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PROTEIN SYNTHESIS (PowerPoint)

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					PROTEIN SYNTHESIS
  PROTEIN SYNTHESIS
 DNA: NUCLEIC        RNA: NUCLEIC
  ACID, DOUBLE         ACID, SINGLE
  STRAND, PO4, DE-     STRAND, PO4,
  OXYRIBOSE SUGAR.     RIBOSE SUGAR.

 BASE PAIRS (N)      BASE PAIRS (N)
 T=THYMINE           U = URACIL
 A=ADENINE           A=ADENINE
 C= CYTOSINE         C=CYTOSINE
 G=GUANINE           G=GUANINE
URACIL (U)
base with a single-ring structure




           phosphate
             group




                                    sugar (ribose)
      POINTS ABOUT
     TRANSCRIPTION
NEED RNA POLYMERASE
CODES FOR 20 AMINO ACIDS
CODON:SERIES OF TRIPLET BASE
 PAIRS.
64 CODONS, 60 FOR AA, OTHERS
 FOR STARTS/STOPS.
INTRONS=NON-CODING
EXONS= CODING FOR RNA
PROTEIN TRANSCRIPTION
 NUCLEUS
 RNA POLYMERASE CODES TO DNA
 DNA TRANSCRIBES TO m-RNA
 INTRONS SNIPPED OUT
 EXONS KEPT IN CODE
                        unit of transcription in a DNA strand
                exon      intron         exon         intron     exon
3’                                                                                     5’




                            transcription into pre-mRNA

                                                                         poly-A
          cap                                                             tail
     5’                                                                           3’

                       (snipped out)             (snipped out)




                5’                                                  3’
                              mature mRNA transcript
                                           transcribed DNA                                     DNA to be
                                           winds up again                                      transcribed
sugar-phosphate backbone of one strand
                                                                                               unwinds
of nucleotides in a DNA double helix




                                           Newly forming                                       The DNA
                                           RNA transcript                                      template at the
                                                                                               assembly site



sugar-phosphate       part of the
backbone of           sequence of                       growing RNA transcript
the other strand      base pairs in DNA                                                   3’
                                                   5’
of nucleotides

                                                   3’                                                 5’
                                                             direction of transcription




                                                   5’                                                 3’

                          RNA polymerase
PROTEIN TRANSLATION
 m-RNA GOES THRU     20 TYPES OF AA
  RIBOSOME.           ANTICODON ON
 RIBOSOME IS r-       ONE END OF t-
  RNA,CODE THREADS     RNA.
  THRU RIBOSOME.      AA ON OTHER END
 AREA OF RIBOSOME     OF t-RNA
  BOUND TO tRNA       AA ATTACH TO
                       EACH OTHER IN
                       PEPTIDE BOND
                      FORM PROTEINS
   Binding site for mRNA




   P
 (first
binding
site for       A
tRNA)      (second
            binding
            site for
            tRNA)
  TRANSCRIPTION         Unwinding of gene regions of a DNA molecule




        Pre mRNA
        Transcript              mRNA                rRNA                tRNA
        Processing

                                    protein
                                    subunits
                      Mature mRNA              ribosomal           mature
                      transcripts              subunits            tRNA




                               Convergence
   TRANSLATION                 of RNAs

                                                                       Cytoplasmic
                                                                       pools of
                                                                       amino acids,
                                                                       tRNAs, and
Synthesis of a                                                         ribosomal
polypetide chain at                                                    subunits
binding sites for
mRNA and tRNA
on the surface of
an intact ribosome



                                                           FINAL PROTEIN
                                                           Destined for use in
                                                           cell or for transport
VALINE                         PROLINE   THREONINE

                     LEUCINE
         HISTIDINE                                   GLUTAMATE GLUTAMATE




VALINE                         PROLINE   THREONINE     VALINE

                     LEUCINE
         HISTIDINE                                              GLUTAMATE
                                                          mRNA transcribed from the DNA



                                                           PART OF PARENTAL DNA TEMPLATE


ARGININE   GLYCINE   TYROSINE   TRYPTOPHAN   ASPARAGINE   resulting amino acid sequence




ARGININE   GLYCINE   LEUCINE     LEUCINE     GLUTAMATE

                                                              altered message in mRNA

                                                              A BASE INSERTION (RED) IN
                                                              DNA

                                                              the altered amino acid
                                                              sequence
Overview: the roles of transcription and translation in the flow of
                       genetic information
The triplet code
  TRANSCRIPTION AND
     TRANSLATION
 C DNA.                ATC-GCG-TAT
 m-RNA.                UAG-CGC-AUA
 t-RNA.                AUC-GCG-UAU
 AMINO ACID            ISO-ALA-TYR

