Chapter 6 Flashcards
The Mechanism of Transcription in Bacteria (333 cards)
1
Q
By 1969, the polypeptides that make up the E. coli RNA polymerase had been identified by
A
SDS polyacrylamid gel electrophoresis
SDS-PAGE
2
Q
In the E. coli RNA polymerase, there are very large subunits:
A
beta and beta-prime, with molecular masses of 150 and 160 kD, respectively.
3
Q
In the E. coli RNA polymerase, there are small subunits:
A
sigma, alpha, and omega, with molecular masses of 70, 40, and 10 kD, respectively.
4
Q
In contrast to the other subunits, the omega-subunit is not required for
A
cell viability, nor for enzyme activity in vitro. It seems to play a role in enzyme assembly.
5
Q
The subunit content of an RNA polymerase holoenzyme is
A
Beta-prime, Beta, Sigma, two Alpha and Omega.
6
Q
From the SDS-PAGE analysis, the remainder of the enzyme, except of the sigma-subunit, is called
A
the core polymerase.
7
Q
Whereas the holoenzyme could transcribe intact phage T4 DNA in vitro quite actively,
A
the core enzyme had little ability to do this.
8
Q
Core polymerase retained
A
its basic RNA polymerizing function because it could still transcribe highly nicked templates very well.
9
Q
Adding sigma back to the core reconstituted the enzyme’s ability to
A
transcribe unnicked T4 DNA.
10
Q
The holoenzyme transcribed only
A
a certain class of T4 genes, but the core showed no such specificity.
11
Q
Not only is the core enzyme indiscriminate about the T4 genes it transcribes, it also transcribes
A
both DNA strands.
12
Q
The core enzyme was found to transcribe both DNA strands by hybridizing
A
the labeled product of the holoenzyme or the core enzyme to authentic T4 phage RNA and then checking for RNase resistance.
13
Q
The two RNAs were attempted to base-pair together and form
A
an RNase-resistant ds-RNA.
14
Q
Because authentic T4 RNA is made asymmetrically, it should not
A
hybridize to T4 RNA made properly in vitro because this RNA is also made asymmetrically and is therefore identical, not complementary, to the authentic RNA.
15
Q
If the RNA is made symmetrically in vitro, up to half of it will be
A
complementary to the in vivo RNA and will be able to hybridize to it and thereby become resistant to RNase.
16
Q
30% of the labeled RNA made by the core polymerase in vitro became
A
RNase-resistant after hybridization to authentic T4 RNA.
17
Q
Depriving the holoenzyme of its sigma-subunit leaves
A
a core enzyme with basic RNA synthesizing capability, but lacking specficity.
18
Q
Adding sigma back restores
A
specificity.
19
Q
The key player in the transcription process is
A
RNA polymerase.
20
Q
The E. coli enzyme is composed of a core, which contains the basic transcription machinery, and
A
a sigma-factor, which directs the core to transcribe specific genes.
21
Q
Nicks and gaps in DNA provide ideal initation sites for
A
RNA polymerase, even core polymerase.
22
Q
When sigma was present, the holoenzyme could recognize
A
the authentic RNA polymerase binding sites on the T4 DNA, called promoters, and begin transcription there.
23
Q
Transcription that begins at promoters in vitro is
A
specific and mimics the initiation that would occur in vivo.
24
Q
Sigma operates by directing
A
the polymerase to initiate at specific promoter sequences.
25
To measure how tightly holoenzyme and core enzyme bind to DNA, these enzymes were isolated and bound to 3H-labeled T7 phage DNA. Then, by adding a great excess of unlabeled T7 DNA, any polymerase that dissociated from a labeled DNA had a much higher chance of rebinding to an unlabeled DNA than to a labeled one. The result was obtained from
passing the mixture through nitrocellulose filters after varying lengths of time. THe labeled DNA would bind to the filter only if it was still bound to polymerase. Thus, this assay measured the dissociation rate of the polymerase-DNA complex.
26
The sigma-factor can promote
tight binding, at least to certain DNA sites.
27
In the assay that allowed the polymerase to bind to unlabeled DNA firstly, measuring the dissociation of the first polymerase, it was revealed that
the holoenzyme, as well as the core, had loose binding sites on the DNA.
28
The holoenzyme finds two kinds of binding sites on T7 DNA:
tight binding sites and loose ones.
29
The core polymerase is capable of binding
only loosely to the DNA.
30
The holoeznyme, but not the core, can recognize
promoters.
31
The tight binding sites are probably
promoters.
32
The loose binding sites represent
the rest of the DNA.
33
The tight complexes between holoenzyme and T7 DNA could initiate transcription immediately on addition of
nucleotides, which reinforces the conclusion that the tight binding sites are indeed promoters.
34
If the polymerase had been tightly bound to sites remote from the promoters, a lag would have occurred while
the polymerases searched for initiation sites.
35
The loose sites are found virtually
everywhere on the DNA and are therefore nonspecific.
36
The inability of the core polymerase to bind to the tight binding sites accounts for its
inability to transcribe DNA specifically, which requires binding at promoters.
37
The effect of temperature on binding of holoenzyme to T7 DNA is that
tighter binding at elavated temperature.
38
RNA polymerase holoenzyme binds loosely to DNA at first. It either binds
initially at a promoter or scans along the DNA until it finds one.
39
The complex with holoenzyme loosely bound at the promoter is called
a closed promoter complex, because the DNA remains in closed ds-form.
40
The holoenzyme can melt a short region of the DNA at the promoter to
form an open promoter complex in which the polymerase is bound tightly to the DNA. This is called an open promoter complex because the DNA has to open up to form it.
41
The sigma-factor allows
initiation of transcription by causing the RNA polymerase holoenzyme to bind tightly to a promoter.
42
The tight binding of RNA polymerase holoenzyme to a promoter depends on
local melting of the DNA to form an open promoter complex and is stimulated by sigma.
43
The sigma-factor can therefore select which genes will be
transcribed.
44
Several E. coli and phage promoters were compared and found a region in common:
a sequence of 6 or 7 bp centered approximately 10 bp upstream of the start of transcription. It is called -10 box.
45
Another short sequence centered approximately 35 bp upstream of the transcription start site is known as
the -35 box.
