DNA Transcription

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• DNA Transcription
• Defined as the transfer of genetic material from
  ds template DNA to a ss RNA molecule.

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• The Central Dogma proposed by James Watson, is
  that genetic information is transferred
• From DNA to DNA through replication during its
  transmission from generation to generation.
• From DNA to RNA to protein during its phenotypic
  expression in an organism.

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• Transcription is catalyzed by the enzyme RNA
  polymerase
• a single strand of RNA is synthesized using a
  double stranded DNA molecule as a template. The
  two strands of the DNA molecule are separated
  from one another, exposing the nitrogenous bases.
  Only one strand is actively used as a template in
  the transcription process, this is known as the
  sense strand, or template strand. The
  complementary DNA strand, the one that is not
  used, is called the nonsense or antisense strand.

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• RNA polymerase
• Is specific for RNA synthesis,
• Will only use ATP, GTP, CTP, and UTP that
  contains a ribose sugar
• Synthsizes in the 5-3 direction
• DNA template 3 ATACTGGAC-5
• RNA product 5 UAUGACCUG-3

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• RNA polymerase requires DNA template , Mg2,
  the 4 ribonucleotide triphosphate

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• Characteristics of RNA polymerase
• Has no proof reading ability
• The enzyme has 5 polypeptide subunits
• two alpha (? ) one beta (ß) one beta prime
  (ß) and omega (?)
• the active enzyme is called the core
  enzyme MW of 450 000 daltons (Da)
• Sigma factor is 85 000 Da small
  proteinase factor
• The core enzyme plus the sigma factor is
  called the
• holoenzyme or complete enzyme

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• The holoenzyme performs 4 functions
• Recognizes a promotor on the ds DNA
• Denatures and unwinds DNA at the promoter
• Must align itself on the template and
  transcribe the complete gene
• Must stop transcription at the terminator
• Unlike in replication where the total length
  of the chromosome is replicated in transcription
  first the gene to be transcribed needs to be
  identified
• Transcription can be divided into 3 stages
• Initiation, Elongation and Termination

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• Initiation
• RNA polymerase must be able to recognize the
  beginning of a gene so that it knows where to
  start synthesizing an mRNA. It is directed to the
  start site of transcription by one of its
  subunits' affinity to a particular DNA sequence
  that appears at the beginning of genes. This
  sequence is called a promoter. It is a
  unidirectional sequence on one strand of the DNA
  that tells the RNA polymerase both where to start
  and in which direction (that is, on which strand)
  to continue synthesis. The bacterial promoter
  almost always contains some version of the
  following elements
• Promoter recognition at
  10bp region Pribnow box
• 
  at 35 bp region,
• 
  these are known asconsensus
• 
  sequences since they are
• 
  similar in diff. Genes in the
• Upstream
  same organism

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• The efficiency and rate of transcription may be
  directly related to differences in these 2
  promoter sequences.
• Elongation The RNA polymerase then stretches
  open the double helix at that point in the DNA
  and begins synthesis of an RNA strand
  complementary to one of the strands of DNA. It is
  the sigma subunit of RNA polymerase that binds to
  the promoter and starts the elongation but after
  adding a few ribonucleotides the s subunit
  dissociates from the holoenzyme and elongation
  proceeds under the direction of the core enzyme.
  In E.coli this process proceeds at the rate of
  about 50 nucleotides/second at 37c
• Topoisomerase II handles the template supercoiling

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• Termination Following elongation of the entire
  gene the enzyme encounters a specific sequence
  that acts as a termination signal. In prokaryotes
  these sequences are about 40 bp in length and are
  followed by six AT base pairs.
• These AT base pairs are transcribed in a poly U
  tail, also two fold symmetry can cause hair pin
  loops, this type of termination is named as Rho
  independent termination.
• Rho dependant terminaton Rho is a large hexmeric
  protein that interacts with the growing RNA
  transcript and disrupts the RNA polymerase/DNA
  template association
• In bacteria groups of genes whose products are
  related are often clustered along the chromosome.
  They are transcribed contiguously and as a result
  a long mRNA is produced. Since genes in bacteria
  are sometimes called cistrons these mRNA are
  named a polycistronic. The products of genes
  transcribed in this fashion are all needed at the
  same time

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• Transcription in Eukaryotes
• The general aspects of the mechanism of the
  process is the same except
• - more than one RNA polymerase
• - promoter and termination sequences are
  different
• - RNA processing step
• The 3 major RNA Polymerases in Eukaryotes
• RNA poly 1 found only in the nucleolus
  catalayses the synthesis of 28S, 18S, 5.8S rRNA
  these molecules are important in protein
  synthesis
• RNA poly II found only in the nucleoplasm ,
  catalyses the synthesis of all mRNAs and some
  small nuclear RNAs (snRNA)
• RNA poly III found in nucleoplasm catalyses
  tRNA, 5S RNA and other small RNA molecules
• All eukaryotic RNA polymerases have several sub
  units (2 large and 4 or more small)
• In eukaryotes transcription occurs within the
  nucleus and the RNA transcript is not free to
  associate with ribosomes prior to the completeion
  of transcription.

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• Some differences between prokaryote and eukaryote
  trascription, in eukaryotes
• Initiation and regulation of transcription
  involve a more extensive interaction between
  upstream DNA sequences and protein factors
  involved in stimulating and and initiating .
• In addition to promoters, other control units
  called enhancers may be located in the 5
  regulatory region upstream from the initiation
  point, but they have been found within the gene
  or even in the 3 downstream region beyond the
  coding sequence.
• Maturation of eukaryotic mRNA from the primary
  RNA transcript involves many complex stages
  referred to as processing.