 PEPTIDE BONDS/POLYPEPTIDES/PROTEINS
             Translation

                                        Nuclear
                                DNA     membrane



               Transcription
                                 Pre-mRNA

Eukaryotic     RNA Processing

Cell                             mRNA

                                      Ribosome

               Translation

                                  Protein
            Translation
 Synthesis of proteins in the cytoplasm

 Involves the following:
  1. mRNA (codons)
  2. tRNA (anticodons)
  3. rRNA
  4. ribosomes
  5. amino acids
         Types of RNA
 Three types of RNA:
 A. messenger RNA (mRNA)
 B. transfer RNA (tRNA)
 C. ribosome RNA (rRNA)

 Remember: all produced in the nucleus!
A. Messenger RNA (mRNA)
 Carries the information for a specific protein.
 Made up of 500 to 1000 nucleotides long.
 Made up of codons (sequence of three bases:
                        AUG - methionine).
 Each codon, is specific for an amino acid.
       A. Messenger RNA (mRNA)
          start
          codon

mRNA    A U G G G C U C C A U C G G C G C A U A A

        codon 1      codon 2    codon 3   codon 4      codon 5    codon 6     codon 7

protein methionine    glycine    serine   isoleucine    glycine    alanine     stop
                                                                               codon



                  Primary structure of a protein
       aa1           aa2         aa3          aa4           aa5              aa6

                           peptide bonds
  B. Transfer RNA (tRNA)
 Made up of 75 to 80 nucleotides long.
 Picks up the appropriate amino acid floating in
  the cytoplasm (amino acid activating enzyme)
 Transports amino acids to the mRNA.
 Have anticodons that are complementary to
  mRNA codons.
 Recognizes the appropriate codons on the
  mRNA and bonds to them with H-bonds.
anticodon




                                                  codon in mRNA
                                                   anticodon




                                        amino acid
                                        attachment site
                                amino
                tRNA MOLECULE    acid                             OH



amino acid attachment site
The structure of transfer RNA (tRNA)
B. Transfer RNA (tRNA)
amino acid
attachment site        methionine   amino acid




                    U A   C
                  anticodon
        C. Ribosomal RNA
              (rRNA)
 Made up of rRNA is 100 to 3000 nucleotides long.

 Important structural component of a ribosome.
 Associates with proteins to form ribosomes.
              Ribosomes
 Large and small subunits.
 Composed of rRNA (40%) and proteins
  (60%).
 Both units come together and help bind the
  mRNA and tRNA.
 Two sites for tRNA
  a. P site (first and last tRNA will attach)
  b. A site
                                    Ribosomes
Origin         Complete Ribosomal   rRNA         Proteins
               ribosome subunit     components
Cytosol        80 S     40 S        18 S         C.30
(eukaryotic             60 S         5S          C.50
ribosome)                           5.8 S
                                    25 S
Chloroplasts   70 S     30 S        16 S         C. 24
(prokaryotic            50 S        4.5 S        C. 35
ribosome)                            5 S
                                    23 S
Mitochondrion 78 S       30 S      18 S         C. 33
(prokaryotic             50 S       5S          C. 35
ribosome)                           26 S
                 Ribosomes

Large
subunit
                  P       A
                 Site    Site


                                    mRNA
                A U G   C U A C U U C G

Small subunit
            Translation
 Three parts:
  1. initiation: start codon (AUG)
  2. elongation:
  3. termination: stop codon (UAG)

 Let’s make a PROTEIN!!!!.
                Translation

Large
subunit
                  P       A
                 Site    Site


                                    mRNA
                A U G   C U A C U U C G

Small subunit
                        Translation
                • Initiation
The inactive 40S and 60S subunits will bind to
 each other with high affinity to form inactive
 complex unless kept apart
This is achieved by eIF3, which bind to the
 40S subunit
mRNA forms an initiation complex with a
 ribosome
A number of initiation factors participate in
 the process.
                                           33
                          Translation
 Cap sequence present at the 5’ end of the
  mRNA is recognized by eIF4
 Subsequently eIF3 is bound and cause the
  binding of small 40S subunit in the complexes
 The 18S RNA present in the 40 S subunit is
  involved in binding the cap sequence
 eIF2 binds GTP and initiation tRNA, which
  recognize the the start codon AUG
 This complex is also bound to 40S subunit
                                             34
                          Translation
 Driven by hydrolysis of ATP, 40S complex
  migrate down stream until it finds AUG start
  codon
 The large 60S subunit is then bound to the 40S
  subunit
 It is accompanied by the dissociation of several
  initiation factor and GDP
 The formation of the initiation complex is now
  completed
 Ribosome complex is able to translate