46
Thousands of promoters have been examined and
a typical, or consensus sequence for each of these boxes has emerged.
47
The probabilities are such that one rarely finds
-10 or -35 boxes that match the consensus sequences perfectly.
48
When such perfect matches are found, they tend to occur in
very strong promoters that initiate transcription unusually actively.
49
Mutations that destroy matches with the consensus sequences tend to be
down mutations. That is, they make the promoter weaker, resulting in less transcription.
50
Mutations that make the promoter sequences more like the consensus sequences usually make the promoters
stronger; these are called up mutations.
51
The spacing between promoter elements is also important, and deletions or insertions that move
the -10 and -35 boxes unnaturally close together or far apart are deleterious.
52
In addition to the -10 and -35 boxes, which we can call core promoter elements, some very strong promoters have
an additional element farther upstream called an UP element.
53
E. coli cells have seven genes called
rrn genes that encode rRNAs.
54
Under rapid growth conditions, when rRNAs are required in abundance, the seven genes of
rrn genes account for the majority of the transcription occurring in the cell.
55
Upstream of the core promoter, there is an UP element between positions
-40 and -60. It stimulates transcription of the gene by a factor of 30 in the presence of RNA polymerase alone.
56
Because it is recognized by the polymerase itself, the UP element is concluded to be
a promoter element.
57
The RNA promoter is also associated with three
Fis sites between positions -60 and -150, which are binding sites for the transcription-activator protein Fis.
58
The Fis sites, because they do not bind to RNA polymerase itself, are not
classical promoter elements, but instead are members of transcription-activating DNA elements class, called Enhancers.
59
The E. coli rrn promoters are also regulated by
a pair of small molecules: the initating NTP and an alarmone, guanosine 5'-diphosphate 3'-disphosphate (ppGpp).
60
An abundance of iNTP indicates that
the concentration of nucleotides is high,and therefore it is appropriate to synthesize plenty of rRNA.
61
iNTP stabilizes
the open promoter complex, stimulating transcription.
62
When cells are starved for amino acids, protein synthesis cannot occur readily and
the need for ribosomes and rRNA decreases.
63
Ribosomes sense the lack of amino acids when uncharged tRNAs bind to
the ribosomal site where aminoacyl-tRNAs would normally bind.
64
Under the amino acid shortage, a ribosome-associated protein called RelA receives the "alarm" and produces
the "alarmone" ppGpp, which stabilizes open promoter complexes whose lifetimes are normally short, thus inhibiting transcription.
65
The protein DskA plays an important role, in which binds to RNA polymerase and reduces
the lifetimes of the rrn open promoters to a level at which they are responsive to changes in iNTP and ppGpp concentrations.
66
DskA is required for the regulation of rrn transcription by
the two small molecules, iNTP and ppGpp.
67
rrn transcription is insensitive to iNTP and ppGpp in
mutants lacking DskA.
68
Bacterial promoters contain two regions centered approximately at
-10 and -35 bp upstream of the transcription start site.
69
In E. coli, these bear a greater or lesser resemblance to two consensus sequences:
TATAAT and TTGACA, respectively.
70
In general, the more closely regions within a promoter resemble these consensus sequences,
the stronger that promoter will be.
71
Some extraordinarily strong promoters contain
an extra element (an UP element) upstream of the core promoter, making the promoters even more attractive to RNA polymerase.
72
Transcription from the rrn promoters responds positively to
increases in the concentration of iNTP, and negatively to the alarmone ppGpp.
73
Incubating E. coli RNA polymerase with DNA bearing a mutant E. coli lac promoter known as the lac UV5 promoter, heparin was added as well to
compete with DNA in binding tightly to free RNA polymerase. The heparin prevented any reassociation between DNA and polymerase released at the end of a cycle of transcription.
74
Measuring the amounts of the oligonucleotides and compared them to the number of RNA polymerases, it was found that
many oligonucleotides every polymerase, because the heparin in the assay prevented free polymerase from reassociating with the DNA.
75
The polymerase makes
many small, abortive transcripts without ever leaving the promoter.
76
The transcription initiation is done in four steps:
1) formation of a closed promoter complex;
2) conversion of the closed promoter complex to an open promoter complex;
3) polymerizing the first few nucleotides (up to 10) while the polymerase remains at the promoter, in an initial transcribing complex;
4) promoter clearance, in which the transcript becomes long enough to form a stable hybrid with the template strand.
77
The transcription initiation helps
to stabilize the transcription complex, and the polymerase changes to its elongation conformation and moves away from the promoter.
78
Because sigma directs tight binding of RNA polymerase to promoters, it places
the enzyme in a position to initiate transcription - at the beginning of a gene.
79
The first nucleotide incorporated into an RNA retains
all three of its phosphates, whereas all other nucleotides retain only their alpha-phosphate.
80
By incubating polymerase core in the presence of increasing amounts of sigma in two separate sets of reactions, some reactions had the labeled nucleotide being
[14C]ATP, which is incorporated throughout the RNA and therefore measures elongation, as well as initiation, of RNA chains.
81
By incubating polymerase core in the presence of increasing amounts of sigma in two separate sets of reactions, other reactions had the labeled nucleotide being
[gamma-32P]ATP or GTP, whose label should be incorporated only into the first position of the RNA, and therefore is a measure of transcription initiation.
82
sigma stimulated the incorporation of both
14C- and gamma-32P-labeld nucleotides, which suggests that sigma enhanced both initiation and elongation.
83
Initiation is the rate-limiting step in
transcription.
84
sigma, could appear to stimulate elongation by stimulating
initiation and thereby providing more initiated chains for core polymerase to elongate.
85
sigma does not accelerate the rate of RNA chain growth, because, when the number of RNA chains is held constant, sigma did not
affect the length of the RNA chains.
86
The number of RNA chains is held constant by allowing
a certain amount of initiation to occur, then blocking any further cahin initation with the antibiotic rifampicin, which blocks bacterial transcription initiation, but not elongation.
87
sigma does not stimulate
elongation, and the apparent stimulation is simply an indirect effect of enhanced initiation.
88
sigma stimulates
initiation, but not elongation, of transcription.
