Transcription and Translation by r900ws

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									 Protein Synthesis or Gene
         Expression:
 Outlining the Processes of
Transcription and Translation
      Adenine (DNA and RNA)
      Cystosine (DNA and RNA)
      Guanine(DNA and RNA)
      Thymine (DNA only)
      Uracil (RNA only)




                                       RNA
                                 polymerase

                                       DNA
                                RNA
             From Genes to Proteins
   The genetic material, DNA, contains
    discrete units, genes, that specify certain
    traits
   How do genes confer specific phenotypes?
       The function of a gene is to dictate the
        production of a specific protein or enzyme
          The enzyme catalyzes a certain chemical reaction
           in the cell
          Differences or mutations in these genes cause
           different or defective proteins leading to a given
           phenotype
     One gene – one enzyme
    hypothesis (Beadle and Tatum)

   Studying the mold Neurospora
       Wild type mold can make its own amino acids
       Treating mold with X-rays damages DNA,
        causing mutations in specific genes
       Mutants can grow on complete media
          Determined mutant gene by growing mutant
           species on minimal media plus one of the 20
           amino acids
          Found 3 classes of mutants that had mutations in
           synthesizing arginine
     Beadle and Tatum’s Evidence for the One gene - One
                    Enzyme hypothesis
                                     Each mutant was defective in a single
                                      gene and lacked a specific enzyme




Conclusion: Mutations were abnormal variations of different genes, each gene dictating
              the production of one enzyme: One Gene – One Protein
THE CENTRAL DOGMA




Transcription   Translation
             Overview: transcription and translation



                                 mRNA
DNA provides the
template for RNA
synthesis. A copy
     of a gene is
produced called a
  messenger RNA
   (mRNA) which
     carries the
 instructions from
DNA to the protein
    synthesizing
     machinery.
               Overview: transcription and translation

 mRNA provides the
template for protein
 synthesis. Nucleic
acids are translated
into amino acids by
    ribosomes.



 In a prokaryotic
cell, the two steps
        occur
  simultaneously
 since there is no
     nucleus to
   separate the
     processes.
              Overview: transcription and translation




In a eukaryotic cell,
     the nucleus
 separates the DNA
from the ribosomes
  in the cytoplasm.


mRNA synthesized
 in the nucleus is
called the primary
     transcript.
             Overview: transcription and translation




   The primary
     transcript
  undergoes RNA
 processing steps,
which modifies it in
certain ways. The
   mRNA is then
translocated from
the nucleus to the
    cytoplasm.
              Overview: transcription and translation




    Once in the
    cytoplasm,
ribosomes bind to
  the mRNA and
  synthesize the
specified protein.
                The Genetic Code
   How does the mRNA nucleotide sequence
    dictate the amino acid sequence of proteins
    during translation?
       4 nucleotides specify 20 amino acids
       Triplets of nucleotides could code for single amino
        acids
            43 = 64 possible code words (amino acids)
   Triplet codon = genetic instructions for a
    polypeptide chain are written in the DNA as a
    series of three-nucleotide words
       example: AGT will end up coding for Serine
   DNA is double stranded – which strand is
    transcribed and translated?
       Template strand
The Triplet Code
           The transcript is
           complementary to the coding
           sequence of the TEMPLATE (or
           SENSE) STRAND.
           The CODING (OR ANTI-
           SENSE) STRAND is not used but
           contains specific sequences that
           help regulate transcription (i.e.
           promoter sequences)



           RNA uses uracil
           (U) in place of
           thymine (T)
The Dictionary of the Genetic Code
                           Nirenberg experiment - The
                           genetic code was solved
                           when a synthetic
                           oligonucleotide of uracil
                           produced a chain of
                           phenylalanine amino
                           acids. (UUU) – Led to
                           discoveries of AAA, GGG,
                           CCC
                           The genetic code is
                           redundant, 64 codons
                           code for 20 amino acids
                       •   3 of the codons do not
                           code for any amino acid
                           (therefore 61 codons)
                       •   What if the amino acid to
                           codon ratio was one-to-
                           one?
        Features of the Genetic Code:
                  Continuity
   DNA encodes a sequence of non-
    overlapping base triplets – codons
       AUGACGAAGAGGGGAUAA
          Read: AUG ACG AAG AGG GGA UAA
          Not read: AUGACGAAG = AUG UGA GAC ACG



   Reading Frame: codons must be read in
    the correct groupings
       AUGACGAAGAGGGGAUAA
          Read: AUG ACG AAG AGG GGA UAA
          Not read: UGA CGA AGA GGG GAU
        Genetic Code is Universal
   This genetic code is shared by
    organisms from the simplest
    bacteria to the most complex
    animals and plants
       This allows the transplanting of
        DNA from one species to another
            Human genes can be inserted into
             bacteria to produce proteins for
             medical uses
   The universality suggests that
    the genetic code must have                  A tobacco plant expressing
    evolved early in the history of             the luminescent genes
                                                transplanted from a firefly
    life                                        genome.
The Stages of Transcription: Initiation, Elongation, and
                    Termination




            Transcription is INITIATED at special
            sequences called PROMOTERS where
             an enzyme, RNA polymerase binds.