                                              35
                               Translation
 Extrachromosomal mRNAs have no cap site
 Plastid mRNA has a special ribosome binding site for
  the initial binding to the small subunit of the ribosome
  (shine-Dalgarno sequence)
 This sequence is also found in bacterial mRNA, but it is
  not known in the mitochondria
 In the prokaryotic, the initiation tRNA is loaded with
  N-formylmethionine
 After peptide formation, the formyl residue is cleaved
  from the methionine

                                                      36
               Initiation
                                  aa2
               aa1




                                2-tRNA
            1-tRNA
                                G A U
anticodon   U A C
 hydrogen   A U G    C U A C U U C G A
 bonds       codon              mRNA
                         Translation
               • Elongation
A ribosome contains two sites where the
 tRNAs can bind to the mRNA.
 P (peptidyl) site allows the binding of the
 initiation tRNA to the AUG start codon.
The A (aminoacyl) site covers the second
 codon of the gene and the first is
 unoccupied
On the other side of the P site is the exit
 (E) site where empty tRNA is released

                                                38
                           Translation
                 • Elongation
 The elongation begins after the corresponding
  aminoacyl-tRNA occupies the A site by forming
  base pairs with the second codon
 Two elongation factors (eEF) play an important
  role
 eEF1 binds GTP and guides the corresponding
  aminoacyl-tRNA to the A site, during which GTP is
  hydrolized to GDP and P.
 The cleavage of the energy-rich anhydride bond in
  GTP enables the aminoacyl-tRNA to bind to codon
  at the A site

                                                  39
                             Translation
                    • Elongation
 Afterwards the GDP still bound to eEF1, is exchange
  for GTP as mediated by the eEF1
 The eEF1 -GTP is now ready for the next cycle
 Subsequently a peptide linkage is form between the
  carboxyl group of methionine and the amino group of
  amino acid of the tRNA bound to A site
 Peptidyl transferase catalyzing the reaction. It
  facilitates the N-nucleophilic attack on the carboxyl
  group, whereby the peptide bond is formed with the
  released of water
                                                    40
                         Translation
                • Elongation
Accompanied by the hydrolysis of one molecule
 GTP to form GDP and P, the eEF2 facilitates
 the translocation of the ribosome along the
 mRNA to three bases downstream
Free tRNA arrives at site E is released, and
 tRNA loaded with the peptide now occupies the
 P Site
The third aminoacyl-tRNA binds to the vacant
 A site and a further elongation cycle can begin
                                            41
  Elongation
                     peptide bond
                                       aa3
               aa1        aa2




                                    3-tRNA

            1-tRNA     2-tRNA       G A A
anticodon   U A C     G A U
 hydrogen   A U G     C U A C U U C G A
 bonds       codon                  mRNA
                     aa1        peptide bond
                                               aa3
                              aa2


1-tRNA

U A C                                      3-tRNA
(leaves)
                           2-tRNA          G A A

                        G A U
               A U G    C U A C U U C G A
                                           mRNA

           Ribosomes move over one codon
             peptide bonds
    aa1                         aa4

             aa2      aa3



                             4-tRNA

          2-tRNA   3-tRNA    G C U

        G A U G A A
A U G   C U A C U U C G A A C U
                             mRNA
                     peptide bonds
             aa1                              aa4
                    aa2

                             aa3



2-tRNA
                                           4-tRNA
G A U
(leaves)                  3-tRNA           G C U

                     G A A
   A U G       C U A C U U C G A A C U
                                           mRNA

           Ribosomes move over one codon
        peptide bonds           aa5
aa1
      aa2
                        aa4
               aa3


                              5-tRNA

                              U G A
            3-tRNA   4-tRNA

        G A A G C U
G C U A C U U C G A A C U
                              mRNA
    aa1           peptide bonds            aa5
            aa2
                   aa3
                           aa4



                                         5-tRNA

3-tRNA                                   U G A
G A A                    4-tRNA

              G C U
G C U A C U U C G A A C U
                                         mRNA

         Ribosomes move over one codon
            aa5
      aa4            aa199           Termination
 aa3 primary                 aa200
     structure
aa2 of a protein


aa1
                                     terminator
                        200-tRNA
                                      or stop
                                      codon
A C U             C A U G U U U A G
 mRNA
                          Translation
                 • Release
 When A site finally binds to a stop codon (UGA,
  UAG, UAA)
 Stop codons bind eRF accompanied by
  hydrolysis GTP to form GDP and P
 Binding of eRF to the stop codon alters the
  specificity the peptidyl transferase
 Water instead amino acid is now the acceptor
  for the peptide chain
 Protein released from the tRNA
                              Translation
                • The difference
• Eukaryotic and prokaryotic translation can react
  differently to certain antibiotics
Puromycin
  an analog tRNA and a general inhibitor of protein
  synthesis
 Cycloheximide
  only inhibits protein synthesis by eukaryotic ribosomes
 Chloramphenicol, Tetracycline, Streptomycin
  inhibit protein synthesis by prokaryotic ribosome
                End Product
 The end products of protein synthesis is a
  primary structure of a protein.

