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46 Cards in this Set
- Front
- Back
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Mutations help define important sites in a promoter region
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The s54 RNA polymerase requires an activator protein
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Numbering rules are +1 +2 +3 for downstream (direction of synthesis) and -1 -2 -3 for upstream (nucleotides before synthesis)
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What does RNA polymerase catalyze the formation of?
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Phosphodiester bonds
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Determines RNA sequence
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DNA template sequence
- complementary pairing - one DNA strand as template, other is sense strand that is same sequence as RNA |
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What 4 nucleotides are required for RNA synthesis?
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ATP, CTP, GTP, UTP
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What direction does RNA chain grow?
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5' to 3'
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Does RNA Polymerase require RNA primer?
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NO
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Bacterial RNA polymerase are divided into what?
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Large, multisubunit enzymes
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alpha subunit
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FUNCTION: required for enzyme assembly, interacts w/ some regulatory proteins
- # of subunits = 2 - rpoA gene |
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beta subunit
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FUNCTION: forms pincer and is site of rafampicin action
- rpoB gene |
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beta' subunit
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FUNCTION: pincer and provides and absolutely conserved -NADFDGD- motif that is essential for catalysis
- rpoC gene |
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omega subunit
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FUNCTION: helps in enzyme assembly but is not required for activity
- rpoZ gene |
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sigma subunit
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FUNCTION: directs enzyme to promotors but is not required for phosphodiester bond formation
- rpoD gene |
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Bacterial RNA polymerases consists of what 2 things
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core enzyme and sigma factor
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Core enzyme characteristics
(a2BB') |
-synthesize RNA from ssDNA and nicked DNA templates
-cannot use intact circular dsDNA as template -can be reassebled in vitro from subunits |
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Holoenzyme characteristcs
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uses intact circular dsDNA as template
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promoter function
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-holoenzyme recognizes and binds the promoter
- the promoter resides at the beginning of a transcription unit |
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Role of Sigma Factor
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-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 |
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Promoter sequences in o70 promoters
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-10 box (pribnow box) centered 10 bp upstream w/ TATAAT
-35 box is centered about 35 bp upstream with TTGACA |
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Different sigma factors recognize different consensus sequences
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-
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omega54 RNA polymerase requires what protein?
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Activator protein
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Footprinting does what two things?
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-Identify protein binding sites on DNA
-analysis of promoter-holoenzyme interaction |
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DNA Melting
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The holoenzyme-promoter complex changes conformation during initiation
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The Thermus aquaticus RNA polymerase structure resembles WHAT?
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A Claw
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Features of RNA polymerase
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-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 |
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Structer of the open complex
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RNA polymerase has mobile modules and conformational flexibility
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First step in transcription initiation
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closed complex formation
- sigma factor interacts with core and DNA |
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Second step in transcription initiation
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intermediate stage
- downstream DNA segment bends across the entrance to the active site channel; DNA begins melting |
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Third step in transcription initiation
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Open complex formation and abortive initiation
-DNA melts to complete the transcription bubble -omega32 loop must be displaced to prevent abortive initiation |
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Fourth step in transcription initiation
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end of abortive initiation
-RNA chain elongates to about 12 nt's and RNA-DNA hybrid forms |
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Fifth step in transcription initiation
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Promoter escape
-movement fo RNA polymerase away from the promoter |
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Three models for how RNA polymerase moves
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Scrunching model
inchworm model transient excursion model |
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Scrunching model
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Correct model
-DNA is pulled int he polymerase holoenzyme as the DNA unwinds to form the open complex |
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Inchworm Model
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-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
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Transient excursion model
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Transien cycles of forward enzyme motion during abortive initiation and backward motion after RNA release
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Scrunching Model
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-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 |
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Transcription elongation model
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The transcription elongation complex
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-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 |
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RNA polymerase does not move at a steady pace
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-chain temporarily delayed at pause sites
-pausing may lead to arrest adn termination -arrest is important step in proofreading |
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RNA poly. proofreads during transcription
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back track and cleave bad RNA
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What helps rescue an arrested complex?
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GreB subunit
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Intrinsic Transcription Terminators
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-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 |
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Rho-dependant termination
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-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 |
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Structure of the Rho factor
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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
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