               Prokaryotic cells have one RNA
                        polymerase.
            Eukaryotic cells have 3 (I, II, and III)
               RNA pol II - synthesizes mRNA
The Stages of Transcription: Initiation, Elongation, and
                    Termination




              The two strands of DNA are
             unwound and separated. RNA
             polymerase initiates synthesis
                at the start codon on the
               template (coding) strand.
The stages of transcription: initiation, elongation, and termination



  upstream
                                                                downstream




  DNA reforms a        The transcript is ELONGATED as the RNA
double helix in the   polymerase moves downstream, unwinding
      wake of             the DNA and adding RNA molecules
   transcription            complementary to the template.
The Stages of Transcription: Initiation, Elongation, and
                    Termination

                                         Transcription is
                                     TERMINATED when
                                      the RNA polymerase
                                    translates a terminator
                                           sequence.


                                     The RNA transcript is
                                       released and the
                                     polymerase detaches
                                         from the DNA
             Transcription “Initiation”
   Promoters
       Transcriptional start site
        (AUG)
       Binding sequence for RNA
        pol (TATA box)
            Located about 25
             nucleotides from the start
             site
       Designates the template
        strand
   Initiation complex
       Eukaryotic transcription
        factors mediate the binding
        of RNA pol to the promoter
       These factors unwind and
        separate the DNA and RNA
        synthesis begins
        Transcription Elongation

   Elongation occurs in the 5’  3’ direction
   As RNA polymerase moves downstream,
    DNA unwinds about 10-20 bases at a time
   Transcription rate = 60 nucleotides/sec
   One mRNA can be transcribed
    simultaneously by several RNA polymerases,
    increasing the amount of mRNA produced
    Transcription Termination
   Transcription continues until a terminator
    sequence (or signal) on the non-transcribed
    strand is reached.
   Prokaryotes terminate immediately following this
    signal
   Eukaryotic transcription proceeds hundreds of
    nucleotides past this signal (most common site
    = AATAAA)
   About 10-35 nucleotides past the signal, the
    mRNA is cleaved
RNA Processing: Bustin’ a 5 cap and Pinnin’ a poly(A) tail

   5’ cap – modified guanine (G)
      Protects mRNA from degradation

      Binding site for ribosomes in the
        cytoplasm
   3’ poly A tail – 50-250 (upwards to 1000)
    adenines (A)
      Protects mRNA from degradation

      Binding site for ribosomes

      Aids in transport of mRNA to cytoplasm
    RNA Splicing – Post Transcriptional
              Modifications
   Splicing
       RNA transcripts have long stretches of non-coding regions
        (introns) interspersed between coding regions (exons)
            Introns are cut out and exons are joined together
       Spliceosome
            Composed of snRNPs and other proteins
            Small nuclear ribonucleoproteins (snRNPs) bind to splice sites (short
             sequences at the end of introns)
The Roles of snRNPs and Spliceosomes in mRNA splicing
Transcription   Translation
               Translation Overview
   Interpreting a genetic message: series of
    codons along an mRNA
       Interpreter = transfer RNA (tRNA)
            Transfers amino acids from cytoplasm to ribosome
       Machinery = ribosome
            Adds amino acid from tRNA to the growing
             polypeptide chain
   As mRNA moves through a ribosome,
    codons are translated into amino acids
                 Translation: The Basic Concept

   Each type of tRNA links
    a particular mRNA
    codon with a particular
    amino acid
       One end binds a specific
        amino acid
       Other end is the
        anticodon, nucleotide
        triplet that is
        complementary and binds
        to the codon on the
        mRNA transcript
            AAG binds UUC in the
             mRNA and adds Phe
         The Structure of Transfer RNA (tRNA)


   The secondary
    structure of tRNA
    looks like a cloverleaf
       There are three stem-
        loop structures
       The anticodon is
        specific for each tRNA
            AAG binds UUC in the
             mRNA and adds Phe
         The Structure of Transfer RNA (tRNA)



  The tRNA
folds into an
 L shape 3D
  structure


 Anticodons
 are written
   3’  5’ to
align with the
mRNA codon.
                    Wobble Theory
   If one tRNA existed for each codon that
    specifies an amino acid, there would be 61
    tRNAs BUT there are only 45.
   The base pairing rules are relaxed for the
    3rd amino acid in a codon/anti-codon
       U can bind with either A or G in mRNA
       The redundancy of the genetic code permits
        this wobble
            UUA and UUG both code for leucine, so a U in the
             anticodon will be able to bind either codon and
             translate it to leucine
An aminoacyl-tRNA synthetase joins a specific amino
                 acid to a tRNA
There are 20 different enzymes that
    catalyze the addition of the 20
 different amino acids to its specific
                 tRNA.