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• Promoters
• Important DNA sequences lie within this region
  that initiate transcription by DNA polymerase.
  Two such elements are present
• Goldberg Hogness or TATA box located at 30
  (30 bp upstream) from the start point of
  transcription, the consensus sequence is a
  heptanucleotide consisting of A and T residues
  TATAAAA( 5-3) similar to pribnow box in
  prokaryotes. It fixes the site of transcription
  initiation by facilitating denaturation of the
  helix.
• The other element is the CAAT box located at 80,
  it contains the consensus sequence of GGCCAATCT
  any mutation in the CAAT element, results in a
  marked reduction in transcription.
• There may be more than one copy of this GC
  elements in the promoter. They help bind the RNA
  polymerase.

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• Enhancers- important for maximal transcription of
  a gene there are no consensus sequences for
  enhancers
• Two types of enhancers (i) Activator (
  activates transcription)
• (2)
  Repressors (also called silencer elements)
• Enhancers may involve Protein binding to the
  enhancers
• DNA
  loop structures forming between the enhancer and
  gene
• 
  sequencers
• Transcription factors Another kind of specific
  proteins that facilitate initiation of
  transcription

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• TF1 for RNA Pol I,
• TFII for RNA Pol II
• TFIII for RNA Pol. III
• Some binds to TATA elements other may bind to
  CAAT or GC elements. In most cases it binds to
  RNA Pol and facilitate initiation of
  transcription
• An overview of RNA Processing
• mRNA (messenger RNA) Transcribed by structural
  genes (protein coding genes) contain the coded
  information for the Amino Acid sequence of a
  protein.
• mRNAs have 3 main parts
• 5 leader sequence- important for the start of
  protein synthesis
• Coding sequence sequence that codes for AA
• 3 trailor sequence- poly A tail

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• Typical structure of a biologically active mRNA
  molecule
• start codon
  stop codon
• 5leader sequence coding sequence poly
  A tail
• The start codon is AUG and stop codons are UAA,
  UAG and UGA.The production of biologically active
  mRNA is different, in prokaryotes and eukaryotes
• In prok. The mRNA transcript functions directly
  in translation. Since theres no nucleus
  translation begins before mRNA is completely
  transcribed.
• In eukaryotes the mRNA transcript must be
  modified in the nucleus by a series of events
  called post transcriptional modifications or
  RNA processing.

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• 5 capping - Involves the addition of a guanine
  (7 methyl guanosine) to the terminal 5
  nucleotide. A capping enzyme is responsible for
  the addition and completing the process. The 5
  cap is required for the ribosome to bind to the
  mRNA as the initial step in translation

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• Addition of 3 poly A tail- This poly A tail is
  usually about 50-250 bp of adenine in length and
  there is no DNA template for this tail. (poly A
  tails are found in most mRNA molecules but not
  all. Ex. Histones mRNA have no poly A tail. Later
  the enzyme endonucleases cleaves the molecule at
  the poly A site to generate 3 OH end.
• The tail is important for the stability of the
  mRNA molecule. In general a eukaryotic mRNA mole.
  Is longer than the required transcript. The
  enzyme endonuclease cleaves the molecule at the
  poly A addition site to generate 3 OH end.
• Introns and exons Eukaryotic mRNAs often
  contain long insertions of non-amino acid coding
  sequences. - These sequences are transcribed into
  initial RNA transcript but they are removed
  before a mature mRNA is developed.
• - First noticed when researchers found that the
  nucleotide sequence of some genes (globin genes)
  were longer than necessary to produce the protein
  product. Eventually it was found that only a few
  genes were with out introns.

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• Introns (intervening sequences) insertions of
  non A.A coding sequences of a pre mRNA that are
  removed in the mature process of mRNA. The genes
  containing introns are called split genes. The
  ovalbumin gene of chickens contain about 7
  introns.
• Exons (expressed sequences) is the nucleotide
  sequence that is translated into an A.A sequence.
• Both are copied into a primary pre- mRNA
  transcript.
• The final mature mRNA is very much short than the
  initial RNA transcript.
• Splicing mechanism This is the mechanism by
  which introns are excised and exons are spliced
  back together. It appears that somewhat different
  mechanisms work for diff. Types of RNA and also
  RNA produced in mitochondria and chloroplasts.
  During the process, first a molecular complex
  known as spliceosome forms and Then, after a
  two-step enzymatic reaction, the intron is
  removed and two neighboring exons are joined
  together. The enzyme RNA lygase joins the two
  exons

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• The small nuclear RNAs are the most essential
  component in splicesomes

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• The RNAs
• Four different classes of RNA are transcribed
• mRNA, tRNA, rRNA and small nuclear RNA (SnRNA)
• The first 3 are found both in prokaryotes and
  eukaryotes, but SnRNAs are limited only to
  eukaryotes.
• Stability- Except for mRNAs others are relatively
  long lived, mRNA are very short lived
• Genes that code for mRNAs are structural genes
  that code for proteins, but for other RNAs it is
  not structural genes since they produce only a
  RNA molecule
• In prokaryotes only one RNA polymerase
  transcribes all genes, but in eukaryotes 3
  different RNA polymerases are involved.

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