Transcription

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Transcription

• M.PRASAD NAIDU
• MSc MEDICAL BIOCHEMISTY,
• Ph.D.RESEARCH SCHOLAR

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• Definition
• Synthesis of RNA using ssDNA as a template by
  DNA
• dependent RNA polymerase
• Similar to replication in terms of chemical
  mechanism,
• polarity, and use of template but differ
  in
• -does not require primers
• -only a short segment of
  DNA is transcribed

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• All 3 types of cellular RNAs are copied during
  transcription
• Messenger RNAs (mRNAs) encode the amino acid
  sequence of one or more polypeptides specified by
  a gene or set of genes.
• Transfer RNAs (tRNAs) read the information
  encoded in the mRNA and transfer the appropriate
  amino acid to a growing polypeptide chain during
  protein synthesis.
• Ribosomal RNAs (rRNAs) are constituents of
  ribosomes, the intricate cellular machines that
  synthesize proteins.

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• Basic Requirements of Transcription--prokaryotic
• Template ------ssDNA
• Enzyme-----RNA polymerase
• Regulatory proteins
• Ribonucleoside triphosphates ( A, G,C,U )
• Energy uses the energy released from the
  cleavage of pyrophosphates to two phosphates by
  pyrophosphotase

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• Steps involved in Transcription
• Initiation
• -recognition of specific DNA sequence
  ( promoter region )
• -beginning of bond formation process
• Elongation
• Termination release

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• Initiation
• Starts with the recognition of promoter sequence
  on the DNA coding ( anti-template ) strand by RNA
  polymerase
• Promoter sequence
• Present on the DNA
• Two common sequences are present on the
  upstream ( 5 ) side of the start site
• Start site is denoted by 1

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• Contd--
• -10 sequence -35 sequence
• 5------TTGACA--------TATAAT--------start site
  --------terminator 3
• (-35 ) ( -10)
  1
• Sequences are 6 bp long
• Distance b/n these two promoters is conserved
  is a
• separation of 17-19 nucleotides in normal
• -10 sequence is called Pribnow box ( TATA box )

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• Contd
• Genes with strong promoters cause frequent
  initiation of transcription as often as every two
  seconds ( E coli ) (strong promoters have
  sequence that correspond closely to the consensus
  sequence )
• Genes with weak promoters are transcribed about
  once in 10 minute ( weak promotes tend to have
  multiple substitution at that site )

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• RNA Polymerase ( E.Coli )
• Multisubunit enzyme
• DNA dependent RNA polymerase
• Very large molecule ( 500 kd ) and complex enzyme
  consisting of four kinds of subunits
• Prokaryotes have single RNA pol that transcribes
  all the three RNAs ( mRNA, t RNA, r RNA )
• a2 ß ß O s ------holoenzyme
• RNA polymerase with out s subunit is called
  core enzyme
• Core enzyme contains the catalytic activity.

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Subunit Gene Number Mass ( kd ) Role
a rpo A 2 37 Binds regulatory proteins
ß rpo B 1 151 Forms phosphodieter bond
ß rpo C 1 155 Binds DNA template
s rpo D 1 70 Recognizes promoter initiates synthesis
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• Contd--
• RNA polymerases lack a separate proofreading 3
  to 5 exonuclease active site (such as that of
  many DNA polymerases)
• The error rate for transcription is higher than
  that for chromosomal DNA replication
  approximately one error for every 10 4 to
  10 5 ribonucleotides incorporated into RNA.