   These enzymes are termed
 aminoacyl-tRNA synthetases.


  The addition of the amino acid
  requires energy (ATP) and this
  process activates the tRNA for
 delivery of the amino acid to the
 ribosome for attachment to the
      growing peptide chain.
              The Anatomy of a Functioning Ribosome

   Ribosomes – complex of
    proteins and ribosomal RNA
    (rRNA)
       Large subunit (60S)
            Binding sites for tRNAs
                  E (exit site)
                  P (peptidyl-tRNA site)
                  A (aminoacyl-tRNA site)
       Small subunit (40S)
            Binding site for mRNA
   Ribosome holds the mRNA
    and the tRNA in close
    proximity
       Addition of the new aa to the
        carboxyl end of the chain
       Catalyze formation of peptide
        bond
                      The Initiation of Translation

   Small subunit binds to the mRNA through a special binding
    site that recognizes a specific sequence
       An initiator tRNA binds which carries Met
       The large subunit binds, placing the tRNA in the P site
   Energy (GTP) and initiation factors are required for this
    assembly
The Elongation Cycle of Translation

                            Incoming tRNA
                            binds the codon
                              in the A site




                              The ribosome
                               catalyzes the
                              formation of a
                               peptide bond
          The Elongation Cycle of Translation

                                        Incoming tRNA
                                        binds the codon
                                          in the A site




                        The tRNA in
                        the P site is
                        translocated
                        to the E site
                         where it is
                          released




 The tRNA in the A                       The ribosome
 site is translocated                     catalyzes the
to the P site, taking                    formation of a
  the mRNA along                          peptide bond
          The Termination of Translation
   Elongation continues until a stop codon in the
    mRNA reaches the A site of the ribosome
       UAA, UAG, UGA
       A release factor protein binds directly to the stop
        codon in the A site and causes the addition of water,
        which hydrolyzes and releases the polypeptide chain
       The assembly dissociates
                      Polyribosomes
   An mRNA molecule is translated by several
    ribosomes simultaneously
       The strings of ribosomes are called polyribosomes
       Many copies of a protein can be synthesized quickly
            A single ribosome can make a protein in less than a minute
                 Targeting Proteins
       Once proteins are formed, they must reach
        their ultimate destination
         Cytosolic proteins
             synthesized on FREE ribosomes in the cytoplasm
         ER, Golgi, lysosomal, plasma membrane and
          secreted proteins
             synthesized on ribosomes BOUND to the cytosolic side of
              the ER
       Protein synthesis starts in the cytosol and
        continues there unless the protein itself directs
        the ribosome to the ER
         Proteins contain a signal peptide
             20 aa near the leading end of the polypeptide
         Recognized by a signal-recognition particle
          (SRP)
             Brings the ribosome to a receptor in the ER membrane
The Signal Mechanism for Targeting Proteins to the ER
    SRP binding causes synthesis to stop
    Once the ribosome is set up at the ER membrane, the SRP
     leaves and synthesis continues
    The growing chain is translocated through a pore in the
     ER membrane
    An enzyme cleaves off the signal sequence
              The Types and Roles of RNA Molecules

                                                              RNA



                                                             can be



         Messenger RNA                                  Ribosomal RNA                               Transfer RNA




also called      which functions to            also called       which functions to   also called          which functions to


                                                                                                                 Bring
                                                                        Combine
 mRNA                Carry instructions          rRNA                                    tRNA               amino acids to
                                                                      with proteins
                                                                                                              ribosome



              from                        to                          to make up



              DNA                   Ribosome                          Ribosomes
                Prokaryotes vs. Eukaryotes

   Transcription                  Coupled transcription and
                                    translation in bacteria
       Different RNA polymerase
       Eukaryotes depend on
        transcriptions factors
       Termination different
       Eukaryotes have RNA
        processing steps
   Translation
       Different ribosomes
       Eukaryotes target
        proteins to appropriate
        compartments
   Compartmentalization
A Summary of Transcription and Translation
          in a Eukaryotic Cell

								
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