89
When the transcription reaction is ran at low ionic strength, RNA polymerase core is prevented from
dissociationg from DNA template at the end of a gene.
90
The prevention of RNA polymerase core to dissociate from DNA template at the end of a gene caused
transcription initiation (as measured by the incorporation of gamma-32P-labeled purine nucleotides into RNA) to slow to a stop.
91
When a new core polymerase is added with the old RNA polymerase still bound to DNA, transcription began
anew, associating with sigma that had been released from the original holoenzyme.
92
The new transcription could occur on
a different kind of DNA added along with the new core polymerase.
93
sigma cycles from one core to another is called
"sigma cycle"
94
When rifampicin added, along with the core polymerase, which came from a rifampicin-resistant mutant, transcription still occurred, because
the sigma was from the original rifampicin-sensitive polymerase. The rifampicin resistance in the renewed transcrption must have been conferred by the newly added core.
95
At some point after sigma has participated in initiation, it appears to
dissociate from the core polymerase, leaving the core to carry out elongation.
96
sigma can be reused by different core polymerase, and the core, not sigma, governs
rifampicin sensitivity or resistance.
97
Sigma dissociates from the core as
the polymerase undergoes promoter clearance and switches from initiation to elongation mode. It is known as the obligate release version of the sigma-cycle model.
98
sigma is involved in pausing at position +16/+17 downstream of the late promoter (Pr') in lambda phage, implying that
sigma is still attached to core polymerase at the position, well after promoter clearance has occurred. This supports the stochastic release model.
99
The stochastic release model holds that sigma is indeed released from the core polymerase, but there is
no discrete point during transcription at which this release is required; rather, it is released randomly.
100
To test the obligate release hypothesis, a technique called
Fluorescence Resonance Energy Transfer (FRET) was adopted.
101
FRET allows the position of sigma relative to a site on the DNA to be measured without
using separation techniques that might temselves displace sigma from core.
102
The FRET technique relies on the fact that
two fluorescent molecules close to each other will engage in transfer of resonance energy, and the efficency of this energy transfer will decrease as the two molecules move apart.
103
FRET was measured with fluorescent probes on both sigma and DNA, in which
the probe on sigma serves as the fluorescence donor, and the probe on the DNA serves as the fluorescence acceptor.
104
Sometimes the probe on the DNA was at the 5', or upstream end, called
trailing edge FRET, which allowed the investigators to observe the drop in FRET as the polymerase moved away form the promoter and the 5'-end of the DNA.
105
Sometimes the probe on the DNA was at the 3', or downstream end, called
leading edge FRET, which allowed the investigators to observe the increase in FRET as the polymerase moved toward the downstream end.
106
The trailing-edge FRET strategy does not distinguish between
one model in which sigma dissociates from the core, and a second model in which sigma does not dissociate, after promoter clearance.
107
In both cases of the trailing-edge FRET strategy, the donor probe on sigma gets farther away from
the acceptor probe at the upstream end of the DNA after promoter clearance and the FRET efficiency therefore decreases.
108
In the trailing-edge FRET, the FRET efficiency does decrease with time when
the probe on the DNA is at the upstream end.
109
The leading-edge FRET strategy can distinguish between
the two models of sigma dissociating and not dissociating
110
In the leading-edge FRET strategy, if sigma dissociates from the core, then FRET efficiency should
decrease. But if sigma is not released from the core, it should move closer to the probe at the downstream end of the DNA with time, and FRET efficiency should increase.
111
The magnitude of the FRET efficiency increase suggests that
100% of the complexes after promoter clearance still retained their sigma-factor.
112
Open promoter complexes are formed in solution. Then, heparin is added to bind to
any uncompexed polymerase, which is subjected to nondenaturing electrophoresis in a PAAM gel.
113
The complexes in the gel is isolated and added
transcription buffer. FRET effiency on RP0 is measured.
114
By adding three nucleotides, the complex is allowed to
move the polymerase downstream, measuring FRET efficiency on the elongation complex.
115
Complexes between holoenzyme and a DNA containing one promoter was added with
three out of four nucleotides to allow the polymerase to move to position +32.
116
The elongation complex called EC23 was purified by
annealing the upstream end of the elongating RNA to a complementary oligonucleotide attached to resin beads. This is because they are the only ones with a nascent RNA that can bind to the complementary oligonucleotide.
117
The elongation complexes purified and released from the beads with nuclease, and performed
an immunoblot to identify the proteins associated with the complexes.
118
While showing that the purified EC32 complexes contained at least some sigma, it was shown that
complexes isolated from stationary phase cells contained 33% of the full complement of sigma per complex, and complexes isolated from exponential phase cells contained 6% of the full complement of sigma per complex.
119
sigma could aid considerably in
reinitiation of transcription, because the association of core with sigma is the rate-limiting step in transcription initiation.
120
Stochastic release version of the sigma-cycle is consistent with the findings of
sigma release at multiple points throughout transcription.
121
Elongation complexes after only 32-nt of transcription, which could be too early in transcription to see complete sigma release, is collected; and, while there is
no significant sigma dissociation after 50 nt of transcription observed, a DNA template that contributed to this phenomenon was used.
122
The promoter containing a second -10-like box just downstream of the transcription start site causes
pausing that depends on sigma, and indeed appears to aid in sigma retention.
123
When the second-10-like box was mutated, the FRET signal
decreased, and sigma dissociation increased more than 4-fold.
124
When fluorescent labels were on sigma and core, rather than sigma and DNA, their FRET signal
decreased with increasing transcript length.
125
Some sigma was dissociating from core during the transcription process, and that the DNA sequence can
influence the rate of such dissociation.
126
To probe further the validity of the sigma-cycle hypothesis,
leading and trailing edge single-molecule FRET analysis with alternating-laser excitation was used.
127
For leading edge FRET, the leading edge of sigma was tagged with the donor fluorophore and a downstream DNA site with the acceptor.
For trailing edge FRET, the trailiing edge of sigma was tagged with the donor and an upstream DNA site with the acceptor fluorophore.
128
In the FRET, fluorescence efficiency and "stoichiometry," or the presence of one or both of the fluorophores (donor and acceptor) in a small excitation volume, which should have
at most one copy of the elongation complex at any given time.
