Chapter Eleven Transcription of the Genetic Code_ The Biosynthesis by hcj

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									         Chapter Eleven
Transcription of the Genetic Code:
     The Biosynthesis of RNA
            Chapter 11
                 Transcription
• Overview of Transcription
  • synthesized on a DNA template, catalyzed by DNA-
    dependent RNA polymerase
  • ATP, GTP, CTP, and UTP are required, as is Mg2+
  • no RNA primer is required
  • the DNA base sequence contains signals for initiation
    and termination of RNA synthesis; the enzyme binds
    to and moves along the DNA template in the 3’ -> 5’
    direction
  – the RNA chain is synthesized in the 5’ -> 3’ direction
  • the DNA template is unchanged
       Transcription in Prokaryotes
• E. coli RNA Polymerase:
   – molecular weight about 500,000
   – four different types of subunits: ￿ , ￿and s
                                        , ￿ ’,
   – the core enzyme is ￿ ’2￿￿
   – the holoenzyme is ￿ ’s
                          2￿￿
   – the role of the s subunit is recognition of the promoter locus;
                                                              locus
     the s subunit is released after transcription begins
   – of the two DNA strands, the one that serves as the template for
     RNA synthesis is called the template strand or antisense strand;
                                                               strand
     the other is called the coding (or nontemplate) strand or sense
     strand
   – the holoenzyme binds to and transcribes only the template
     strand
The Basics of Transcription
           Promoter Sequence
• Simplest of organisms contain a lot of DNA that is
  not transcribed

• RNA polymerase needs to know which strand is
  template strand, which part to transcribe, and
  where first nucleotide of gene to be transcribed
  is

• Promoters-DNA sequence that provide direction
  for RNA polymerase
Promoter Sequence
   How does RNA polymerase know
    where to begin transcription?
• Polymerase moves along template strand
  from 3’-5’ and RNA is formed from 5’-3’
• Binding site for polymerase is upstream of
  start of transcription – away from 5’ of coding
  strand
• Promoter sequence is based on coding strand
  and RNA polymerase is binding to template
  strand
   How does RNA polymerase know
    where to begin transcription?
• Promoter are upstream towards 5’ of coding
  and 3’ template strand
• The first base to be incorporated is at position
  + 1 = transcription start site
• All nucleotides upstream from this start site
  are given –ve numbers
• The first promoter element is about 10 bases
  upstream is – 10 region or pribnow box
   How does RNA polymerase know
    where to begin transcription?
• After PB 16-18 bases are variable
• Next promoter element is about 35 bases
  upstream of TSS = -35 region or -35 element
• Area from -35 element to TSS = core promoter
• Upstream of core element is UP element =
  enhances binding of RNA polymerase
• Region from UP element to TSS = extended
  promoter
• BASE SEQUENCE of promoter is A and T
               Chain Initiation
• First phase of transcription is initiation

• Initiation begins when RNA polymerase binds
  to promoter and forms closed complex

• After this, DNA unwinds at promoter to form
  open complex, which is required for chain
  initiation
Initiation and Elongation Transcription
Chain Elongation (Cont’d)
             Chain Elongation
• After strands separated, transcription bubble of
  ~17 bp moves down the DNA sequence to be
  transcribed

• RNA polymerase catalyzes formation of
  phosphodiester bonds between the incorp.
  ribonucleotides

• Topoisomerases relax supercoils in front of and
  behind transcription bubble
             Chain Termination
• Two types of termination mechanisms:
  • intrinsic termination- controlled by specific
  sequences, termination sites
• Termination sites characterized by two inverted
  repeats
             Chain Termination
• Other type of termination involves rho () protein
• Rho-dependent termination sequences cause
  hairpin loop to form
       Transcription Regulation in
              Prokaryotes

• In prokaryotes, transcription regulated by:

  • alternative s factors
  – enhancers
  – operons
  – transcription attenuation
Alternative s factors

           • Viruses and bacteria
             exert control over
             which genes are
             expressed by producing
             different s-subunits
             that direct the RNA
             polymerase to different
             genes.
                 Enhancers
• Certain genes include sequences upstream of
  extended promoter region
• These genes for ribosomal production have 3
  upstream sites, Fis sites
• Class of DNA sequences that do this are called
  enhancers
• Bound by proteins called transcription factors
Elements of a Bacterial Promoter
                     Operon
• Operon: a group of operator, promoter, and
  structural genes that codes for proteins
  – the control sites, promoter, and operator genes
    are physically adjacent to the structural gene in
    the DNA
  – the regulatory gene can be quite far from the
    operon
  – operons are usually not transcribed all the time
     Example of Operon system

