MECHANISM OF TRANSCRIPTION IN BACTERIA Flashcards

1
Q

(RNA POLYMERASE STRUCTURE)

1969 - Polypeptides that make up the E.coli RNA polymerase had been identified by __________

A

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

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2
Q

(RNA POLYMERASE STRUCTURE)

Enzyme Preparation contained two very large sub-units:

A

ß (150kD) and ß’ (160 kD)

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3
Q

(RNA POLYMERASE STRUCTURE)

Other RNA Polymerase Subunits (Visible):

A

Sigma (σ) 70 kD - lower one on the gel
Alpha (α) 40 kD - 2 copies present in holoenzyme

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4
Q

(RNA POLYMERASE STRUCTURE)

  • Was not clearly visible in SDS-PAGE, but seen in other experiments (too small to see in the gel)
  • Not required for cell viability or in vivo enzyme activity 
  • Appears to play a role in enzyme assembly
A

Omega (ω) 10 kD

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5
Q

(RNA POLYMERASE STRUCTURE)

________ - is an enzyme complex composed of multiple enzymes/subunits.

A

Polymerase

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6
Q

2 Forms of Polymerase:
__________ - All subunits
__________ - Lacks sigma (apoenzyme)

A

Holoenzyme
Core polymerase

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7
Q

Could transcribe intact phage T4 DNA in vitro quite actively.

A

Holoenzyme

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8
Q
  • Had little ability to transcribe intact phage T4 DNA
  • But could still transcribe highly nicked templates very well.
A

Core enzyme

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9
Q

(Sigma (σ) as a Specificity Factor)

Adding ___ back to the core reconstituted the enzyme’s ability to transcribe unnicked T4 DNA.

A

σ

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10
Q

Showed that the holoenzyme transcribed only a certain class of T4 genes (called immediate early genes), but the core showed no such specificity.

A

Ekkehard Bautz and colleagues

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11
Q

Also transcribes both DNA strands.

A

Core enzyme

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12
Q
  • The key player in the transcription process is ________
  • The ______ enzyme is composed of a core containing the basic transcription machinery.
  • ______, which directs the core to transcribe specific genes
A

RNA polymerase ; E. coli ; σ factor

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13
Q

(PROMOTERS)

_____ and ____ in DNA provide ideal initiation sites for RNA polymerase.

When σ was present, the holoenzyme could recognize the authentic RNA polymerase binding sites on the _____ and begin transcription there.

A

Nicks and gaps ; T4 DNA

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14
Q

(PROMOTERS)

Polymerase binding site is called ________

Transcription that begins at promoters in vitro is ______ and mimics the initiation that would occur in vivo.

A

PROMOTERS ; specific

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15
Q

(Binding of RNA Polymerase to Promoters)

Used nitrocellulose filters

A

David Hinkle and Michael Chamberlin

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16
Q

(Binding of RNA Polymerase to Promoters)

ow tightly does core enzyme and holoenzyme bind to DNA?

______ binds much more tightly to the T7 DNA (stays stuck to the radioactive DNA-high affinity)

_______ binding is more transient ( binds initially, but weak affinity and it comes off easily)

A

Holoenzyme ; Core enzyme

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17
Q

(Binding of RNA Polymerase to Promoters)

Holoenzyme finds two kinds of binding sites on T7 DNA:

A

Tight Binding sites and loose binding sites.

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18
Q

(Binding of RNA Polymerase to Promoters)

Capable of of binding only loosely to the DNA

A

Core polymerase

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19
Q

(Binding of RNA Polymerase to Promoters)

  • Had shown that holoenzyme can recognize promoters and not the core.
  • Tight-binding sites are probably promoters
  • Loose binding sites represent the rest of the DNA
A

Butz and Coworkers

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20
Q

Showed that the tight complexes between holoenzyme and T7 DNA could initiate transcription immediately.