129
In the FRET, the excitation was switched rapidly between the two fluorophore, while stalling the elongation complex at various points by
coupling the E. coli lacUV5 promoter to various G-less cassettes and leaving out CTP in the transcription reaction.
130
By measuring both fluorescence efficiency and stoichiometry for the same elongation complex, they could tell:
1) how far transcription had progressed (by the fluorescence efficiency, which grows weaker in trailing edge FRET, and stronger in leading edge FRET, as transcription progresses); and
2) whether or not sigma had dissociated from core (by the stoichiometry, which should be approximately 0.5 for holoenzyme, but nearer 0 for core alone and 1.0 for sigma alone).
131
sigma did indeed remain associated with the great majority of
elongation complexes that had achieved promoter clearance (with transcripts 11 nt long).
132
About half of halted elongation complexes with longer transcripts had lost
their sigma-factors, in accord with the stochastic release model.
133
Some elongation complexes may retain their
sigma-factors throughout the transcription process. If that is true, these elongation complexes are avoiding the sigma cycle altogether.
134
The sigma-factor appears to be released from the core polymerase, but
not usually immediately upon promoter clearance. Rather, sigma seems to exit from the elongation complex in a stochastic manner during the elongation process.
135
Local melting of DNA occurs on
tight binding to polymerase, because such DNA melting is essential with exposing bases of the template strand for basepairing.
136
The bound E.coli RNA polymerase to a restriction fragment containing three phage T7 early promoters caused
the increase in the DNA's absorbance of 260-nm light, indicating the number of base pairs that are opened.
137
Each polymerase caused
a separation of about 10 bp.
138
End-labeled promoter DNA was added with RNA polymerase to form an open promoter complex. Then, when the strands separate, the N1 of adenine - normally involved in hydrogen bonding to a T in the opposite strand - becomes
sensible to attack by dimethyl sulfate to methylate the exposed adenines. When the RNA polymerase is removed and the melted region closed up again, the methyl groups prevented proper base-pairing between these N1-methyl-adenines and the thymines in the opposite strand and thus preserved at least some of the ss-character of the formerly melted region.
139
The regionally ss-DNA is treated with
S1 nuclease, which specifically cuts ss-DNA. The enzyme should therefore cut wherever an adenine had been in a melted region of the promoter and had become methylated.
140
A series of end-labeled fragments is produced, each one terminating at
an adenine in the melted region.
141
The labeled DNA fragments can be electrophoresed to
determine their precise lengths, and calculate the position of the melted region.
142
There is a blur of several fragments extending from position +3 to -9, where the each of multiple methylation in the melted region introduced a positive charge and therefore weakened base pairing so much that
few strong pairs could re-form; the whole melted region retained at least partially ss-character and therefore remained open to cutting by S1 nuclease.
143
SV40 DNA was bound with RNA polymerase in the absence of nucleotides to for binary complexes, or in the presence of nucelotides to form ternary complexes. Under the conditions of the experiments,
each polymerase initiated only once, and no polymerase terminated transcription, so all polymerases remained complexed to the DNA. This allowed an accurate assessment of the number.
144
After binding a known number of E. coli RNA polymerases to the DNA, any supercoils that had formed with a crude extract from human cells were
relaxed and the polymerases from the relaxed DNA were removed.
145
The removal of polymerases from the relaxed DNA left
melted regions of DNA, which meant that the whole DNA was underwound.
146
Because the DNA was still a covalently closed circle, the underwinding introduced
strain into the circle that was relieved by forming supercoils.
147
The higher the superhelical content,
the greater the double helix unwinding that has been caused by the polymerase.
148
A polymerase binds at the promoter, melts
17 bp of DNA to form a transcription bubble, and a bubble of this size moves with the polymerase as it transcribes the DNA. It can also increase and decrease within a range of approximately 11-16 nt, according to conditions, including the base sequence within the bubble.
149
On binding to a promoter, RNA polymerase causes melting that has been estimated at
10-17 bp in the vicinity of the transcription start site. This moves with the polymerase, exposing the template strand so it can be transcribed.
150
RNA polymerases cannot work if they do not recognize promoters, so they have evolved to
recognize and bind strongly to them.
151
The polymerase cannot move enough downstream to make a 10-nt transcript without doing one of three things:
1) moving briefly downstream and then snapping back to the starting position (transient excursion); 2) stretching itself by leaving its trailing edge in place while moving its leading edge downstream (inchworming); or
3) compressing the DNA without moving itself (scrunching).
152
The E. coli RNA polymerase achieves abortive transcription by
scruching: drawing downstream DNA into the polymerase without actually moving and loosing its grip on promoter DNA.
153
The scrunched DNA could store enough energy to allow the polymerase to
break its bonds to the promoter and begin productive transcription.
154
Each bacterium has a primary
sigma-factor that transcribes its vegetative genes - those required for everyday growth.
155
The primary sigma's in E coli and B subtilis are called
sigma70 and sigma43, respectively, and called sigmaA because of their primary nature.
156
The sigma's have four common regions, suggesting that they are
important in the function of sigma, and in fact they are all involved in binding to core and positively or negatively, in binding to DNA.
157
Region 1 is found only in
the primary sigma's, preventing from binding by itself to DNA. This is important because free sigma binding to promoters could inhibit holoenzyme binding and thereby inhibit transcription.
158
Region 2 is found in
all sigma-factors and is the most highly conserved sigma region. It can be subdivided into four parts, 2.1-2.4.
159
Region 2.4 is responsible for
a crucial sigma activity, recognition of the promoter's -10 box.
160
Since sigma region 2.4 does recognize the -10 box, the sigma's with similar specificities have
similar regions 2.4, recognizing speicific promoter sequences, including -10 boxes.
161
Region 2.4 of the sigma-factor contains an amino acid sequence that suggests
it can form an alpha-helix, which is a favourite DNA-binding motif, which is consistent with a role of this part of the sigma in promoter binding.
162
Region 3 is
involved in both core and DNA binding.
163
Region 4 is like region 2, which can be
subdivided into subregions. It is playing a key role in promoter recognition.
164
Subregion 4.2 contains a helix-turn-helix DNA-binding domain, which
suggests that it plays a role in polymerase-DNA binding.
165
Subregion 4.2 appears to govern
binding to the -35 box of the promoter.
