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					   UNIT 3
 Transcription
       and
Protein Synthesis
Objectives
   Discuss the flow of information from DNA to RNA to Proteins
   Explain transcription
   Differentiate introns and exons
   State functions of the noncoding regions of mRNA
   Describe post transcriptional modification of pre-RNA
   Distinguish between mRNA, tRNA and rRNA
   Distinguish the roles of ribosomes, tRNA and mRNA in protein
    synthesis
   Give detailed description of the process and steps of translation
   Describe application of SDS-Page gel electrophoresis to protein
    analysis
DNA to RNA to Proteins
     DNA to RNA to Proteins
    Eukaryotes vs. Prokaryotes
In prokaryotes, transcription and translation are
  coupled
 translation begins while the mRNA is still being
  synthesized.
mRNA in Prokaryotes
               DNA to RNA to Proteins
              Eukaryotes vs. Prokaryotes
   In Eukaryotes,
    transcription and
    translation are spatially
    and temporally separated

       transcription occurs in the
        nucleus to produce a pre-
        mRNA molecule.
        pre-mRNA is processed
        to produce the mature
        mRNA, which exits the
        nucleus and is translated
        in the cytoplasm.
mRNA in Eukaryotes
 DNA to RNA to Proteins
Eukaryotes vs. Prokaryotes
                   Transcription
   occurs in four main stages:
    1) binding of RNA polymerase to DNA at a promoter

    2) initiation of transcription on the template DNA
    strand

    3) elongation of the RNA chain

    4) termination of transcription along with the release of
    RNA polymerase and the completed RNA product
    from the DNA template.
                        Transcription
   Binding of polymerases to the initiation site at the promoter.
       Prokaryotic RNA polymerases can recognize the promoter and bind to it
        directly, but eukaryotic RNA polymerases have to rely on other proteins
        called transcription factors.
   Unwinding of the DNA double helix by helicase.
       Prokaryotic RNA polymerases have the helicase activity, but eukaryotic
        RNA polymerases do not.
       Unwinding of eukaryotic DNA is carried out by a specific transcription
        factor.
   Synthesis of RNA based on the sequence of the DNA template
    strand.
       RNA polymerases use nucleoside triphosphates (NTPs) to construct a
        RNA strand.
   Termination of synthesis.
       Prokaryotes and eukaryotes use different signals to terminate
        transcription.
                       Promoter
   The DNA promoter region in prokaryotes is a stretch
    of about 40 base pairs adjacent to and including the
    transcription start point.

   The essential features of the promoter are the start
    point (designated +1 and usually an A), the six-
    nucleotide -10 sequence, and the six-nucleotide -35
    sequence.

   The two key sequences are located approximately 10
    nucleotides and 35 nucleotides upstream from the start
    point.
Prokaryotic promoter
                  Elongation
   During elongation, RNA polymerase binds to
    about 30 base pairs of DNA
   At any given time, about 18 base pairs of DNA
    are unwound, and the most recently synthesized
    RNA is still hydrogen-bonded to the DNA,
    forming a short RNA-DNA hybrid (12 bp long,
    but it may be shorter)
   The total length of growing RNA bound to the
    enzyme and/or DNA is about 25 nucleotides.
                        Termination
   Requires a termination sequence that triggers the
    end of transcription.
   Two classes exist:
       rho dependent
            a short complementary GC-rich sequence (followed by
             several U residues) will form a "brake" that will help
             release the RNA polymerase from the template.
       rho independent.
            binding of rho to the mRNA releases it from the
             template.
                  Termination
 rho-dependent –
requires a protein called
  rho to bind to specific
  sequence.
                   Termination
   rho-independent -
    depends on a seqence in
    mRNA which forms a
    stem loop.
Termination
        Transcription in Eukaryotes
   Although transcription in eukaryotes is similar to that in
    prokaryotes, the process is more complex.

   Instead of one RNA polymerase, there are three :
       RNA polymerase I (localized to the nucleolus) transcribes
        the rRNA precursor molecules.
       RNA polymerase II produces most mRNAs and snRNAs.
       RNA polymerase III is responsible for the production of
        pre-tRNAs, 5SrRNA and other small RNAs.
       The mitochondria and chloroplasts have their own RNA
        polymerases.
         Transcription complex
   http://highered.mcgraw-
    hill.com/sites/0072437316/student_view0/chap
    ter18/animations.html#
      Transcription in Eukaryotes

   Eukaryotic nuclear genes have three classes of
    promoters which are individual for the three
    types of RNA polymerases
      Transcription in Eukaryotes
   Termination signals end the transcription of
    RNA by RNA polymerase I and RNA
    polymerase III without the activity of hairpin
    structures as seen in prokaryotes.
   mRNA is cleaved 10 to 35 base-pairs
    downstream of a AAUAAA sequence (which
    acts as a poly-A tail addition signal).
    RNA processing in Eukaryotes
   Ribosomal RNA processing
       involves cleavage of multiple rRNAs from a
        common precursor, with nontranscribed spacers
        separate the units.

