Transcription

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Transcription
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Central Dogma
from Purves et al., Life The Science of
Biology, 4th Edition, by Sinauer Associates
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Types of RNA

• messenger RNA (mRNA). This will later be
  translated into a polypeptide.
• ribosomal RNA (rRNA). This will be used in the
  building of ribosomes
• transfer RNA (tRNA). RNA molecules that carry
  amino acids to the growing polypeptide.
• small nuclear RNA (snRNA). DNA transcription of
  the genes for mRNA, rRNA, and tRNA produces large
  precursor molecules ("primary transcripts") that
  must be processed within the nucleus to produce
  the functional molecules for export to the
  cytosol. Some of these processing steps are
  mediated by snRNAs.
• small nucleolar RNA (snoRNA). These RNAs help
  process ribosomal RNA (rRNA) molecules.

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Ribosomal RNA (rRNA)

• There are 4 kinds. In eukaryotes, these are
• 18S rRNA. One of these molecules, along with some
  30 different protein molecules, is used to make
  the small subunit of the ribosome.
• 28S, 5.8S, and 5S rRNA. One each of these
  molecules, along with some 45 different proteins,
  are used to make the large subunit of the
  ribosome.

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Transfer RNA (tRNA)

• There are 32 different kinds of tRNA in a typical
  eukaryotic cell.
• each is the product of a separate gene
• many of the bases in the chain pair with each
  other forming sections of double helix. the
  unpaired regions form 3 loops
• each kind of tRNA carries (at its 3' end) one of
  the 20 amino acids (thus most amino acids have
  more than one tRNA responsible for them) at one
  loop, 3 unpaired bases form an anticodon

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Messenger RNA (mRNA)

• Messenger RNA comes in a wide range of sizes
  reflecting the size of the polypeptide it
  encodes. Most cells produce small amounts of
  thousands of different mRNA molecules, each to be
  translated into a peptide needed by the cell.
• Many mRNAs are common to most cells, encoding
  "housekeeping" proteins needed by all cells (e.g.
  the enzymes of glycolysis). Other mRNAs are
  specific for only certain types of cells. These
  encode proteins needed for the function of that
  particular cell (e.g., the mRNA for hemoglobin in
  the precursors of red blood cells).

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Transcription
from Purves et al., Life The Science of
Biology, 4th Edition, by Sinauer Associates
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Transcription in Prokaryotes

• Requires a promoter region, with which RNA
  polymerase interact.
• The RNA-coding sequence is the DNA that is to be
  transcribed and later translated.
• Requires a terminator, downstream of the end of
  the RNA-coding sequence.

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Promoter

• Specific sequences are critical for the
  recognition by the polymerase.
• Generally found at 35 and 10 that is they are
  centered at 35 and 10 bp upstream of 1,
  transcription start site.
• Consensus sequence for 35 region (-35 box) is
  5-TTGACA-3
• Consensus sequence for 10 region (Pribnow box)
  is 5-TATAAT-3.

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RNA Polymerase

• A form of RNA polymerase called holoenzyme
  (Complete enzyme) must bind to the promoter.
• Holoenzyme consists of the the core enzyme (made
  up of four polypeptides) bound with another
  polypeptide called the sigma factor, necessary to
  recognize the 35 and 10 regions of the
  promoter.
• Without the sigma factor, the core enzyme doesnt
  start transcription efficiently.

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Binding to the promoter

• Binds in two steps
• Binds loosely to the 35 box while DNA is double
  stranded.
• Binds more tightly to DNA as DNA untwists for
  about 17 bp centered around the 10 box.

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Promoter sequences and regulation of
transcriptional rate

• Promoters differ in their sequences slightly, so
  the binding efficiency of the promoter varies.
• A 10 region sequence of 5-GATACT-3 has a lower
  rate of transcription initiation than
  5-TATAAT-3.

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Sigma factor and transcriptional regulation

• There are different sigma factors sigma 70 is
  the most common
• sigma32 is used for conditions of heat shock
  sequences that are recognized by sigma32 has
  CCCCC at 39 and TATAAATA at 15.