 A sequence of amino acid bonded together by
  peptide bonds.
                            aa5
          aa3       aa4
aa2                                   aa199

aa1                                            aa200
                    Polyribosome
• Groups of ribosomes reading same mRNA simultaneously
  producing many proteins (polypeptides).



incoming
large
subunit

                1    2    3     4      5   6   7
                                                   mRNA

incoming
small subunit            polypeptide
   TYPES OF PROTEINS
 ENZYMES/HELICASE
 CARRIER/HEMOGLOBIN
 IMMUNOGLOBULIN/ANTIBODIES
 HORMONES/STEROIDS
 STRUCTURAL/MUSCLE
 IONIC/K+,Na+
 all regulate things put together ”critter”
                       Protein Sorting
 Vast majority of protein within the cell are synthesized
  within the cytoplasm, but the final sub-cellular location
  can be in one of a whole array of membrane-bound
  compartment
 Protein is subjected to be sorted for special targeted
  organelles
                       Protein Sorting
 Vast majority of protein within the cell are synthesized
  within the cytoplasm, but the final sub-cellular location
  can be in one of a whole array of membrane-bound
  compartment
 Protein is subjected to be sorted for special targeted
  organelles:
 Plastids
 Mitochondria
 Peroxisomes
 Vacuoles
                                Mitochondria
 More than 95% of mitochondrial proteins in plant are encoded in the
  nucleus and translated in the cytosol
 Proteins are generally equipped with targeting signals ( a signal
  sequence of 12-70 amino acids at the amino terminal)
 Protein import occurs at translocation site
 In most cases, protein destined for the mitochondrial inner
  membrane after transport through outer membrane are guided
  directly to the location by internal targeting sequence
 Protein destined for the inner mitochondrial membrane contain pro-
  sequence that guides first into the mitochondrial matrix. After
  removal of the pro-sequence by processing peptidase, the proteins
  are directed by second targeting signal sequence into the inner
  membrane
  Plastids
 ATP is consumed for the phosphorilation of a protein,
  probably the receptor OEP86
 The protein transport is regulated by the binding of the
  GTP to OEP86 and OEP34
 After the protein is delivered, the pre-sequence is
  removed by a processing peptidase
 The protein destined to thylakoid membrane are first
  delivered into stroma and then directed by internal
  targeting signal into thylakoid membrane
  Peroxisomes
 Small membrane-bound cytoplasmic organelle
  containing oxidizing enzymes
 They can be found in leaf cells where they contain
  some of the enzymes of glycolytic pathway
 All protein have to be delivered from the cytosol
 The transport is accompanied by ATP hydrolysis
 Targeting sequence SKL (serine-lysine-leucine) has
  been observed in C terminus, but this sequence is not
  removed after uptake
  Vacuole
 Proteins are transferred during their synthesis to the lumen of ER
 This is aided by a signal sequence at the terminus of the
  synthesized protein, which binds with a signal recognition particle
  to a pore protein present in the ER membrane and thus directs the
  protein to the ER lumen
 In such cases, ribosome is attached to the ER membrane during
  protein synthesis and the synthesized protein appears immediately
  in the ER lumen. It is called co-translational protein transport
 This protein is then transferred from the ER by vesicles transfer
  across the golgi apparatus to the vacuole or are exported by
  secretory vesicles from the cell
Coupled transcription and translation in bacteria
         original                                                            a base
         base triplet                                                        substitution
         in a DNA                                                            within the
         strand                                                              triplet (red)
                         As DNA is replicated, proofreading
                         enzymes detect the mistake and
                         make a substitution for it:

                                           POSSIBLE OUTCOMES:


                                                      OR

                         One DNA molecule                                  The other DNA
                         carries the original,                             molecule carries
                         unmutated sequence                                a gene mutation




VALINE                                 PROLINE     THREONINE   VALINE

                           LEUCINE
             HISTIDINE                                                  GLUTAMATE
A summary of transcription and translation in a eukaryotic cell

				
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