166
Regions 2.4 and 4.2 of sigma are capable of binding to
promoter regions on their own, but other domains in sigma interferes with this binding.
167
Region 1.1 prevents sigma from binding to
DNA in the absence of core.
168
When sigma associates with core, it changes
conformation, unmasking its DNA-binding domains, so it can bind to promoters.
169
Region 4 protein binds weakly to
nonspecific DNA, but not strongly to tac promoter-containing DNA.
170
The binding between the GST-sigma region 4 proteins and the promoters involves
the -35 box, but not the -10 box.
171
The polymerase holoenzyme can recognize
promoters and form an open promoter complex by melting a short region of the DNA, approximately between positions -11 and +1.
172
One feature of open complex formation is binding of
polymerase to the nontemplate strand in the -10 region of the promoter.
173
The fragment of B' containing amino acids 1-550 caused binding between
sigma and the nontemplate strand DNA (but not the template strand), whereas sigma by itself showed little binding.
174
The very smal 262-309 fragment, with only 48 amino acids, could stimulate
binding very actively, even at room temperature.
175
Mutations in three amino acids in this region (R275, E295, and A302) were known to
interfere with sigma binding to promoters.
176
Comparison of the sequences of different sigma genes reveals
four regions of similarity among a wide variety of sigma-factors.
177
Subregions 2.4 and 4.2 are involved in
promoter -10 and -35 box recognition, respectively.
178
The sigma-factor by itself cannot
bind to DNA, but interaction with core unmasks a DNA-binding region of sigma.
179
In particular, the region between amino acids 262 and 309 of B' stimulates
sigma binding to the nontemplate strand in the -10 region of the promoter.
180
RNA polymerase itself can recognize
an upstream promoter called an UP element.
181
The sigma-factor recognizes
the core promoter elements.
182
The alpha-subunit of the core polymerase recognizes
the UP element.
183
When E. coli strains with mutations in the alpha-subunit were made, some of these were incapable of
responding to the UP element - they gave no more transcription from promoters with UP elements than from those without UP elements.
184
The wild-type polymerase made a footprint in the core promoter and the UP element, but that the mutant polymerase lacking the C-terminal domain of the alpha-subunit made
a footprint in the core promoter only. It indicates that the alpha-subunit C-terminal domain is required for interaction between polymerase and UP elements.
185
Limited proteolysis analysis is used to show that
the alpha-subunit N-terminal and C-terminal domains (the alpha-NTD and alpha-CTD) fold independently to form two domains that are tethered together by a flexible linker.
186
A protein domain is
a part of a protein that folds independently to form a defined structure.
187
Because of their folding, domains tend to resist
proteolysis, so limited digestion with a proteolytic enzyme will attack unstructured elements between domains and leave the domains themselves alone.
188
The alpha-subunit folds into two domains:
a large N-terminal domain encompassing amino acids 8-241, and a small C-terminal domain including amino acids 249-329.
189
The N-terminal and C-terminal domains are joined by
an unstructured linker that can be cleaved in at least three places by the protease used, which must be at least 13 amino acids long (239-251)
190
RNA polymerase binds to a core promoter via
its sigma-factor, with no help from the C-terminal domains of its alpha-subunits, but it binds to a promoter with an UP element using sigma plus the alpha-subunit C-terminal domains.
191
The RNA polymerase alpha-subunit has
an independently folded C-terminal domain that can recognize and bind to a promoter's UP element. This allows very tight binding between polymerase and promoter.
192
After initiation of transcription is accomplished, the core continues to
elongate the RNA, adding one nucleotide after another to the growing RNA chain.
193
The core polymerase contains the RNA synthesizing machinery, so the core is the central player in
elongation.
194
The B- and B'-subunits are involved in
phosphodiester bond formation and DNA binding.
195
The alpha-subunit has several activities, including
assembly of the core polymerase.
196
When the alpha, beta' and sigma subunits from a rifampicin-sensitive bacterium with the beta subunit from a rifampicin-resistant bacterium were combined,
the resulting polymerase was antibiotic-resistant.
197
When the beta-subunit came from an antibiotic sensitive bacterium, the reconstituted enzyme was
antibiotic-sensitive, regardless of the origin of the other subunits.
198
The beta-subunit is the determinant of
rifampicin sensitivity or resistance.
199
Streptolydigin blocks
RNA chain elongation.
200
The beta-subunit also governed
streptolydigin sensitivity or resistance.
201
Rifampicin actually blocks
early elongation, preventing the RNA from growing more than 2-3 nucleotides long. Thus, strictly speaking, it blocks initiation, because initiation is not complete until the RNA is up to 10 nt long, but its effect is really on the elongation that is part of initiation.
202
The beta-subunit is the only core subunit labeled by
any of the affinity reagents, suggesting that this subunit is at or very near the site where phosphodiester bond formation occurs.
203
Some labeling of sigma suggests that
it too may lie near the catalytic center.
204
The core subunit beta lies near
the active site of the RNA polymerase where phosphodiester bonds are formed.
205
The sigma-factor may also be near
the nucleotide-binding site, at least during the initiation phase.
206
The B and B[ subunits are involved in
DNA binding.
207
The RNA product forms
an RNA-DNA hybrid with the DNA template strand for a few bases before peeling off and exiting from the polymerase.
208
Using a transcript walking technique, with RNA-DNA cross-linking, an RNA-DNA hybrid occur within
the elongation complex with 8-9 bp long.
209
The transcript walking technique works as follwos:
using gene cloning techniques to engineer an RNA polymerase with six extra histidines at the C-terminus of the Beta-subunit.
210
The string of histidines allowed to tether the polymerase to a nickel resin, because of
its affinity for divalent metals such as nickel.
211
The substrates were changed rapidly by washing the resin, with the polymerase stably attached, and then adding fresh reagents of nucleotides,
the polymerase could walk to a particular position on the template. Then the first set of nucleotides were washed aaway and added a second subset to walk the polymerase to a defined position further downstream.
212
The RNA-DNA hybrid within the E. coli elongation complex extends from
position -1 to position -8 or -9 with respect to the 3'-end of the nascent RNA. The T7 hybrid apepars to be 8 bp long;
213
The overall shape of the polymerase resembles
an open crab claw.
