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MOLGEN > Chapter 17 - Prokaryotic Transcription > Flashcards

Chapter 17 - Prokaryotic Transcription Flashcards

1
Q

Transcription proceeds in a … direction down a template strand that is oriented …

A

5’ to 3’
3’ to 5’

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2
Q

RNA polymerase

A

An enzyme that synthesizes a complementary RNA using a DNA template

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3
Q

Transcription unit

A

Stretch of DNA that codes for an RNA molecule and any sequences needed for transcription

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4
Q

RNA polymerase separates the two strands of DNA in a

A

transient transcription bubble

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5
Q

Bacterial RNA polymerase transcribes how many nucleotides per second

A

40-50

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6
Q

a single type of RNA polymerase produces what in bacteria

A

all RNA

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7
Q

holoenzyme

A

The form of RNA polymerase that is used to initiate transcription

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8
Q

holoenzyme contents

A

five subunits of the core polymerase and the sigma factor

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9
Q

RNA polymerase catalysis is derived from

A

the beta and beta prime subunits

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10
Q

the c-terminal domain in RNA polymerase

A

has the alpha subunits
is involved in stimulating transcription in prokaryotes

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11
Q

RNA polymerase has a general affinity for DNA because of

A

electrostatic interactions

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12
Q

The core polymerase can synthesize RNA, but cannot recognize

A

promotors

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13
Q

The sigma factor is used to initiate transcription at

A

specific sites

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14
Q

promotor strength

A

Efficiency of individual promoters in initiating transcription
Frequency of initiation varies from 1/sec to 1/30 minutes

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15
Q

first way RNAP finds promotors

A

Random diffusion and nonspecific binding to short sequences
Rapid dissociation of enzyme and repositioning
Very, very inefficient mechanism

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16
Q

second way RNAP finds promotors

A

Nonspecific binding to genome and then movement along genome to specific promoter(s)
Sliding
Intersegment transfer
Intradomain association and dissociation (hopping)

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17
Q

holoenzyme structure - initiation

A
  1. When the holoenzyme slides into a promoter it transitions to a closed binary complex
  2. If the sigma factor interacts strongly with the promoter, the holoenzyme transitions to an unstable open complex
  3. Several rNTPs are incorporated into the transcript, forming the ternary complex
  4. The RNAP transitions into a ternary elongation complex
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18
Q

a closed binary complex is

A

reversible

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19
Q

open complex

A

DNA duplex is bent 90° in order to place it into the active site
Promoter is denatured from -11 to +3 with assistance from sigma
Transcription bubble increases to 22-24 nt in length
Jaws close around downstream sequence
Transition to open state is irreversible

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20
Q

ternary complex

A

Additional rNTPs are added to the transcript while the RNAP remains tightly bound to the promoter
RNAP is not able to move down the template
RNAP pulls upstream DNA into the active site via a “scrunching” mechanism
Scrunching creates considerable stress that results in release of these short transcripts
Abortive initiation transcripts of 15-20 nucleotides
Energy of successive abortive initiation events is used to eventually break RNAP free from the promoter and transition to elongation

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21
Q

ternary elongation complex

A

Sigma factor is released
Bubble returns to 10-12 nt in length
RNAP coverts into the core enzyme

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22
Q

promoters are

A

cis-acting control elements

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23
Q

In a typical bacterial genome, a … bp sequence would be the minimum length required to have a unique recognition site