• b-Galactosidase, an inducible protein
  – coded for by a structural gene, lacZ
  – structural gene lacY codes for lactose permease
  – structural gene lacA codes for transacetylase
  – expression of these three structural genes is
    controlled by the regulatory gene lacI that codes
    for a repressor
Transcription in Eukaryotes is complex
•   Three RNA polymerases are known - each
    transcribes a different set of genes and
    recognizes a different set of promoters:
    • RNA Polymerase I- found in the nucleolus and
    synthesizes precursors of most rRNAs
    • RNA Polymerase II- found in the nucleoplasm
    and synthesizes mRNA precursors
    • RNA Polymerase III- found in the nucleoplasm
    and synthesizes tRNAs, other RNA molecules
    involved in mRNA processing and protein
    transport
            RNA Polymerase II
• Most studied in
  the polymerases
• Consists of 12
  subunits
• RPB- RNA
  Polymerase B
   How does Pol II Recognize the Correct
                   DNA?
• Four elements of the Pol II promoter .
            Pol II promoters
• Variety of upstream elements – activators and
  silencers
- GC box (-40) – Consensus sequence –
  GGGCGG
- CAAT box (extending to – 110) – Consensus
  sequence - GGCCAATCT
            Pol II promoters
• Second element found at -25 = TATA box has
  consensus sequence of TATAA (T/A)
• Transcription start site at position + 1
  surrounded by a sequence called initiator
  element (Inr)
• Inititator and TATA box = core promoter
• Fourth element – downstream regulator -
  rare
Initiation of Transcription
              • Transcription factor -
                Any protein regulator of
                transcription that is not
                a subunit of Pol II

              • Initiation begins by
                forming the
                preinitiation complex -
                Transcription control is
                based here
      Transcription Order of Events
• The phosphorylated
  Pol II synthesizes
  RNA
• Leaves the promoter
  region behind
• GTFs are left at the
  promoter or
  dissociate from Pol II
     Elongation and Termination
• Elongation is controlled by:
  – pause sites - where RNA Pol will hesitate
  – positive transcription elongation factor (P-TEF)
    and negative transcription elongation factor (N-
    TEF)
• Termination
  – begins by stopping RNA Pol; the eukaryotic
    consensus sequence for termination is AAUAAA
               Gene Regulation
• Enhancers and silencers- regulatory sequences that
  augment or diminish transcription, respectively
• DNA looping brings enhancers into contact with
  transcription factors and polymerase
Response elements
         • Response elements are
           enhancers that respond
           to certain metabolic
           factors
           • heat shock element
           (HSE)
           • glucocorticoid response
           element (GRE)
           • metal response element
           (MRE)
           • cyclic-AMP response
           element (CRE)
      Structural Motifs in DNA-Binding
                  Proteins
• Most proteins that activate or
  inhibit RNA Pol II have two
  functional domains:
  – DNA-binding domain
  – transcription-activation domain
• DNA-Binding domains have
  domains that are either:
  • Helix-Turn-Helix (HTH)
  • Zinc fingers
  • Basic-region leucine zipper
            Helix-Turn-Helix Motif
Hydrogen bonding between amino acids and DNA
               Zinc Finger Motif

• Motif contains 2
  cysteines and 2 His –
  after every 12 amino
  acids

• Zinc binds to the
  repeats
Basic Region Leucine Zipper Motif
                 • Many transcription
                   factors contain this motif
                   - CREB

                 • Half of the protein
                   composed of basic region
                   of conserved Lys, Arg, and
                   His

                 • Half contains series of Leu
Post Transcriptional RNA Modification
• tRNA, rRNA, and mRNA are all modified after transcription to
  give the functional form
   – the initial size of the RNA transcript is greater than the
     final size because of the leader sequences at the 5’ end
     and the trailer sequences at the 3’ end
• Modifications
   – trimming of leader and trailer sequences
   – addition of terminal sequences (after transcription)
   – modification of specific bases (particularly in tRNA)
            Modification of tRNA
• Transfer RNA-
  – trimming, addition of
    terminal sequences, and
    base modification - take
    place
  – methylation and
    substitution of sulfur for
    oxygen are the two most
    usual types of base
    modification
         Modification of rRNA
• Ribosomal RNA

  – processing of rRNA - methylation and trimming to
    the proper size
         Modification of mRNA
• Capping of the 5’ end with an N-methylated
  guanine
• A polyadenylate “tail” that is usually100-200
  nucleotides long, is added to the 3’ end before
  the mRNA leaves the nucleus
Organization of Split Genes in Eukaryotes
       Modification of mRNA
– Eukaryote genes frequently contain intervening
  base sequences that do not appear in the final
  mRNA of that gene product

– Expressed DNA sequences are called exons

– Intervening DNA sequences that are not
  expressed are called introns

– These genes are often referred to as split genes
           The Splicing Reaction
• Exons are
  separated by
  intervening intron

• When the exons are
  spliced together -
  lariat forms in the
  intron
Ribozymes
     • The first ribozymes
       discovered included
       those that catalyze their
       own self-splicing
     • ribozymes have been
       discovered that are
       involved in protein
       synthesis
     • Group I and II
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