214
Half of the claw is composed primarily of
the beta-subunit, and the other half is composed primarily of the beta-subunit.
215
The two alpha-subunits lie at
the "hinge" of the claw, with one of them aI associated with the beta-subunit, and the other aII associated with the beta'-subunit. The small omega-subunit is at the bottom, wrapped around the C-terminus of Beta'.
216
The polymerase contains a channel, about 27A wide, between
the two parts of the claw, and the template DNA presumably lies in this channel.
217
The catalytic center of the polymerase is marked by
the Mg2+ ion.
218
An invariant string of amino acids (NADFDGD) occurs in
the beta'-subunit from all bacteria examined so far, and it contains three aspartate residues (D) suspected of chelating a Mg2+ ion.
219
Mutations in any of the Asp residues are
lethal. These Asp residues are essential for catalytic activity, but not for tight binding to DNA.
220
The side chains of the three Asp residues are indeed coordinated to
a Mg2+ ion. Thus, the three Asp residues and a Mg2+ ion are at the catalytic center of the enzyme.
221
Rifampicin-binding site in the part of the beta-subunit forms
the ceiling of the channel through the enzyme.
222
Rifampicin allows RNA synthesis to begin, but blocks
elongation of the RNA chain beyond just a few nucleotides.
223
Rifampicin has no effect on elongation once
promoter clearance has occurred.
224
Rifampicin bound in the channel blocks
the exit through which the growing RNA should pass, and thus prevents growth of a short RNA.
225
Once an RNA reaches a certain length, it might block access to
the rifampicin-binding site, or at least prevent effective binding of the antibiotic.
226
The antibiotic lies in the predicted site in such a way that it would block
the exit of the elongating transcript when the RNA reaches a length of 2 or 3 nt.
227
X-ray crystallography on the RNA polymerase core has revealed
an enzyme shaped like a crab claw designed to grasp DNA.
228
A channel through the enzyme includes
the catalytic center (a Mg2+ ion coordinated by three Asp residues), and the rifampicin-binding site.
229
To generate a homogeneous holoenzyme-DNA complex, the T. aquaticus holoenzyme was bound to
the "fork-junction" DNA, that is mostly double-stranded, including the -35 box, but has a single-stranded projection on the nontemplate strand in the -10 box region, beginning at position -11.
230
The fork-junction DNA stimulates the character of the promoter in
the open promoter complex, and locks the complex into a form resembling RP0.
231
The DNA stretches across the top of the polymerase where
the sigma-subunit is located. In fact, all of the specific DNA-protein interactions involve sigma, not core.
232
sigma region 2.4 recognizes
the -10 box of the promoter.
233
Mutations in Gln 437 and Thr 440 of E. coli sigma70 can suppress
mutations in position -12 of the promoter, suggesting an interaction between these two amino acids and the bases at position -12.
234
Gln 437 and Thr 440 in E. coli sigma70 correspond to
Gln 260 and Asn 263 of T. aquaticus sigmaA, expecting these two amino acids to be close to the base at position -12 in the promoter.
235
While Gln 260 is indeed close enough to contact base -12, Asn 263 is
too far away to make contact in this structure, but a minor movement, which could easily occur in vivo, would bring it close enough.
236
Three highly conserved aromatic residues in E. coli sigma70 have been implicated in
promoter melting. These amino acids presumably bind the nontemplate strand in the -10 box in the open promoter complex.
237
The amino acids are indeed in position to interact with the ss-nontemplate strand in
the RF complex.
238
Trp 256 is neatly positioned to stack with
base pair 12, which is the last base pair before the melted region of the -10 box.
239
Trp 256 would substitute for a base pair in
position -11 and help met that base pair.
240
Two invariant basic residues in sigma regions 2.2 and 2.3 are known to participate in
DNA binding, because they are well positioned to bind to the acidic DNA backbone by electrostatic interaction.
241
Region 3 of sigma in DNA binding is implicated to
bind to the extended (upstream) -10 box.
242
Glu 281 was found to be important in recognizing
the extended -10 box, while His 278 was implicated in more general DNA-binding in this region.
243
Both Glu 281 and His 278 are exposed on
an alpha-helix, and face the major groove of the extended -10 box.
244
Glu 281 is probably close enough to contact
a thymine at position -13
245
His 278 is probably close enough to contact
the extended -10 box that it could interact nonspecifically with the phosphodiester bond linking the nontemplate strand residues -17 and -18.
246
Specific residues in sigma region 4.2 are instrumental in binding to
the -35 box of the promoter.
247
The -35 box DNA in the RF structure is pushed out of
its normal position relative to sigma4.2 by crystal packing forces.
248
The two Mg2+ ions are held by
the same three aspartate side chains that hold the single Mg2+ ion, in a network involving several nearby water molecules.
249
The crystal structure of a T. aquaticus holoenzyme-DNA complex mimicking an open promoter complex reveals several things.
First, the DNA is bound mainly to the sigma-subuni, which makes all the important interactions with the promoter DNA. Second, the predicted interactions between amino acids in region 2.4 of sigma and the -10 box of the promoter are really possible. Third, three highly conserved aromatic amino acids are predicted to participate in promoter melting, and they really are in a position to do so. Fourth, two invariant basic amino acids in sigma are predicted to participate in DNA binding and they are in a position to do so. A higher resolution crystal structure reveals a form of the polymerase that has two Mg2+ ions, in accord with the probable mechanism of catalysis.
250
THe x-ray crystal structure of the T. thermophilus RNA polymerase elongation complex contained
24 bp of downstream ds-DNA that had yet to be melted by the polymerase, 9 bp of RNA-DNA hybrid, and 7 nt of RNA product in the RNA exit channel.
251
In the elongation complex, a valine residue in the beta' subunit inserts into
the minor groove of the downstream DNA, which could prevent the DNA from slipping backward or forward in the enzyme, or could induce the screw-like motion of the DNA through the enzyme.
252
In the elongation complex, the downstream DNA is ds up to and including the +2 base pair, where
+1 is the position at which the new nucleotide is added. This means that only one base pair is melted and available for base-pairing with an incoming nucleotide, so only one nucleotide at a time can bind specifically to the complex.