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Elongation

• Transcription bubble forms.
• Once 8 or 9 RNA nucleotides are linked together,
  the sigma factor disassociates from the RNA
  polymerase core enzyme.
• Core enzyme completes the transcription. As it
  moves, it untwists the DNA double helix producing
  torsion, DNA double helix reforms behind the
  enzyme.
• Within the untwisted region a temporary RNA-DNA
  hybrid is formed, the rest of the RNA is
  displaced away from the DNA.

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Termination

• Requires terminator sequences.
• Rho-independent termination. These sequences
  consists of sequences with a two-fold symmetry
  that makes a hairpin loop, followed by 4-8 AT
  basepairs, leading to termination when
  encountered.
• Rho-dependent termination. These sequences lack
  AT string and many cannot form hairpin
  structures. Rho factor (a protein) is needed to
  recognize a specific sequence and face the
  polymerase by moving along the DNA using ATP.

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Transcription in Prokaryotes
Enzyme RNA polymerase
Promoter is the DNA sequence where RNA polymerase
binds to initiate transcription
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Transcription in E.Coli
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The Operon Model

• Groups of genes coding for related proteins are
  arranged in units known as operons.
• An operon consists of an operator, promoter,
  regulator, and structural genes.

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The Operon Model

• The regulator gene codes for a repressor protein
• that binds to the operator, obstructing the
  promoter (thus, transcription) of the structural
  genes.
• The regulator does not have to be adjacent to
  other genes in the operon. If the repressor
  protein is removed, transcription may occur.

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Eukaryotic Transcription
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RNA Processing pre-mRNA -gt mRNA

• Synthesis of the cap. This is a stretch of three
  modified nucleotides (7methylguanosine) attached
  to the 5' end of the pre-mRNA. This structure
  aligns mRNAs on the ribosome during translation

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Capping
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RNA Processing pre-mRNA -gt mRNA

• Synthesis of the poly(A) tail. This is a stretch
  of adenine nucleotides attached to the 3' end of
  the pre-mRNA.
• This regulates both translation and mRNA
  stability. It is also important in early
  development.

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Polyadenylation
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RNA Processing pre-mRNA -gt mRNA

• Splicing. Step-by-step removal of introns present
  in the pre-mRNA and splicing of the remaining
  exons.

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Splicing
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Splicing

• Three critical sequence elements of pre-mRNA
• Sequences at the 5 splice site
• sequences at the 3 splice site
• sequences within the intron at the branch point

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Spliceosomes

• Composed of proteins and RNAs.
• The RNA components of the spliceosome are small
  nuclear RNAs (snRNAs) called, U1, U2, U4, U5, and
  U6. These range between 50 to 200 nucleotides
  complexed with six to ten protein molecules to
  form small nuclear ribonucleoprotein particles
  (snRNPs).

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Self-splicing

• Some RNAs are capable of self-splicing.
• They can catalyze the removal of their own
  introns in the absence of other RNA or proteins.
• 28S RNA of the protozoan Tetrahymena.
• Self-splicing is catalyzed by the intron itself,
  which acts as a ribozyme.

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Alternative splicing
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The RNA polymerases

• RNA polymerase I (Pol I). Located in nucleolus.
  It transcribes the rRNA genes for the precursor
  of the 28S, 18S, and 5.8S molecules (and is the
  busiest of the RNA polymerases). S values are
  derived from the rate at which the rRNA molecules
  sediment during sucrose gradient centrifugation.

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The RNA polymerases

• RNA polymerase II (Pol II). Found in nucleoplasm.
  It transcribes the mRNA and snRNA genes.
• RNA polymerase III (Pol III). Found in
  nucleoplasm. It transcribes the 5S rRNA genes and
  all the tRNA genes.

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mRNA transcription by RNA PolII

• RNA polyII produces a precursor mRNA (pre-mRNA).
• Promoters contain basal promoter elements and
  promoter proximal elements, having general
  activity.
• Basal promoter elements TATA box is located at
  about position 25, and a pyrimidine-rich
  sequence near the transcription start site is
  called the initiator element (Inr). TATA box
  consensus is TATAAAA.
• Promoter proximal elements Located upstream of
  TATA box, about 50-200 nucleotides from the start
  of transcription. Examples are CAAT (cat) box,
  which is located at about 75 GC box with a
  consensus of GGGCGG, located at about 90.

