Lecture 1, Fall 2003 Eucaryotic transcription units and the general transcriptional machinery'

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Lecture 1, Fall 2003Eucaryotic transcription
units and the general transcriptional machinery.
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Mature mRNA
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Stop codons
Polycistronic
Start codons
Start codon (AUG)
Poly-A tail
Monocistronic
3 UTR
Stop codon
5 UTR
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NELF-E gene Coding sequence red 5 and 3 UTR
blue Introns lower case embedded in red Exons
blue and red
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Characterizing eucaryotic transcription units.

• Transcription start - promoter
• Exons and introns
• 3 end of the transcript - usually the poly-A
  addition site

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Genomic library contains transcribed and
nontranscribed sequences. Buried within this
library are all the sequences contributing to the
production of the primary transcript.
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• DNA libraries
• Vast collection of insert DNA molecules, each
  contained in a separate vector molecule.
• The inserts are selected so they contain
  sequences of interest, eg. cDNA library is
  enriched in protein-coding sequences.
• Vector is designed so that individual molecules
  can be propagated in individual cells - examples
  include bacterial plasmids, bacteria phage
  lambda, and yeast vectors.

Genomic DNA library introns, exons, promoters,
regulatory regions, UTRs, enhancers, lots of
nontranscribed sequence.
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cDNA library is enriched in protein coding
sequences.
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Analysis of cDNA clones is usually sufficient to
identify the 3 end of mRNA because the
construction of cDNA clones often begins with
extension of an oligo-dT primer annealled to the
poly-A tail. The selection of poly-A mRNA has
a significant advantage. 90 of the RNA is
ribosomal RNA, which lacks poly-A, so rRNA
sequences are depleted from the final preparation
of nucleic acid.
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Comparison of cDNA and genomic sequences
identifies introns.
Sequence comparison of these two would show an
interruption of the red sequence.
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• Mapping the transcription start site
• Primer extension and S1 nuclease digestion.

Lodish 7-35
Often, the 5 end of the transcription unit is
under-represented in a cDNA library because
reverse transcription is incomplete. It is
important to locate the sequences encoding the 5
end in order to identify the promoter and to
predict the correct translation start.
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5 RACE (Rapid Amplification of cDNA Ends)
By using a gene specific primer (GSP) close to
the putative 5 end, one can increase the chances
of RT extending all the way to the 5 end.
http//molecool.wustl.edu/krolllab/PDFs/PCR/5'20R
ACE20-20Gibco.pdf
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Components of the general transcriptional
machinery responsible for transcribing
protein-encoding genes (ie Pol II transcription).
General transcription factors (GTFs) consist of
a core collection of proteins required to
reconstitute promoter-specific transcription.
GTFs consist of TFIIA, TFIIB, TFIID, TFIIE,
TFIIF, TFIIH. RNA polymerase II could also be
considered a GTF, since it is the enzyme
responsible for producing the mRNA. The names
IIA, IIB etc. originated as names for fractions
eluting from a phosphocellulose column.
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• Three distinct RNA polymerases.
• Distinguished by their sensitivities to the
  mushroom toxin alpha amanitin pol II gt pol III gt
  pol I
• pol II - protein-encoding genes
• pol III - tRNA, 5S RNA, and some small nuclear
  RNA (snRNA)
• pol I - ribosomal RNA
• Some subunits are shared but many are specific
  for one type type of RNA polymerase.

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Alpha-amanitin sensitivity can be used as a way
to determine if a response in a eukaryotic cell
depends on transcription.

• Other inhibitors commonly used to investigate
  cellular responses
• Transcriptional inhibitors
• procaryotes rifamycin
• eucaryotes alpha-amanitin
• both actinomycin D
• Translational inhibitors
• procaryotes (mitochondria, chloroplasts)
  chloramphenicol
• eucaryotes cycloheximide
• both puromycin

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• RNA polymerase II
• 12 subunits, 0.5 MD
• Significant similarity to the structure of
  bacterial polymerase.
• Significant homology between subunits of pol II
  and alpha, beta and beta of bacterial polymerase
• Note, pol II lacks a subunit with homology to
  sigma. (why might this be significant?)

• Pictured is the structure of pol II
• green - structure similar to bacteria
• blue - Zn serving as structural components
• purple - Mg serving as a cofactor in catalysis.

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Lodish 10-26
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Comparison of E.coli RNA polymerase and RNA
polymerase II.

• Similarities
• Initiates polymerization without a primer.
• Synthesizes RNA 5 to 3 using ribonucleoside
  triphophates (ie. ribonucleotides)
• Highly processive - processivity is a measure of
  the ability of the enzyme to remain associated
  with the template through successive cycles of
  chemical reactions.
• Unable to resume elongation if transcript is
  released.

Differences

• Pol II
• Requires GTFs to recognize promoter.
• Requires GTF and ATP hydrolysis to melt DNA.

• E.coli pol
• Intrinsic ability to recognize promoter.
• Intrinsic capacity to melt DNA.

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• GTFs
• TFIIA, TFIIB, TFIID, TFIIF, TFIIE, TFIIH are
  distinct multisubunit (except IIB) proteins.
• In vitro, they can be assembled on the promoter
  in a particular order.
• TFIID recognizes promoter DNA.
• TFIIB and TFIIA join
• Pol II/TFIIF complex joins
• TFIIE and TFIIH join
• If this stepwise assembly model is correct, each
  step is a potential target for regulation.
• The model may simply be a consequence of the way
  these factors were first identified?

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• TFIID
• TFIID TBP TAFs (TBP associated factors)
• TBP recognizes the TATA box via the minor groove
  and introduces a severe bend in the DNA.
• TAFs recognize sequences in the promoter located
  downstream from the TATA box (initiator and DPE)
  - functionally analogous to sigma factor in
  procaryotes.
• Various TAFs appear to be contacted by regulatory
  proteins and could be the targets of these
  regulators.
• TAF250 has kinase activity, acetyl transferase
  activity and monoubiquitin-conjugating activity.

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• TFIIH contains 2 DNA helicases and a protein
  kinase.
• DNA helicase uses ATP hydrolysis to power
  promoter melting.
• Protein kinase phosphorylates serines in the
  carboxyl-terminal domain (CTD) of the largest
  subunit of RNA polymerase II.

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Carboxyl-terminal domain of the largest subunit
of pol II. In humans, there are 52 repeats of
the sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser. CTD of
polymerase is not phosphorylated when polymerase
joins the initiation complex and becomes highly
phosphorylated during or shortly after
transcriptional initiation.
CTD
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Amino acid sequence of pol II CTD from various
species. from Corden (1990) TIBS 15, 383-387
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• Function of the CTD?
• CTD is not required for transcription by purified
  polymerase so it doesnt appear to be directly
  involved in the enzymatic reaction.
• Probably involved in regulation!
• Dephosphorylated CTD exhibits affinity for TBP so
  this might contribute to the binding of
  polymerase to the promoter. Phosphorylation
  decreases the affinity.
• P-CTD serves to couple transcription and RNA
  processing - appears to recruit or control RNA
  processing factors involved in capping, splicing
  and poly-A addition.
• P-CTD renders transcription elongation resistant
  to inhibition by the proteins NELF and DSIF - ie
  elongation is stimulated in vivo.