces the rate of (p)ppGpp synthesis.
•
(p)ppGpp degradation is catalyzed by the spoT gene
product.
Eukaryotic RNA polymerases
1. RNA polymerase I (RNAP I, Pol I, RNAP A)-located in
nucleoli, synthesizes rRNA precursors.
2. RNA polymerase II (RNAP II, Pol II, RNAP B)- in the
nucleoplasm, synthesizes mRNA precursors.
3. RNA polymerase III (RNAP III, Pol III, RNAP C)- alos
in nucleoplasm, synthesizes precursors of 5S rRNA,
tRNAs, and other small nuclear and cytosolic RNAs.
Have greater subunit complexity than prokaryotic RNAP.
Molecular masses up to 600 kD
Page 1232
Table 31-2
RNA Polymerase Subunitsa.
RNAP II
• RNAP II is regulated by it’s C-terminal domain (CTD).
•
Contains 52 highly conserved repeats of the heptad
PTSPSYS in mammals (26 in yeasts).
•
Subject to phosphyorylation/dephosphorylation by CTD
kinases and CTD phosphatases.
•
RNAP II initiates transcription when CTD is
unphosphorylated.
•
RNAP II commences elongation only after the CTD is
phosphorylated.
•
Crystal structures shows it is similar to the Taq RNA
polymerase.
•
RNAP II binds 2 Mg2+ ions in the active site.
•
Several subunits not observed in Taq RNA polymerase.
Page 1233
Figure 31-20a
The X-Ray structure of yeast RNAP
II that lacks its Rpb4 and Rpb7 subunits.
Page 1233
Figure 31-20b
The X-Ray structure of yeast RNAP
II that lacks its Rpb4 and Rpb7 subunits. (b) View of the
enzyme from the right in Part a showing its DNA binding
cleft.
RNAP II
• Eukaryotic RNAPs cannot independently bind to their
target DNA.
•
They must be recruited to target promoters through
complexes of transcription factors.
•
RNAP II can initiate transcription on a dsDNA with a
3’ single-stranded tail at one end.
Page 1234
Figure 31-21a
Secondary structure of an RNAP II
elongation complex. Template DNA cyan, nontemplate
DNA green, and newly synthesized RNA red.
Page 1234
Figure 31-21c
Cutaway schematic diagram of the
transcribing RNAP II elongation complex.
Amatoxins
• Amanita phalloides (death cap) mushroom produces
bicyclic octapeptides known as amatoxins. -amanitin
is shown:
• Forms a tight 1:1 complex
with RNAP II (K = 10-8 M)
and RNAP III (K = 10-6 M).
•
Binding slows RNAP
synthesis from 1000s to a
few nt per min.
•
RNAP I, mitochondrial,
chloroplast and
prokaryotic RNAPs are
insensitive.
Page 1235
Figure 31-22 The proposed transcription cycle and
translocation mechanism of RNAP. (a) Nucleotide
addition cycle. (b) RNA · DNA complex in RNAP II.
Eukaryotic promoters
• Mammalian RNA polymerase I has a bipartite promoter
consisting of a core promoter element (-31 to +6) and
upstream promoter element (-187 to -107) ; GC-rich,
recruits transcription factors.
•
RNA polymerase II promoters are longer than
prokaryotic promoeters.
•
Constitutively expressed genes have 1 or more copies
of GGGCGG (GC box) located upstream of
transcription start site.
Eukaryotic promoters
• Most structural genes have a conserved AT-rich
sequence 25-30 bp upstream from transcription start
site.
•
TATA box (sometimes called Hogness box)-resembles 10 region of prokaryotic promoters.
•
Deletion of TATA box does not eliminate transcription;
instead generates differences in transcription start site.
•
-50 to -110 also contains promoter elements, example:
globin genes have a conserved CCAAT box -70 to -90.
•
Globins also have the CACCC box upstream from the
CCAAT box.
Page 1236
Figure 31-23 The promoter
sequences of selected
eukaryotic structural genes.
Enhancers in eukaryotes
• Enhancers are transcriptional control regions that can
be located several thousand base pairs upstream or
downstream from the transcription start site.
•
Enhancers must be associated with promoters to trigger
site-specific and strand -specific transcription initiation.
•
Required for full activities from promoters.
•
Enhancers are recognized by specific transcription
factors that stimulate RNA polymerase II to bind to the
corresponding but distant promoters.
•
Mediate selective gene expression in eukaryotes.
RNA processing
• Most (all in eukaryotes) primary transcripts (the RNA
molecule as encoded in the DNA) function after being
altered covalently by one or more of the following
processing steps: removal of 5’ and/or 3’ nucleotides,
addition of nucleotides at the 5’ and/or 3’ ends, covalent
modification of the bases, or “editing” of the nucleotide
sequence (changing the information content of the RNA).