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• Contd--
• After recognizing binding to the promoter
  region RNA pol synthesizes complimentary RNA
  sequence to the DNA template strand ( U instead
  T is paired with A )
• RNA synthezised from 5 to 3 ( template read
  from 3 to 5 )
• 5 end of new RNA chain is highly distinctive a
  molecule starts with either pppG or pppA ( DNA
  synthesis Primers )
• Template strand is determined by the location of
  the promoter region for that gene

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• Contd--
• RNA pol interacts with activator repressor
  proteins that modulate the rate of transcription
• In bacteria one species of RNA pol can synthezise
  all the RNA molecules ( mRNA, tRNA, rRNA ) except
  short RNA primers needed for DNA replication (
  primase )

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• Elongation
• RNA polymerase elongates an RNA strand by adding
  ribonucleotide units to the 3-hydroxyl end
• The 3-hydroxyl group acts as nucleophile,
  attacking the phosphate of the incoming
  ribonucleoside triphosphate and releasing
  pyrophosphate.
• By the time 10 nucleotides are added ,the s
  factor dissociates the core enzyme continues
  the elongation of the transcript

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• Contd
• The template DNA strand is copied in the 3 to 5
  direction (antiparallel to the new RNA strand),
  just as in DNA replication.
• Each nucleotide in the newly formed RNA is
  selected by Watson-Crick base-pairing interaction
• RNA pol
• (NMP)n NTP
  (NMP)n1 PPi
• RNA
  Lengthened RNA

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• Termination
• E. coli has at least two classes of termination
  signals
• one class relies on a protein factor called ?
  (rho)
• the other is ? independent.
• Most ? independent terminators have two
  distinguishing features.
• The first is a region that produces an RNA
  transcript with self complementary sequences,
  permitting the formation of a hairpin structure
  centered 15 to 20 nucleotides before the
  projected end of the RNA strand.

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• Contd--
• The second feature is a highly conserved string
  of A residues in the template strand that are
  transcribed into U residues near the 3 end of the
  hairpin.
• When a polymerase arrives at a termination site
  with this structure, it pauses
• Formation of the hairpin structure in the RNA
  disrupts several AUU base pairs in the RNA-DNA
  hybrid segment and may disrupt important
  interactions

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• Contd
• Rho (? ) dependent termination
• Rho factor is an ATP dependent RNA-DNA helicases
• Recognizes and bind to the termination signals
  and disrupts the nascent RNA/DNA complex

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• Transcription Is Regulated at Several Levels
• Requirements for any gene product vary with
  cellular conditions or developmental stage, and
  transcription of each gene is carefully regulated
  to form gene products only in the proportions
  needed.
• Regulation can occur at any step in
  transcription, including initiation ,elongation
  and termination.
• Much of the regulation is directed at the
  polymerase binding and transcription initiation

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• The binding of proteins to sequences both near to
  and distant from the promoter can also affect
  levels of gene expression.
• Protein binding can activate transcription by
  facilitating either RNA polymerase binding or
  steps further along in the initiation process, or
  it can repress transcription by blocking the
  activity of the polymerase.
• In E. coli, one protein that activates
  transcription is the cAMP receptor protein (CRP),
  which increases the transcription of genes coding
  for enzymes that metabolize sugars other than
  glucose when cells are grown in the absence of
  glucose.

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• Contd--
• Repressors are proteins that block the synthesis
  of RNA at specific genes.
• In the case of the Lac repressor , transcription
  of the genes for the enzymes of lactose
  metabolism is blocked when lactose is unavailable

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• Inhibitors
• Rifampicin ( antitubercular drug)
• -semisynthetic derivative
• ( sterptomyces)
• -specifically inhibit initiation not by blocking
  the binding of RNA pol but rather by interfering
  with the formation of the first phosphodiester
  bond
• -Site of action is on ß subunit of RNA pol
• -some mutants having an altered ß subunit are
  resistant to rifampacin

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• Actinomycin D
• -polypeptide derived from streptomyces
• -binds tightly specifically to double stranded
  DNA prevents it from being an effective
  template for RNA synthesis
• -at low concentration inhibits transcription
  without appreciably affecting replication
  protein synthesis
• -inhibitor of both prokaryotic eukaryotic
  cell
• -effective therapeutic agent in the treatment of
  some cancers

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Thank you