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Transcription Factors

• Basal transcriptional factors are the proteins
  that are required for the function of RNA
  polymerase II
• They bind to the promoter area, TATA Box
• General Transcription Factors
• TFIID
• TFIIB
• TFIIF
• TFIIF
• TFIIH

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Transcription Factors

• TFIID binds to the TATA box to form the initial
  committed complex. TFIID has a subunit called
  TATA-binding protein (TBP) that recognizes the
  TATA box, and a number of other proteins called
  TBP-associated factors (TAFs).
• TFIID-TATA box complex acts as a binding site for
  TFIIB, which recruits RNA PolII and TFIIF to
  produce the minimal transcription initation
  complex.
• Next, TFIIE and TFIIH bind to produce the
  complete transcription initiation complex (or
  preinitiation complex, PIC).

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Other transcription factors

• Activators bind to enhancers, sequences required
  for maximal transcription.
• Activator binds to an enhancer, and through an
  interaction with another protein called an
  adapter forms a bridge to the preinitation
  complex.
• In most cases, enhancers are upstream of the
  gene.
• Silencer elements have repressing ability for the
  transcription upon binding with a repressor.

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Transcription by RNA Pol I

• Transcription of rRNA genes.
• rRNA genes are found in tandem repeats.
• A large 45S pre-RNA is transcribed, then
  processed to yield 28S, 18S, and 5.8S rRNAs.
• Promoter spans about 150 bp upstream of the
  initiation site.
• UBF (upstream binding factor) and SL1
  (selectivity factor) are transcription factors
  that recognize the promoter.
• TBP is a component of SL1 and is essential.
• rDNA promoter does not contain TATA box.

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rRNA gene
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rDNA transcription
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Transcription by RNA Pol III

• Synthesizes tRNA and 5S rRNA, and some small RNAs
  involved in splicing and protein transport.
• Promoter of 5S rRNA is downstream of the
  transcription initiation site!
• TFIIIA, TFIIIC, TFIIIB, and the polymerase bind
  to the promoter, sequentially.
• tRNA promoters do not contain a binding site for
  TFIIIA.
• TFIIIC initiates transcription.
• The promoter of U6 snRNA is upstream of the start
  site and contains a TATA box, recognized by TBP
  subunit of TFIIIB.

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Transcription of polymerase III genes
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RNA processing
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RNA processing

• Three of the Eukaryotic rRNAs (28S, 18S, and 5.8S
  rRNAs) are cleaved from a single transcript.
• Bacterial rRNAs (23S, 16S, and 5S) are cleaved
  from a single transcript.
• Cleavage involves several steps are somewhat
  different in eukaryotes and prokaryotes.

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Processing of rRNAs
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Processing of tRNAs

• tRNAs are synthesized as longer molecules,
  pre-tRNAs, some of which contain multiple tRNAs.
• The 5 end of pre-tRNA is cleaved by an enzyme
  called RNaseP.
• RNaseP contains RNA and proteins, and RNA itself
  can do the cleavage.
• RNaseP is a ribozyme.

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Processing of tRNAs

• The 3 of tRNAs is generated by Rnase.
• The CCA is added into the terminus, as the site
  of amino acid attachment, and is required for
  tRNA activity during translation.
• Some bases (10) are modified at characteristics
  places, e.g., methylguanosine, inosine, etc. They
  alter the base pairing properties of the tRNA
  molecule.

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Processing of tRNA
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Processing of tRNA
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Nonsense-mediated mRNA decay

• A quality control system
• Degredation of mRNAs that lack complete open
  reading frames.
• Takes place in cytoplasm in yeast.
• If a ribosome encounters a premature stop codon,
  this system is triggered.
• In mammals, it may take place in nucleus.

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RNA degradation in cytoplasm

• rRNA and tRNA are stable they are found in high
  levels in the cell.
• Bacterial mRNA have a half life of 2-3 minutes.
• Why?
• In eukaryotes, different mRNAs have different
  life spans (30 min to 20 hours).

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How mRNA is degraded?

• Degradation is initiated by shortening of their
  polyA tails.
• 5 cap is removed.
• RNA is degraded by nucleases from both ends.
• Which RNAs are expected to be short lived?

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RNA degradation

• Shortening of poly A tails
• Removal of 5 cap
• degradation of RNA by nucleases.
• IRE Iron-response element.
• If iron is available, nuclease degrade the mRNA.
• If iron is scarce, IRE-BP binds to a sequence
  near 3 protect the mRNA.