Chapter 13 Bacterial Rna Polymerase

Front 1

Back 1

Mutations help define important sites in a promoter region

Front 2

Back 2

The s54 RNA polymerase requires an activator protein

Front 3

Back 3

Numbering rules are +1 +2 +3 for downstream (direction of synthesis) and -1 -2 -3 for upstream (nucleotides before synthesis)

Front 4

What does RNA polymerase catalyze the formation of?

Back 4

Phosphodiester bonds

Front 5

Determines RNA sequence

Back 5

DNA template sequence
- complementary pairing
- one DNA strand as template, other is sense strand that is same sequence as RNA

Front 6

What 4 nucleotides are required for RNA synthesis?

Back 6

ATP, CTP, GTP, UTP

Front 7

What direction does RNA chain grow?

Back 7

5' to 3'

Front 8

Does RNA Polymerase require RNA primer?

Back 8

NO

Front 9

Bacterial RNA polymerase are divided into what?

Back 9

Large, multisubunit enzymes

Front 10

alpha subunit

Back 10

FUNCTION: required for enzyme assembly, interacts w/ some regulatory proteins
- # of subunits = 2
- rpoA gene

Front 11

beta subunit

Back 11

FUNCTION: forms pincer and is site of rafampicin action
- rpoB gene

Front 12

beta' subunit

Back 12

FUNCTION: pincer and provides and absolutely conserved -NADFDGD- motif that is essential for catalysis
- rpoC gene

Front 13

omega subunit

Back 13

FUNCTION: helps in enzyme assembly but is not required for activity
- rpoZ gene

Front 14

sigma subunit

Back 14

FUNCTION: directs enzyme to promotors but is not required for phosphodiester bond formation
- rpoD gene

Front 15

Bacterial RNA polymerases consists of what 2 things

Back 15

core enzyme and sigma factor

Front 16

Core enzyme characteristics
(a2BB')

Back 16

-synthesize RNA from ssDNA and nicked DNA templates
-cannot use intact circular dsDNA as template
-can be reassebled in vitro from subunits

Front 17

Holoenzyme characteristcs

Back 17

uses intact circular dsDNA as template

Front 18

promoter function

Back 18

-holoenzyme recognizes and binds the promoter
- the promoter resides at the beginning of a transcription unit

Front 19

Role of Sigma Factor

Back 19

-essential for promoter recognition
-does not bind to promoter DNA on its own
-marjor E. coli sigma factor is o70
-this sigma factor recognizes promoters for housekeepin genes

Front 20

Promoter sequences in o70 promoters

Back 20

-10 box (pribnow box) centered 10 bp upstream w/ TATAAT
-35 box is centered about 35 bp upstream with TTGACA

Front 21

Different sigma factors recognize different consensus sequences

Back 21

-

Front 22

omega54 RNA polymerase requires what protein?

Back 22

Activator protein

Front 23

Footprinting does what two things?

Back 23

-Identify protein binding sites on DNA
-analysis of promoter-holoenzyme interaction

Front 24

DNA Melting

Back 24

The holoenzyme-promoter complex changes conformation during initiation

Front 25

The Thermus aquaticus RNA polymerase structure resembles WHAT?

Back 25

A Claw

Front 26

Features of RNA polymerase

Back 26

-internal channel is formed b/t the B and B' subunits
-N-terminal domains of the alpha subunits allow them to dimerize
-alpha subunit N-terminal domains bind ot the B and B' subunits
-B and B' subunits interact extgensively with each other and together form the catalytic site
-the B' subunit binds the active site Mg2+ and is required for phosphodiester bond formation

Front 27

Back 27

Structer of the open complex

Front 28

Back 28

RNA polymerase has mobile modules and conformational flexibility

Front 29

First step in transcription initiation

Back 29

closed complex formation
- sigma factor interacts with core and DNA

Front 30

Second step in transcription initiation

Back 30

intermediate stage
- downstream DNA segment bends across the entrance to the active site channel; DNA begins melting

Front 31

Third step in transcription initiation

Back 31

Open complex formation and abortive initiation
-DNA melts to complete the transcription bubble
-omega32 loop must be displaced to prevent abortive initiation

Front 32

Fourth step in transcription initiation

Back 32

end of abortive initiation
-RNA chain elongates to about 12 nt's and RNA-DNA hybrid forms

Front 33

Fifth step in transcription initiation

Back 33

Promoter escape
-movement fo RNA polymerase away from the promoter

Front 34

Three models for how RNA polymerase moves

Back 34

Scrunching model
inchworm model
transient excursion model

Front 35

Scrunching model

Back 35

Correct model
-DNA is pulled int he polymerase holoenzyme as the DNA unwinds to form the open complex

Front 36

Inchworm Model

Back 36

-Leading edge of RNA polymerase advances during the early stage fo initation to move the active site forward while the other end of the enzyme remains anchored

Front 37

Transient excursion model

Back 37

Transien cycles of forward enzyme motion during abortive initiation and backward motion after RNA release

Front 38

Scrunching Model

Back 38

-RNA poly. holoenzyme unwinds adjacent DNA segments
-unwound DNA is pulled into active site during initial transcription
-unwound DNA re-winds when RNA poly. holoenzyme leaves the initiation site and moves down the DNA
-energy stored during scrunching is used during promoter escape to break interactions b/t the holoe. and initiation site to allow RNA poly. to move forward

Front 39

Transcription elongation model

Back 39

Front 40

The transcription elongation complex

Back 40

-More stable than initiation complex
-14 bp form transcription bubble
-first 8 nucleotides within bubble are paired w/ RNA chain
-dsDNA opens in front of bubble and bloses behind as RNA poly. moves
-transcription bubble extends from -12 to +2

Front 41

RNA polymerase does not move at a steady pace

Back 41

-chain temporarily delayed at pause sites
-pausing may lead to arrest adn termination
-arrest is important step in proofreading

Front 42

RNA poly. proofreads during transcription

Back 42

back track and cleave bad RNA

Front 43

What helps rescue an arrested complex?

Back 43

GreB subunit

Front 44

Intrinsic Transcription Terminators

Back 44

-nucleotide sequences are present in DNA
-the nascent RNA triggers termination
- G/C rich inverted repeat allows the RNA to form a stem loop that reaches to within 7-9 nt of the 3' end of RNA
-U rich stretch immediately after stem loop causes pausing and release

Front 45

Rho-dependant termination

Back 45

-requires Rho factor, a hexameric ATP-dependant helicase
-Rho factor releases RNA from and RNA-DNA hybrid
-Rho factor loads onto the RNA chain at ta C-rich region called the Rho utilization (rut) site

Front 46

Structure of the Rho factor

Back 46

In the open ring form of Rho factor, the opening is sufficiently large for RNA to enter. The center cavity of the ring is large enough to fit ssRNA molecule inside