E.coli RNA polymerase

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• E. coli RNA Polymerase

• M.Prasad Naidu
• MSc Medical Biochemistry,
• Ph.D.Research Scholar

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RNA Polymerase

• Catalyzes the formation of the phosphodiester
  bonds between the nucleotides (sugar to
  phosphate)
• Uncoils the DNA, adds the nucleotide one at a
  time in the 5 to 3 fashion
• Uses the energy trapped in the nucleotides
  themselves to form the new bonds

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Differences in DNA and RNA Polymerases

• RNA polymerase adds ribonucleotides (rNTPs) not
  deoxynucleotides (dNTPs)
• RNA polymerase does not have the ability to
  proofread what they transcribe
• RNA polymerase can work without a primer
• RNA will have an error 1 in every 10,000 nt (DNA
  is 1 in 10,000,000 nt)

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Forms of RNA polymerases (RNAPs)
Bacteriophages - large, single subunit RNA
polymerases - make specificity factors
that alter the promoter recognition of
host bacterial enzymes Bacteria - 4 or
more core subunits, with exchangeable
specificity factors (sigmas) E. coli has ß, ß,
a2, ?, s Archaea - multiple subunits
related to both bacteria and eukaryotic

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Eukaryotes Three RNA polymerases many with
subunits pol I - only the large ribosomal RNA
subunit precursors pol II - all pre-mRNAs,
some small nuclear RNAs (snRNAs),
most small nucleolar RNAs (snoRNAs)
used in rRNA processing pol III - tRNAs, 5S
rRNA, U6 snRNA, 7SL RNA (in SRP),
and other small functional RNAs Mitochondria and
Chloroplasts - combination of phage-like
(single subunit) and bacterial-like
(multi-subunit) Eukaryotic viruses - can
take over host RNAP or encode own in some
large viruses (e.g. vaccinia)
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Bacterial RNA polymerase

• Isolated in bacterial extracts in 1960 by
  independent groups Samuel Weiss and Jerard
  Hurwitz
• Responsible for synthesis of all 3 types of RNA
  species mRNA, rRNA and tRNA
• RNAP is a huge enzyme (460 kD) made of five
  subunits

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E. coli RNA polymerase

• Five subunits
• 2 a subunits
• 1 b subunit
• 1 b subunit
• 1 ? subunit
• s factor

Core enzyme
Holoenzyme
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E. coli RNA polymerase 2a, 1ß, 1ß, 1? and s
factor
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• a subunit Mol wt is 36.5 kDa, encoded by rpoA
  gene. Required for core protein assembly, and
  also play a role in promoter recognition.
  Assembly of ß and ß.
• ß subunit Mol wt is 151 kDa, encoded by rpoB
  gene. DNA-binding active center. Rifampicin is
  shown to bind to the ß subunit and inactivates.
• ß subunit Mol wt is 155 kDa, encoded by rpoC
  gene. Responsible for binding to the template
  DNA. Uses 2 Mg2 ions for catalytic function of
  the enzyme.
• ? subunit Mol wt 91 kDa, encoded by rpoZ gene.
  restores denatured RNA polymerase to its
  functional form in vitro. It has been observed to
  offer a protective/chaperone function to the ß'
  subunit in Mycobacterium smegmatis.

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E. coli RNA polymerase

• The processivity of E. coli RNA polymerase is
  around 40 nt/sec at 37ºC, and requires Mg2
• (RNA polymerase of T3 and T7 are single
  polypeptides with a processivity of 200 nt/sec)
• The enzyme has a nonspherical structure with a
  projection flanking a cylindrical channel
• The size of the channel suggests that it can bind
  directly to 16 bp of DNA
• The enzyme binds over a region of DNA covering
  around 60 bp

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s (sigma) Factor

• Binds the core enzyme to convert it to the
  holoenzyme
• It is encoded by rpoD gene (s70 )
• It has a critical role in promoter recognition,
  but is not required for transcription elongation
• It recognizes the correct promoter site by
  decreasing the affinity of the enzyme at the
  nonspecific DNA sequences
• The amount is only 30 to amount of the enzyme

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• Each s factor recognizes a particular sequence
  of nucleotides upstream from the gene
• s70 looks for -35 sequence TTGACA and
  
• -10 sequence TATAAT
• Other s factors look for other sequences
• The match need not always be exact
• The better the match, the more likely
  transcription will be initiated

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Alternative Sigma Factors

• Alternative sigma factors can be classified into
  two structurally unrelated families
• s70 and s54
• Although no sequence conservation exists between
  s70 and s54like family members, both types bind
  to core RNA polymerase.
• Promoter structures recognized by s54RNAP differ
  from those recognized by s70RNAP.
• s54 RNAP recognizes -24 and -12
• s70 RNAP recognizes -35 and -10

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Sigma factors have four main regions that are
generally conserved N-terminus
--------------------- C-terminus
1.1 2 3 4 The regions are further
subdivided (e.g. 2 includes 2.1, 2.2,
etc.) The exception to this organization is
in s54-type sigma factors. Proteins homologous
to s54/RpoN are functional sigma factors, but
they have significantly different primary
amino acid sequences.
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E. coli Sigma Factors
s70 (RpoD) - the "housekeeping" sigma factor or
also called as primary sigma factor, transcribes
most genes in growing cells. Makes the proteins
necessary to keep the cell alive. s54 (RpoN) -
the nitrogen-limitation sigma factor s38 (RpoS)
- the starvation/stationary phase sigma
factor s32 (RpoH) - the heat shock sigma factor,
it is turned on when exposed to heat s28
(RpoF) - the flagellar sigma factor s24 (RpoE) -
the extracytoplasmic/extreme heat stress sigma
factor s19 (FecI) - the ferric citrate sigma
factor, regulates the fec gene for iron
transport