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MOLBIO OFFSEM MIDTERM > MECHANISM OF TRANSCRIPTION IN BACTERIA > Flashcards

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
Q

(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.

A

promoter ; closed promoter complex

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26
Q

(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.

A

open promoter complex ; sigma

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27
Q

The σ-factor allows the initiation of _________ by causing the RNA polymerase holoenzyme to bind tightly to a promoter.

A

transcription

28
Q

This tight binding depends on the local melting of the DNA to form an open promoter complex and is stimulated by __.

A

σ

29
Q

The σ-factor can therefore select which genes will be _________.

A

transcribed

30
Q

(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.

A

David Pribnow ; Pribnow box

31
Q

(Promoter Structure)

_________ Noticed another short sequence centered approximately 35 bp upstream of the transcription start site.

The start site known as the ________

A

Mark Ptashne and colleagues ; -35 box

32
Q

(Promoter Structure)

Represent probabilities

The probabilities are such that one rarely finds -10 or -35 boxes that match the consensus sequences perfectly.

A

Consensus Sequence

33
Q

(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.

A

TATAAT and TTGACA ; Capital letters ; Lowercase letters

34
Q

Mutation that destroys matches with the consensus sequences

A

Down Mutations

34
Q

Mutation that makes the promoter stronger

A

Up Mutations

35
Q

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 _________

A

UP element ; true promoter ; Fis sites.

36
Q

Enhancers that bind the transcription activator protein Fis.

A

Fis Sites

37
Q

__________ contain two regions centered approximately at -10 and -35 bp upstream of the transcription start site.

A

BACTERIAL PROMOTERS

38
Q

Two consensus sequences: _______ and _________

A

TATAAT and TTGACA

39
Q

The more closely regions within a promoter resemble these consensus sequences, the _________ that promoter will be.

A

stronger

40
Q

Some extraordinarily strong promoters contain an extra element (an _________) upstream of the core promoter

A

UP element

41
Q

(Transcription Initiation)

1980

Transcription initiation was assumed to end as RNA polymerase formed the first __________

A

phosphodiester bond.

42
Q

_________ and ________
Reported that initiation is more complex

  • they found several very small oligonucleotides, ranging in size from ______ to _______ (2-6 nt long)
A

Agameomnon Carpousis and Jay Gralla ; dimers to hexamers

43
Q

Transcription Initiation represented in four steps:

A
  • 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
Q

(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
A

σ ; transcription

45
Q

(Sigma Stimulates Transcription Initiation)

Transcribed T4 DNA in vitro with E. coli RNA polymerase core plus increasing amount of σ

A

Travers and Burgess

46
Q

(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 ______

A

initiation ; A or G (purine) ; elongation and initiation

46
Q

(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).

A

Initiation

47
Q

(Sigma Stimulates Transcription Initiation)

___ could appear to stimulate elongation by stimulating initiation and thereby providing more initiated chains for core polymerase to elongate.

A

σ

48
Q

(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

A

rifampicin ; length ; speed ; length ; did not increase

49
Q

(Reuse of σ)

Demonstrated that σ can be recycled

A

1969 - Travers and Burgees

50
Q

(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.

A

low ionic strength

51
Q

(Reuse of σ)

The ___ curve shows the initiation of RNA chains

A

red

52
Q

(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

A

rifampicin-resistant core polymerase

53
Q

(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

A

immediate rise

54
Q

(Reuse of σ)

_______ occurred even in the presence of rifampicin showed that the new core

The ______, not the σ, determines rifampicin resistance or sensitivity.

A

Transcription ; core

55
Q

After σ has participated in initiation, it appears to ______ from the core polymerase, leaving the core to carry out _______.

A

dissociate ; elongation

56
Q

__ can be reused by different core polymerases, and the ____, not σ, governs rifampicin sensitivity or resistance

A

σ ; core

57
Q

(The Stochastic σ-Cycle Model)

“Stochastic” means “______” ; Greek: “stochos”, meaning “______”

A

random ; guess

58
Q

(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.

A

dissociation ; elongation

59
Q

This has come to be known as the obligate release version of the σ-cycle model.

A

(The Stochastic σ-Cycle Model)

60
Q

(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.

A

does not fit

61
Q

(The Stochastic σ-Cycle Model)

Ebright and coworkers used this technique

A

Fluorescence resonance energy transfer (FRET)

62
Q

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

A

Fluorescence resonance energy transfer (FRET)

63
Q

_____ 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

A

FRET

64
Q

(The Stochastic σ-Cycle Model)

The probe on sigma is a _______________

A

fluorescence donor

65
Q

(The Stochastic σ-Cycle Model)

The probe on DNA is a ______________

A

fluorescence acceptor

MOLBIO OFFSEM MIDTERM (9 decks)

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