• Require one or more of the following activities:
capping enzyme, polyA polymerase, specific
ribonuclease, RNA ligase, spliceosomes-specialized RNA
processing complexes consisting RNA and protein, and
catalytic RNA-ribozymes
Messenger RNA processing in
eukaryotes
•
•
•
•
1. Capping
2. Polyadenylation
3. Splicing
4. Introns early or introns late?
A gene is not necessarily co-linear with its encoded
protein (for eukaryotic genes only)
UAG
AUG …
5’ UTR
3’ UTR
Green=ORF(open reading frame)
The linear order is never violated; it is simply interrupted
Eukaryotic mRNA is capped
• 5’ cap is a reversed
guanosine residue so
there is a 5’-5’ linkage
between the cap and
the first sugar in the
mRNA.
• Guanosine cap is
methylated. (cap-0)
• First (cap-1)and
second nucleosides
(cap-2) in mRNA may
be methylated
Capping mRNA-cont.
•
1.
Capping involves several enzymatic reactions
Removal of the leading phsophate group from the 5’ terminal
triphosphate group by RNA triphosphatase
2. Guanylation of the mRNA by capping enzyme; requires GTP and yields
the 5’-5’ triphosphate bridge and PPi
3. Methylation of guanine by guanine-7-methyltransferase (methyl from
SAM).
4. The O2’ methylation of mRNAs first and maybe second nucleotide by a
SAM requiring 2’-O-methyltransferase.
Capping enzyme and guanine-7-methyltransferase bind to phosphorylated
CTD of RNAPII.
Poly (A) tails
• Eukaryotic mRNAs are monocistronic.
• Sequences signaling transcriptional termination not
identified; not precise.
• Mature mRNAs have well defined 3’ ends of poly A tails
(~250 in mammals and ~80 in yeast).
• Added in two reactions by a complex of at least 6 proteins.
Polyadenylation of eukaryotic mRNA
•
•
•
Transcript is cleaved to yield a free 3’OH group at a specific site 15-25 nt past
an AAUAAA site and within 50 nt before
U-rich or G-U rich sequence
Endonuclease that cleaves RNA
uncertain but requires cleavage factors
(CFI and CFII).
Poly(A) tail is made from ATP by
poly(A) polymerase (PAP) which is
recruited by the cleavage and
polyadneylation specificity factor
(CPSF).
Polyadenylation -cont.
• Polyadenylation is correlated with messenger
half-life:
• No/short polyA>short mRNA life-time; long
polyA>long life-time of mRNA.
• Short half-life:little protein; long half-life, more
protein
Purification of messenger RNA
using oligo dT columns
Total cellular RNA;
apply at room temperature to anneal polyA tail to oligo(dT)
Oligo(dT)
attached to
cellulose
AAAA..
TTTT
polyA binds to
oligo(dT) on
column
} non-polyA
RNA flows
through
•Why does polyA anneal to oligo(dT) at room temp?
5’
TTTT
65oC
AAAAA..
polyA mRNA elutes at
high temperature
Messenger RNA splicing
•
•
•
Pulse-chase labeling studies
indicated two important
characteristics of eukaryotic RNA:
1. Most of the rapidly-synthesized
RNA in the nucleus never reached the
cytoplasm.
2. The rapidly-synthesized nuclear
RNA was much larger on average
than cytoplasmic RNA.
A pulse-chase experiment
Cells
32P-RNA
32P-RNA
+ 32P phosphate for
30 seconds (“pulse”)
+31P for 10
minutes (“chase”)
Heteroduplex analysis-the first
evidence of splicing
Viral DNA
Virus-infected cells
denature
Isolate mRNA
• The
annealing of
viral DNA
also
occurred
Anneal, shadow with heavy metals,
analyze in EM
Heteroduplex analysiscontinued
Expected:
• The appearance of Dloops (displacement
loops) in the DNARNA hybrid indicated
the presence of
regions of DNA that
were transcribed, but
later discarded from
the RNA product.
Intron sequences not present in
mature mRNA
Observed:
Eukaryotic genes: alternating expressed
and unexpressed sequences
• Most eukaryotic genes are intersperesed with unexpressed regions.
• Primary sequences vary greatly in length (~2000 - 20,000 nt); much
larger than expected based on the proteins encoded-heterogeneous
nuclear RNA (hnRNA).
• premRNAs are processed by the excision of internal sequences
(introns) which can be 4-10 times longer in aggregate length than the
expressed seqeuences (exons).
DNA-RNA
heteroduplexes
• Annealing RNA from
virus-infected cells
with viral DNA
revealed the
Interpretation of the EM image:
existence of seven
introns-transcribed
regions of the DNA
removed from the
mature mRNA. How
would you prove the loops were
DNA and not RNA?