MECHANISM OF TRANSCRIPTION IN BACTERIA

Question 1

(RNA POLYMERASE STRUCTURE)

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

Answer 1

SDS polyacrylamide gel electrophoresis (SDS-PAGE)

Question 2

(RNA POLYMERASE STRUCTURE)

Enzyme Preparation contained two very large sub-units:

Answer 2

ß (150kD) and ß’ (160 kD)

Question 3

(RNA POLYMERASE STRUCTURE)

Other RNA Polymerase Subunits (Visible):

Answer 3

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

Question 4

(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

Answer 4

Omega (ω) 10 kD

Question 5

(RNA POLYMERASE STRUCTURE)

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

Answer 5

Polymerase

Question 6

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

Answer 6

Holoenzyme
Core polymerase

Question 7

Could transcribe intact phage T4 DNA in vitro quite actively.

Answer 7

Holoenzyme

Question 8

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

Answer 8

Core enzyme

Question 9

(Sigma (σ) as a Specificity Factor)

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

Answer 9

σ

Question 10

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

Answer 10

Ekkehard Bautz and colleagues

Question 11

Also transcribes both DNA strands.

Answer 11

Core enzyme

Question 12

• 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

Answer 12

RNA polymerase ; E. coli ; σ factor

Question 13

(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.

Answer 13

Nicks and gaps ; T4 DNA

Question 14

(PROMOTERS)

Polymerase binding site is called ________

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

Answer 14

PROMOTERS ; specific

Question 15

(Binding of RNA Polymerase to Promoters)

Used nitrocellulose filters

Answer 15

David Hinkle and Michael Chamberlin

Question 16

(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)

Answer 16

Holoenzyme ; Core enzyme

Question 17

(Binding of RNA Polymerase to Promoters)

Holoenzyme finds two kinds of binding sites on T7 DNA:

Answer 17

Tight Binding sites and loose binding sites.

Question 18

(Binding of RNA Polymerase to Promoters)

Capable of of binding only loosely to the DNA

Answer 18

Core polymerase

Question 19

(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

Answer 19

Butz and Coworkers

Question 20

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

Answer 20

Chamberlin and Hinkle

Question 21

(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)

Answer 21

Hinkle and Chamberlin

Question 22

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.

Answer 22

unlabeled T7 DNA ; nitrocellulose ; holoenzyme ; polymerase

Question 23

(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)

Answer 23

• T7 DNA ; elevated temperature.
• E.coli RNA polymerase holoenzyme ; 3H-labeled T7 DNA

Question 24

(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 ___________

Answer 24

• dissociated
• 15°C
• RNA polymerase holoenzyme and T7 DNA.

Question 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.

Answer 25

promoter ; closed promoter complex

Question 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.

Answer 26

open promoter complex ; sigma

Question 27

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

Answer 27

transcription

Question 28

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

Answer 28

σ

Question 29

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

Answer 29

transcribed

Question 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.

Answer 30

David Pribnow ; Pribnow box

Question 31

(Promoter Structure) _________ Noticed another short sequence centered approximately 35 bp upstream of the transcription start site. The start site known as the ________

Answer 31

Mark Ptashne and colleagues ; -35 box

Question 32

(Promoter Structure) Represent probabilities The probabilities are such that one rarely finds -10 or -35 boxes that match the consensus sequences perfectly.

Answer 32

Consensus Sequence

Question 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.

Answer 33

TATAAT and TTGACA ; Capital letters ; Lowercase letters

Question 34

Mutation that destroys matches with the consensus sequences

Answer 34

Down Mutations

Question 34

Mutation that makes the promoter stronger

Answer 34

Up Mutations

Question 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 _________

Answer 35

UP element ; true promoter ; Fis sites.

Question 36

Enhancers that bind the transcription activator protein Fis.

Answer 36

Fis Sites

Question 37

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

Answer 37

BACTERIAL PROMOTERS

Question 38

Two consensus sequences: _______ and _________

Answer 38

TATAAT and TTGACA

Question 39

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

Answer 39

stronger

Question 40

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

Answer 40

UP element

Question 41

(Transcription Initiation) 1980 Transcription initiation was assumed to end as RNA polymerase formed the first __________

Answer 41

phosphodiester bond.

Question 42

_________ and ________ Reported that initiation is more complex - they found several very small oligonucleotides, ranging in size from ______ to _______ (2-6 nt long)

Answer 42

Agameomnon Carpousis and Jay Gralla ; dimers to hexamers

Question 43

Transcription Initiation represented in four steps:

Answer 43

- 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.

Question 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

Answer 44

σ ; transcription

Question 45

(Sigma Stimulates Transcription Initiation) Transcribed T4 DNA in vitro with E. coli RNA polymerase core plus increasing amount of σ

Answer 45

Travers and Burgess

Question 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 ______

Answer 46

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

Question 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).

Answer 46

Initiation

Question 47

(Sigma Stimulates Transcription Initiation) ___ could appear to stimulate elongation by stimulating initiation and thereby providing more initiated chains for core polymerase to elongate.

Answer 47

σ

Question 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

Answer 48

rifampicin ; length ; speed ; length ; did not increase

Question 49

(Reuse of σ) Demonstrated that σ can be recycled

Answer 49

1969 - Travers and Burgees

Question 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.

Answer 50

low ionic strength

Question 51

(Reuse of σ) The ___ curve shows the initiation of RNA chains

Answer 51

red

Question 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

Answer 52

rifampicin-resistant core polymerase

Question 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

Answer 53

immediate rise

Question 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.

Answer 54

Transcription ; core

Question 55

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

Answer 55

dissociate ; elongation

Question 56

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

Answer 56

σ ; core

Question 57

(The Stochastic σ-Cycle Model) “Stochastic” means “______" ; Greek: “stochos”, meaning "______"

Answer 57

random ; guess

Question 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.

Answer 58

dissociation ; elongation

Question 59

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

Answer 59

(The Stochastic σ-Cycle Model)

Question 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.

Answer 60

does not fit

Question 61

(The Stochastic σ-Cycle Model) Ebright and coworkers used this technique

Answer 61

Fluorescence resonance energy transfer (FRET)

Question 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

Answer 62

Fluorescence resonance energy transfer (FRET)

Question 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

Answer 63

FRET

Question 64

(The Stochastic σ-Cycle Model) The probe on sigma is a _______________

Answer 64

fluorescence donor

Question 65

(The Stochastic σ-Cycle Model) The probe on DNA is a ______________

Answer 65

fluorescence acceptor