253
In the elongation complex, one amino acid in the beta subunit is situated in
a key position right at the site where nucleotides are added to the growing RNA chain. This is Arg 422 of the beta fork 2 loop.
254
The Arg 422 of the beta fork 2 loop makes a hydrogen bond with
the phosphate of the +1 template nucleotide, and van der Waals interactions with both bases of the +2 base pair.
255
The proximity of these amino acids to the active site, and their interactions with key nucleotides there, suggest that
they play a role in molding the active site for accurate substrate recognition. Because this is true, changing Arg 422 increased fidelity.
256
In the elongation complex, the enzyme can accommodate
nine base pairs of RNA-DNA hybrid. At the end of this hybrid, a series of amino acids of the beta' lid stack on base pair -9, stabilizing it, and limiting any further base-pairing.
257
The hybrid vary between
8-10 bp in length, and the beta' lid appears to be flexible enough to handle that kind of variability.
258
Other forces that are at work in limiting the length of the hybrid are:
the tendency of the two DNA strands to reanneal and the trapping of the first displaced RNA base (-10) in a hydrophobic pocket of a beta loop known as switch 3.
259
Five amino acids in a hydrophobic pocket of a beta loop known as swtich 3 make
van der Waals interactions with the displaced RNA bases, stabilizing the displacement.
260
The RNA product in the exit channel is twisted into
the shape it would assume as one-half of an A-form ds-RNA, ready to form a hairpin that will cause pausing, or even termination of transcription.
261
The fit of an RNA hairpin in the exit channel can be accomplished with
only minor alterations of the protein structure.
262
The RNA hairpin could fit with
the core enzyme in much the same way as the sigma-factor fits with the core in the initiation complex.
263
In the absence of streptolydigin, the trigger loop (1221-1266 of the beta' subunit) is fully folded into
two alpha-helices with a short loop in between. This brings the substrate into the active site in a productive way, with two metal ions close enough together to collaborate in forming the phosphodiester bond that will incorporate the new substrate into the growing RNA chain.
264
In the presence of streptolydigin, the antibiotic forces a change in the trigger loop conformation:
the two alpha-helices unwind somewhat to form a larger loop in between. This in turn forces a change in the way the substrate binds to the active site: the base and sugar of the substrate bind in much the same way, but the triphosphate part extends a bit farther away from the active site, taking with it one of the metal ions required for catalysis. Consequently, catalysis is impossible to occur, blocking transcription elongation.
265
The two states of the elongation complex revealed by streptolydigin correspond to two natural states:
a preinsertion state and an insertion state.
266
The substrate normally binds first in
the preinsertion state, and this allows the enzyme to examine it for correct basepairing and for the correct sugar before it switches to the insertion state, where it can be examined again for correct base-pairing with the template base.
267
Structural studies of the elongation complex involving the T. thermophilus RNA polymerase have revealed the following features:
A valine residue in the beta' subunit inserts into the minor groove of the downstream DNA. In this position, it could prevent the DNA from slipping, and it could induce the screw-like motion of the DNA through the enzyme.
268
Only one base-pair of DNA (at position +1) is melted and available for
base-pairing with an incoming nucleotide, so only one nucleotide at a time can bind specifically to the complex.
269
Several forces limit the length of the RNA-DNA hybrid.
One of these is the length of the cavity in the enzyme that accommodates the hybrid. Another is a hydrophobic pocket in the enzyme at the end of the cavity that traps the first RNA base displaced from the hybrid.
270
The RNA product in the exit channel assumes the shape of
one-half of a ds-RNA. Thus, it can readily form a hairpin to cause pausing, or even termination of transcription.
271
Structural studies of the enzyme with an inactive substrate analog and the antibiotic streptolydigin have identified
a preinsertion state for the substrate that is catalytically inactive, but could provide for checking that the substrate is the correct one.
272
When added nucleotides to an open promoter complex, allowing the polymerase to move down the DNA as it began elongating an RNA chain, the same degree of
melting persisted. Furthermore, the crystal structure of the polymerase-DNA complex showed clearly that the two DNA strands feed through separate channels in the holoenzyme.
273
The DNA double helix opens up in front of the moving "bubble" of melted DNA and
closing up again behind.
274
The polymerase moves in
a straight line, with the template DNA rotating in one direction ahead of it to unwind, and rotating in the opposite direction behind it to wind up again, introducing the DNA strain.
275
The strain of unwinding the DNA is relaxed through
a class of enzymes called topoisomerases that can introduce transient breaks into DNA strands.
276
Strain due to twisting a double-helical DNA causes the helix to
tangle up like a twisted rubber band, a process called supercoiling.
277
The supercoiled DNA is called
a supercoil or superhelix.
278
Unwinding due to the advancing polymerase causes
a compensating overwinding ahead of the unwound region.
279
The supercoiling due to overwinding is by convention called
positive.
280
Positive supercoils build up in
front of the advancing polymerase.
281
Negative supercoils form
behind the polymerase.
282
Topoisomerase mutations that cannot relax supercoils result
positive supercoils building up in DNA that is being transcribed AND negative supercoils accumulating during transcription
283
Elongation of transcription invovles
the polymerization of nucleotides as the RNA polymerase travels along the template DNA.
284
As the RNA polymerase advances, the polymerase maintains
a short melted region of template DNA.
285
The DNA unwind ahead of and close up again behind of
the advancing polymerase is required for it to maintain a short melted region of template DNA.
286
The introduction of strain due to winding and unwinding results
the template DNA that is relaxed by topoisomerases.
287
The process of elongation is far from uniform. Instead, the polymerase
repeatedly pauses, and in some cases backtracks, while elongating an RNA chain.
288
Repeated short pauses significantly slow
the overall rate of transcription.
289
Pausing is physiologically important for at least two reasons:
first, it allows translation, an inherently slower process, to keep pace with transcription. second, the first step in termination of transcription is related.
290
Sometimes the polymerase even backtracks by reversing
its direction and thereby extruding the 3'-end of the growing transcript out of the active site of the enzyme.
291
The polymerase backtracking is due to a special condition:
when nucleotide concentrations are severely reduced, or when the polymerase has added the wrong nucleotide to the growing RNA chain.