       The transcription unit is transcribed by RNA
        polymerase I into a single long transcript (pre-
        rRNA)
    RNA processing in Eukaryotes
   Every tRNA gene is transcribed as a precursor
    that must be processed into a mature tRNA
    molecule by:
     removal of the leader sequence at the 5΄ end
     replacement of two nucleotides at the 3 ΄ end by the
      sequence CCA (with which all mature tRNA
      molecules terminate)
     chemical modification of certain bases

     excision of an intron.
    RNA processing in Eukaryotes
   Transcription of eukaryotic pre-mRNAs often
    proceeds beyond the 3΄ end of the mature
    mRNA.
   An AAUAAA sequence located slightly
    upstream from the proper 3΄ end then signals
    that the RNA chain should be cleaved about 10-
    35 nucleotides downstream from the signal site,
    followed by addition of a poly-A tail catalyzed
    by poly(A) polymerase.
    RNA processing in Eukaryotes
   Messenger RNA in eukaryotes
        is first made as heterogeneous nuclear mRNA /
        pre-mRNA then processed into mature mRNA
        through:
          the addition of a 5 prime cap - a guanosine nucleotide
           methylated at the 7th position
          addition of poly-A tails

          the splicing out of introns.
              Introns and Exons
   Eukaryotic genes have interrupted coding sequences.

   There are long sequences of bases within the protein-
    coding sequences of the gene that do not code for
    amino acids in the final protein product.

   The nocoding regions within the gene are called
    introns (intervening sequences).

   The exons (expressed sequences) are part of the
    protein-coding sequence.
How introns are removed
         How introns are removed
   The intron loops out as
    snRNPs (small nuclear
    ribonucleoprotein particles,
    complexes of snRNAs and
    proteins) bind to form the
    spliceosome.
   The intron is excised, and the
    exons are then spliced
    together.
   The resulting mature mRNA
    may then exit the nucleus and
    be translated in the
    cytoplasm.
              Animation –
         How introns are removed
   http://highered.mcgraw-
    hill.com/sites/0072437316/student_view0/chap
    ter15/animations.html#
            Introns and Exons
   A typical eukaryotic gene may have multiple
    exons and introns and the numbers are quite
    variable.
   In many cases the lengths of the introns are
    much greater than those of the exon sequences.
   For instance the ovalbumin gene contains about
    7700 base pairs, 1859 of them in exons.
Ovalbumin gene
            Functions of Introns
   Evidence now exists that introns have many
    functions, including for regulation and structural
    purposes, and that many of the roles now
    hypothesized for introns are plausible but need
    further elucidation.
Please read at least three of the
 following articles for tutorial
http://www.sciencedaily.com/releases/2009/05/090528203730.htm

http://www.sciencedaily.com/releases/2007/07/070712143308.htm

http://www.sciencedaily.com/releases/2006/11/061113180029.htm

http://www.sciencedaily.com/releases/2009/06/090606105203.htm

http://www.sciencedaily.com/releases/2006/04/060404090831.htm

http://www.sciencedaily.com/releases/2005/10/051020090946.htm

http://www.sciencedaily.com/releases/2009/05/090520140408.htm

http://www.sciencedaily.com/releases/2008/11/081104180928.htm

http://www.sciencedaily.com/releases/2008/10/081017080145.htm
Roles of ribosomes, mRNA and
 tRNA in Protein Synthesis
             Protein Synthesis
   http://highered.mcgraw-
    hill.com/sites/0072437316/student_view0/chap
    ter15/animations.html#
    References/ sources of images
   http://evolution.berkeley.edu/evolibrary/article/0_0_0/mutations_03
   usmlemd.wordpress.com/2007/07/14/dna-replication/
   http://employees.csbsju.edu/hjakubowski/classes/ch331/dna/centraldogma.jpg
   http://www.usask.ca/biology/rank/demo/replication/cons.rep.gif
   http://click4biology.info/c4b/3/images/3.4/SEMICON.gif
   http://www.bio.miami.edu/~cmallery/150/gene/sf12x16.jpg
   http://publications.nigms.nih.gov/findings/sept08/images/hunt_gene_big.jpg
   http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg
   http://images.google.com.jm/imgres?imgurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication.jpg
    &imgrefurl=http://ghr.nlm.nih.gov/handbook/illustrations/duplication&usg=__BgKRLXXos-
    xRaUqN5EyP7qchszc=&h=400&w=370&sz=38&hl=en&start=2&tbnid=ZfARmmvAKG02xM:&tbnh=124
    &tbnw=115&prev=/images%3Fq%3Dduplication%2Bmutation%26gbv%3D2%26hl%3Den%26client%3Dfir
    efox-a%26rls%3Dorg.mozilla:en-US:official%26sa%3DG
   http://images.google.com.jm/imgres?imgurl=http://www.phschool.com/science/biology_place/biocoach/im
    ages/transcription/euovrvw.gif&imgrefurl=http://www.phschool.com/science/biology_place/biocoach/trans
    cription/tctlpreu.html&usg=__-
    hoX0ehn3x9zc2nUeciZ8gLYLqQ=&h=288&w=261&sz=20&hl=en&start=10&um=1&tbnid=X11pPXboE
    KhRIM:&tbnh=115&tbnw=104&prev=/images%3Fq%3Dtranscription%26ndsp%3D18%26hl%3Den%26sa
    %3DG%26um%3D1
   www.asa3.org/ASA/PSCF/2001/PSCF9-01Bergman.html
   http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/CB21.html
   http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-21/2101.jpg

				
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