A

12

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24
Q

the sequence of the promotor does not need to be

A

contiguous

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25
important conservation is localized in
short consensus sequences
26
promoter structure
1. purine at transcription start site 2. -10 element 3. -35 element 4. Space between the -10 and -35 elements 5. base pairs between -10 and +1 6. Extended -10 element 7. UP element
27
the space between -10 and -35 elements
Is 16-18 bp in 90% of promoters Sequence is unimportant Determines the separation between interacting regions of RNA polymerase Also determines the geometric orientation of DNA helix with respect to interactions with RNAP holoenzyme
28
base pairs between -10 and +1
Discriminator sequence Base pairs downstream of -10 on the nontemplate strand Interactions between these base pairs and 2 are important for stability of the open complex
29
extended -10 element
Upstream of the -10 element TGN sequence at this position can compensate for a -35 element that does not closely match the -35 consensus sequence
30
UP element
20 bp region upstream of the -35 element Interacts with the CTD When the UP closely matches the consensus, transcription is greatly increased
31
down mutations
Usually reduce conformance to the consensus sequences Decrease promoter efficiency
32
up mutations
increase conformance to the consensus sequence increase promoter effiency
33
Mutations in the -35 sequence can affect
initial binding of RNA polymerase
34
Mutations in the -10 sequence can affect
Binding of RNA polymerase The efficiency of the melting reaction that converts closed to open complex
35
Some promoters lack recognizable -10 and -35 elements and Require
ancillary activator proteins to recruit RNA polymerase and initiate transcription
36
Different sigma factors bind to the core polymerase in a similar way Even though
different sigma factors have different amino acid sequences
37
sigma 70
associated with basal gene expression
38
alpha helical domains of sigma 70 associate with
the promoter
39
N-terminal domain of sigma is
autoinhibitory
40
N-terminal domain of sigma functions
Normally masks the DNA binding domain of sigma Prevents sigma from nonspecifically binding to and blocking promoters Swings out of the way once sigma binds to the core RNAP N-terminal domain also blocks the DNA-binding domain of the holoenzyme until an open complex is formed
41
The consensus sequences at -35 and -10 provide most of
the contact points for RNA polymerase in the promoter
42
a nonspecific interaction
occurs between sigma2 and only the phosphodiester backbone in the closed binary RNAP complex
43
a specific interaction
between sigma 2 and base pairs of the -10 and discriminator facilitates the melting that leads to the irreversible transition to the open RNAP complex
44
A portion of sigma must be displaced to accommodate RNA synthesis
The sigma3-sigma4 linker mimics RNA Lies in the middle of the RNA exit channel Must be ejected in order to produce a transcript > 20 nt in length Loss of interaction between sigma3-sigma4 and RNAP is often associated with release of entire sigma and transition to core RNAP and elongation
45
The DNA unwound during scrunching is rewound
Energy of DNA rewinding is used to break -core RNAP and promoter-RNAP interactions Scrunching allows for storage and use of energy needed to transition to elongation
46
All RNA polymerases share
similar structures and mechanisms
47
shared RNA polymerase structures and mechanisms
DNA-binding primary channel lined with positive charges active site with Mg2+ ssDNA is bent at 90 degree angle
48
RNA polymerase moves forward using a
Brownian ratchet mechanism
49
Brownian motion leads to
random fluctuations of structures in the active site
50
Binding of the correct rNTP will stabilize the
active site structure in the active conformation
51
A “trigger loop” becomes ... when a correct rNTP enters the active site
folded
52
Any event that displaces the 3’-OH of the RNA transcript from the active site will interrupt
core polymerase action
53
The core polymerase has an ... function that is able to remove mispaired nucleotides in order to restore correct RNA placement and activity
exonuclease
54
The exonuclease function is stimulated by accessory factors
Accessory factors insert a narrow domain into the RNA polymerase Domain closely approaches the active site Allows a second Mg2+ to enter the active site Converts active site from polymerase function to exonuclease function
55
The core RNA polymerase does not move at a constant rate
Can pause or backtrack Movement determined by context of template strand sequence
56
at a true terminator
Extended pause All hydrogen bonds of RNA-DNA hybrid are broken DNA duplex reforms
57
extrinsic terminators
require Rho
58
Intrinsic terminators have two sequence features
G+C rich hairpin loop formation in RNA transcript Up to seven uracil nucleotides in the RNA transcript following the hairpin region
59
the hairpin induces
a change in RNA polymerase activity that leads to a disruption in the active site and dissociation of U-A base pairs
60
readthrough transcripts
Transcripts that are not stopped by the terminator Can be facilitated by factors that interact with RNA polymerase and/or the RNA transcript Antitermination
61
Mutational studies have shown the importance of the
G-C base pairs of the hairpin and U-rich downstream region
62
Other upstream and downstream sequences also influence the
effiecency of termination
63
extrinsic termination
1. Rho binds to rut sites in the RNA transcript upstream of the termination site 2. Rho moves downstream along the RNA transcript 3. The RNA polymerase will pause 4. Rho invades the active site and uses its intrinsic helicase function to break the RNA-DNA hybrid
64
Rho
Hexameric ATP-dependent helicase
65
How does Rho work
RNA is wound from the 3’ end around the N-terminal RNA binding domains 5’ end of RNA pushed through interior of ring to ATP binding domains ATP hydrolysis used to translocate Rho in a 3’ direction down RNA
66
heat shock response
is a conserved pathway that protects cells from temperature stress Increased denaturing of proteins
67
products of heat shock response
Chaperones that refold proteins Proteases that degrade denatured proteins
68
sigma factor for heat shock
sigma 32
69
Unfolded protein levels decrease after a heat shock response has activated
Concentration of unoccupied proteases and chaperones increases Proteases degrade free sigma32 at a greater rate Relative concentration of sigma32 decreases Relative concentration of sigma70 increases RNA polymerases are less likely to complex with sigma32

MOLGEN (28 decks)

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