292
Backtracking is part of a proofreading process in which
auxiliary proteins known as GreA and GreB stimulate an inherent RNase activity of the polymerase to cleave off the end of the growing RNA, removing the misincorporated nucleotide, and allowing transcription to resume.
293
GreA produces only short RNA end fragments 2-3 nt long, and can prevent, but not reverse
transcription arrest.
294
GreB produces RNA end fragments up to
18 nt long, and can reverse arrested transcription.
295
The nascent RNA itself appears to
participate in its own proofreading.
296
By mixing RNA polymerase with a piece of ss-DNA and an RNA that was either perfectly complementary to the DNA or had a mismatched base at its 3'-end; when Mg2+ was added, the mismatched RNA lost a dinucleotide from
its 3'-end, including the mismatched nucleotide and the penultimate (next-to-last) nucleotide. This proofreading did not occur with the perfectly matched RNA. The fact that two nucleotides were lost suggests that the polymerase had backtracked one nucleotide in the mismatched complex.
297
In the backtracked complex, the mismatched nucleotide, because it is not base-paired to the template DNA, is flexible enough to
bend back and contact metal II, holding it at the active site of the enzyme.
298
It is expected to enhance phosphodiester bond cleavage, because
metal II is presumably involved in the enzyme's RNase activity.
299
The mismatched nucleotide can orient a water molecule to make it a better nucleophile in
attacking the phosphodiester bond that links the terminal dinucleotide to the rest of the RNA.
300
RNA polymerase frequently pauses, or even backtracks, during
elongation.
301
Pausing allows ribosomes to
keep pace with RNA polymerase, and it is aso the first step in termination.
302
Backtracking aids proofreading by
extruding the 3'-end of the RNA out of the polymerase, where misincorporated nucleotides can be removed by an inherent nuclease activity of the polymerase, stimulated by auxiliary factors.
303
The polymerase can carry out proofreading, even without auxiliary factors:
the mismatched nucleotide at the end of a nascent RNA plays a role in this process by contacting two key elements at the active site: metal II and a water molecule.
304
When the polymerase reaches a terminator at the end of a gene,
it falls off the template, releasing the RNA.
305
Intrinsic terminators function with
the RNA polymerase by itself without help from other proteins.
306
The other kind of termination depend on
an auxiliary factor called rho, calling rho-dependent terminators.
307
Rho-independent, or intrinsic, termination depends on terminators consisting of two elements:
an inverted repeat followed immediately by a T-rich region in the non-template strand of the gene. The model of termination depends on a "hair-pin" structure in the RNA transcript of the inverted repeat.
308
The E. coli trp operon contains a DNA sequence called
an attenuator that causes premature termination of transcription.
309
The trp attenuator contains the two elements (an inverted repeat and a string of T's in the nontemplate DNA strand) suspected to be
vital parts of an intrinsic terminator.
310
The inverted repeat in the trp attenuator is not perfect, but 8 bp are still possible, and
7 of these are strong G-C pairs, held together by three hydrogen bonds.
311
When the string of eight T's in the nontemplate strand of the terminator was altered to
the sequence TTTGCAA, creating the mutant trp a1419, attenuation was weakened. It is consistent with the weak rU-dA pairs are important in termination, because half of them would bereplaced by strong base pairs in this mutant.
312
Using the trp attenuator as a model terminator, intrinsic terminators have two important features:
1) an inverted repeat that allows a hairpin to form at the end of the transcript; 2) a string of T's in the nontemplate strand that results in a string of weak rU-dA base pairs holding the transcript to the template strand.
313
Hairpins are found to destabilize
elongation complexes that are stalled artificially.
314
Terminators in which half of the inverted repeat is missing still stall at
the strings of rU-dA pairs, even though no hairpin can form.
315
The rU-dA pairs cause the polymerase to pause, allowing
the hairpin to form and destabilize the already weak rU-dA pairs that are holding the DNA template and RNA product together.
316
The destabilization of the weak rU-dA pairs results in
dissociation of the RNA from its template, terminating transcription.
317
The essence of a bacterial terminator is twofold:
1) base-pairing of something to the transcript to destabilize the RNA-DNA hybrid; and 2) something that causes transcription to pause.
318
A normal intrinsic terminator satisfies the base-pairing of something to the transcript to destabilize the RNA-DNA hybrid by
causing a hairpin to form in the transcript
319
A normal intrinsic terminator satisfies something that causes transcription to pause by
causing a string of U's to be incorporated just downstream of the ahirpin.
320
Rho is a protein that caused
an apparent depression of the ability of RNA polymerase to transcribe certain phage DNAs in vitro.
321
Whenever rho causes a termination event, the polymerase has to
reinitiate to begin transcribing again.
322
Because initiation is a time-consuming event, less net
transcription can occur.
323
Rho had little effect on initiation; if anything, the rate of initiation went up. But rho caused
a significant decrease in total RNA synthesis, consistent with the notion that rho terminates transcription.
324
Rho is causing the synthesis of
much smaller RNAs, consistent with the role of rho in terminating transcription.
325
The transcripts made without rho cosedimented with
the DNA template, indicating that they had not been released from their association with the DNa.
326
The transcripts made with rho sedimented at
a much lower rate, independent of the DNA.
327
rho seems to release
RNA transcripts from the DNA template.
328
rho is able to bind to RNA at
rho loading site, or rho utilization (rut) site, and has ATPase activity that can provide the energy to propel it along an RNA chain.
329
rho has RNA-DNA helicase activity that
can unwind an RNA-DNA hybrid; thus, when rho encounters the polymerase stalled at the terminator, it can unwind the RNA-DNA hybrid within the transcription bubble, releasing the RNA and terminating transcription.
330
Rho-dependent terminators consist of
an inverted repeat, which can cause a hairpin to form in the transcript, but no string of T's.
331
Rho binds to the RNA polymerase in
an elongation complex.
332
When the RNA transcript has grown long enough, rho binds to it via
a rho loading site, forming an RNA loop between the polymerase and rho.
333
RHo continues to feed the growing transcript through itself until
the polymerase pauses at a terminator, allowing rho to tighten the RNA loop and trap the elongation complex. Rho then dissociates the RNA-DNA hybrid, terminating transcript.