A

Chamberlin and Hinkle

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21
Q

(Sigma stimulates tight binding between RNA polymerase and promoter)

Allowed 3H-labeled T7 DNA to bind to E.coli core polymerase (blue) or holoenzyme (red)

A

Hinkle and Chamberlin

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22
Q

Hinkle and Chamberlin

  • They added an excess of ________
  • They filtered the mixtures through _______ at various times to monitor the dissociation of the labeled T7 DNA-polymerase complexes
  • The much slower dissociation rate of the ________ (red) relative to the core ______ (blue) shows much tighter binding between T7 and holoenzyme.
A

unlabeled T7 DNA ; nitrocellulose ; holoenzyme ; polymerase

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23
Q

(The effect of temperature on the dissociation of the polymerase–T7 DNA complex)

Hinkle and Chamberlin:

  • Tested the effect of temperature binding of holoenzyme to _____ and found a striking enhancement of tight binding at ___________
  • Formed complexes between __________ and ____________ at three different temperatures: : 37°C (red), 25°C (green), and 15°C (blue)
A
  • T7 DNA ; elevated temperature.
  • E.coli RNA polymerase holoenzyme ; 3H-labeled T7 DNA
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24
Q

(The effect of temperature on the dissociation of the polymerase–T7 DNA complex)

Hinkle and Chamberlin:

  • Added excess unlabeled T7 DNA to compete with any polymerase that _______
  • The complex formed at 37°C was more stable than that formed which was much more stable than that formed at _____
  • Higher temperature favors tighter binding between __________ and ___________
A
  • dissociated
  • 15°C
  • RNA polymerase holoenzyme and T7 DNA.
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25
(RNA polymerase/promoter binding) The holoenzyme binds and rebinds loosely to the DNA, searching for a ______ The holoenzyme has found a promoter and has bound loosely, forming a “_____________” because the DNA remains in closed double-stranded form.
promoter ; closed promoter complex
26
(RNA polymerase/promoter binding) Then the holoenzyme can melt a short region of the DNA at the promoter to form an ”_____________”. The conversion of loosely to tightly bound polymerase requires _____ that selects promoters to which polymerase will bind and adjacent genes are therefore transcribed.
open promoter complex ; sigma
27
The σ-factor allows the initiation of _________ by causing the RNA polymerase holoenzyme to bind tightly to a promoter.
transcription
28
This tight binding depends on the local melting of the DNA to form an open promoter complex and is stimulated by __.
σ
29
The σ-factor can therefore select which genes will be _________.
transcribed
30
(Promoter Structure) ___________ Compared several E. coli and phage promoters and discerned a region they held in common: A sequence of 6 or 7 bp centered approximately 10 bp upstream of the start transcription. “________” - now usually called the -10 box.
David Pribnow ; Pribnow box
31
(Promoter Structure) _________ Noticed another short sequence centered approximately 35 bp upstream of the transcription start site. The start site known as the ________
Mark Ptashne and colleagues ; -35 box
32
(Promoter Structure) Represent probabilities The probabilities are such that one rarely finds -10 or -35 boxes that match the consensus sequences perfectly.
Consensus Sequence
33
(Consensus Sequence) Two consensus sequences: _________ and _______ ___________ - denote bases found in positions in more than 50% of promoters examined. ____________ - denote bases found in positions in 50% or lower of promoters examined.
TATAAT and TTGACA ; Capital letters ; Lowercase letters
34
Mutation that destroys matches with the consensus sequences
Down Mutations
34
Mutation that makes the promoter stronger
Up Mutations
35
Additional upstream promoter elements that make the promoter even better than just having good -10 and -35 boxes. It is a ________ element because it stimulates the transcription of the rrnB P1 gene. It is also associated with three so-called _________
UP element ; true promoter ; Fis sites.
36
Enhancers that bind the transcription activator protein Fis.
Fis Sites
37
__________ contain two regions centered approximately at -10 and -35 bp upstream of the transcription start site.
BACTERIAL PROMOTERS
38
Two consensus sequences: _______ and _________
TATAAT and TTGACA
39
The more closely regions within a promoter resemble these consensus sequences, the _________ that promoter will be.
stronger
40
Some extraordinarily strong promoters contain an extra element (an _________) upstream of the core promoter
UP element
41
(Transcription Initiation) 1980 Transcription initiation was assumed to end as RNA polymerase formed the first __________
phosphodiester bond.
42
_________ and ________ Reported that initiation is more complex - they found several very small oligonucleotides, ranging in size from ______ to _______ (2-6 nt long)
Agameomnon Carpousis and Jay Gralla ; dimers to hexamers
43
Transcription Initiation represented in four steps:
- Formation of closed promoter complex - Conversion of the closed promoter complex to an open promoter complex - Polymerizing the first few nucleotides (up to 10) while the polymerase remains at the promoter (initial transcribing complex) - Promoter clearance, in which the transcipt becomes long enough to form a stable hybrid with template strand.
44
(Sigma Stimulates Transcription Initiation) - Because __ directs tight binding of RNA polymerase to promoters, it places the enzyme in a position to initiate _________ —at the beginning of a gene. - Therefore, we would expect σ to stimulate initiation of transcription
σ ; transcription
45
(Sigma Stimulates Transcription Initiation) Transcribed T4 DNA in vitro with E. coli RNA polymerase core plus increasing amount of σ
Travers and Burgess
46
(Sigma Stimulates Transcription Initiation) When more sigma was added, more _____ occured. - First nucleotide is usually ____ or _____. - Because all three curves rise with increasing σ concentration, this experiment makes it appear that σ stimulates both ______ and ______
initiation ; A or G (purine) ; elongation and initiation
46
(Sigma Stimulates Transcription Initiation) _________ is the rate-limiting step in transcription (it takes longer to get a new RNA chain started than to extend one).
Initiation
47
(Sigma Stimulates Transcription Initiation) ___ could appear to stimulate elongation by stimulating initiation and thereby providing more initiated chains for core polymerase to elongate.
σ
48
(Sigma Stimulates Transcription Initiation) Experiment to discount the apparent effect of sigma on transcription elongation: - Initiation was allowed to occur and then _____ was added to block any further initiation (i.e. number of RNA chains now held constant) - Now sigma was added, and found that adding more sigma did not help (doesn’t increase the ____ or the _____ of elongation) - The ____ of RNAs was measured in the absence or presence of sigma Result: Sigma _________ the length of the RNAs
rifampicin ; length ; speed ; length ; did not increase
49
(Reuse of σ) Demonstrated that σ can be recycled
1969 - Travers and Burgees
50
(Reuse of σ) The key to this experiment was to run the transcription reaction at ________, which prevents RNA polymerase core from dissociating from the DNA template at the end of a gene.
low ionic strength
51
(Reuse of σ) The ___ curve shows the initiation of RNA chains
red
52
(Reuse of σ) After 10 min (arrow), when most chain initiation had ceased, the investigators added new, __________ in the presence (green) or absence (blue) of rifampicin
rifampicin-resistant core polymerase
53
(Reuse of σ) The ________- of both curves showed that addition of core polymerase can restart RNA synthesis, which implied that the new core associated with σ that had been associated with the original core
immediate rise
54
(Reuse of σ) _______ occurred even in the presence of rifampicin showed that the new core The ______, not the σ, determines rifampicin resistance or sensitivity.
Transcription ; core
55
After σ has participated in initiation, it appears to ______ from the core polymerase, leaving the core to carry out _______.
dissociate ; elongation
56
__ can be reused by different core polymerases, and the ____, not σ, governs rifampicin sensitivity or resistance
σ ; core
57
(The Stochastic σ-Cycle Model) “Stochastic” means “______" ; Greek: “stochos”, meaning "______"
random ; guess
58
(The Stochastic σ-Cycle Model) The σ-cycle model that arose from Travers and Burgess’s experiments called for the ________ of σ from core as the polymerase undergoes promoter clearance and switches from initiation to ________ mode.
dissociation ; elongation
59
This has come to be known as the obligate release version of the σ-cycle model.
(The Stochastic σ-Cycle Model)
60
(The Stochastic σ-Cycle Model) Although this model has held sway for over 30 years and has considerable experimental support, it ________ all the data at hand.
does not fit
61
(The Stochastic σ-Cycle Model) Ebright and coworkers used this technique
Fluorescence resonance energy transfer (FRET)
62
Allows the position of σ relative to a site on the DNA to be measured without using separation techniques. Relies on the fact that 2 fluorescent molecules close together will engage in transfer of resonance energy
Fluorescence resonance energy transfer (FRET)
63
_____ allows the position of sigma relative to a site on the DNA to be measured with using separation techniques that might displace sigma from the core enzyme
FRET
64
(The Stochastic σ-Cycle Model) The probe on sigma is a _______________
fluorescence donor
65
(The Stochastic σ-Cycle Model) The probe on DNA is a ______________
fluorescence acceptor