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Function of RNA polymerase sigma factor
- *prokaryotic subunit
- 1. binds to core enzyme to yield holoenzyme
- 2. sigma 4 binds to BOTH DNA strands. Indirectly binds to template strand thru H2O binding (hydrophobic regions of RNA polymerase and DNA bind and polymerase unwind)
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Holoenzyme importance and role
- Essential in:
- LOCATING and BINDING promoter
- OPENING of helix (unwinding of DNA)
- acts as TARGET for transcription factors
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Eukaryotic Polymerases
- RNA pol I: rRNA
- RNA pol 2: mRNA
- RNA pol 3: tRNA
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RNAP I
- has conserved C-terminal domain (CTD)
- Synthesizes NON-coding RNA, ie rRNA.
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RNAP 2
- Contains 12 subunits.
- Transcribes genes that code for PROTEIN (mRNA)
- Scans for DNA damage & modifies surrounding chromatin
- acts as platform for mRNA processing factors for mRNA modification
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RNAP 3
synthesizes NON-coding RNA, ie tRNA
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Role of CTD (carboxyl tail domain)
- 26-52 repeats of specific amino acids
- Caps and SPLICES RNA transcript
- undergoes PHOSPHORYLATION events on Ser2 and Ser5 repeats
- Hydrophilic
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Ser2 role
- allows for ELONGATION of Transcription (2nd phosphorylation step)
- Phosphorylates solely to TERMINATE process
- undergo phosphorylation by CTD kinase-1, which promotes elongation
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Ser5 role
- Occurs after binding to TFIIH and mediator (start initiation)
- Phosphorylated by TFIIH, initiates transcription (1st phosphorylation step)
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Role of Rpb 1 & 2 in elongation
- form active site of core domain of RNA polymerase II
- nucleotides added in this active site
- has a conserved domain
- Highly POSITIVE charge, helps in stabilizing template DNA entering active site
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Mechanism for elongation at active site
- 3 aspartates in a loop on template DNA
- 1 Mg ion in the middle
- Loops shrinks when RNA pol is absorbing an NTP
- Loop expands when RNAP is completing add'n of nucleotide
- Shrinking loop PUSHES Mg up out of ring & it is now able to access 3'-OH group of nucleotide base
- series of reactions result in phosphodiester bond formation between nucleotides
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Role of Mg ions
- role in catalytic add'n of incoming NTPs during elongation of transcription
- Stabilizes nucleophilic add'n of NTP's
- 1st Mg ion located in Asp triad: frees up Oxygen of the 3'-OH group through H-Bond FORMATION (results in formation of phosphodiester bond)
- 2nd Mg ion associated w/incoming base: stabilizes pyrophosphate that is a biproduct of elongation
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