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GENETIC CODE and PROTEIN SYNTHESIS.ppt

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GENETIC CODE and  PROTEIN SYNTHESIS.ppt Powered By Docstoc
					                          Dental Biochemistry 2013
                                   Lectures 33 & 34




 GENETIC CODE and
PROTEIN SYNTHESIS
    Mukund Modak, Ph.D.




                          Adapted from M. Mathews, Ph.D. 1
                                    .
Proteins are important…
 ~44% of the dry wt. of the human body.
 ~5% of human caloric intake goes for protein synthesis.
 catalyze most of the reactions in living organisms.
 serve many roles (enzymatic, structural, transport, regulation, ...)



                          …in sickness and in health
 protein synthesis is tightly regulated by environmental stimuli
 as well as intrinsic processes (e.g., hormonal, developmental).
 dysregulation can cause disease.
 many antibiotics act at the level of protein synthesis.


                                                                        2   2
I.   INTRODUCTION

     Central Dogma
     Ribosomes and polysomes
     Genetic Code
     Mutations with effects at the translation level

II. TRANSLATIONAL MACHINERY

III. MECHANISM OF TRANSLATION AND INHIBITORS
     OF PROTEIN SYNTHESIS

IV. ENERGETICS AND REGULATION OF TRANSLATION



                                                       3   3
POLYSOMES




  E.M.




            4
                CENTRAL DOGMA
       DNA                RNA                 PROTEIN
The central dogma states that once “information” has passed
into protein it cannot get out again. The transfer of information
from nucleic acid to nucleic acid, or from nucleic acid to protein,
may be possible, but transfer from protein to protein, or from
protein to nucleic acid is impossible. Information means here the
precise determination of sequence, either of bases in the nucleic
acid or of amino acid residues in the protein.
                                       Francis Crick, 1958




                     ’
                 5                    RNA
                                                        3’

    N- or amino-                    protein            C- or carboxy-   5
     terminus                                             terminus
Coupled transcription & translation in bacteria
          [ 5’ to 3’ ] [ N terminus to
                         C terminus ]

           Not so in Eukaryotes
                                                  6
          1st position                 2nd position          3rd position
           (5’ end)                                            (3’ end)
                          U       C           A       G
                          Phe     Ser        Tyr       Cys       U
GENETIC
            U             Phe     Ser        Tyr       Cys       C
 CODE                     Leu
                          Leu
                                  Ser
                                  Ser
                                            STOP
                                            STOP
                                                      STOP
                                                       Trp
                                                                 A
                                                                 G
                          Leu     Pro         His     Arg        U

            C             Leu
                          Leu
                          Leu
                                  Pro
                                  Pro
                                  Pro
                                              HIs
                                              Gln
                                              Gln
                                                      Arg
                                                      Arg
                                                      Arg
                                                                 C
                                                                 A
                                                                 G
                          Ile     Thr         Asn     Ser        U

            A             Ile
                          Ile
                          Met
                                  Thr
                                  Thr
                                  Thr
                                              Asn
                                              Lys
                                              Lys
                                                      Ser
                                                      Arg
                                                      Arg
                                                                 C
                                                                 A
                                                                 G
                           Val    Ala         Asp      Gly       U


            G              Val
                           Val
                           Val
                                  Ala
                                  Ala
                                  Ala
                                              Asp
                                              Glu
                                              Glu
                                                       Gly
                                                       Gly
                                                       Gly
                                                                 C
                                                                 A
                                                                 G

           codon # 5   6   7
                  CCU GAG GAG                             Normal
                         Pro     Glu       Glu            Hb – β

                         CCU GUG GAG                     Sickle cell
                                                                            7
                         Pro     Val       Glu           Hb – βS
GENETIC CODE:

       Co-linear triplet code

       Nearly universal – variations in mitochondria, mycoplasma, ciliates

       Degenerate (or redundant)
       Non-overlapping
       Unpunctuated – although some codons are signals
       Mutations - in coding region can cause various ill-effects, such
       as, change in desired amino acids, early or late stop, insertion,
       etc.




                                                                             8
I.   INTRODUCTION

II. TRANSLATIONAL MACHINERY
        Ribosomes: prokaryotic / eukaryotic
    Messenger RNA
    Transfer RNA
    Aminoacyl-tRNA synthetases; Met-tRNA forms (m, f, i)
    Initiation, elongation and termination enzymes

III. MECHANISM OF TRANSLATION AND INHIBITORS OF
     PROTEIN SYNTHESIS

IV. ENERGETICS AND REGULATION OF TRANSLATION

                                                      9    9
      TRANSLATIONAL COMPONENTS

1. Ribosomes (large and small subunits)

2. Messenger RNA (mRNA)

3. Transfer RNAs (tRNAs)

4. Amino Acids (aa’s)

5. Enzymes (“factors”)

6. Energy (ATP, GTP)


                                          10
1. Ribosome Structure




                        11
                                 Section through 50S ribosomal subunit




 Peptidyl transferase is RNA

 Polypeptide exit tunnel is
      40~50 aa long


C: Central protuberance
PT: Peptidyl tranferase center
Red, yellow, etc.: rRNA
Blue: Ribosomal proteins                                             12   12
White: Nascent polypeptide
                             2. mRNA
 Eukaryotic: Monocistronic (spliced)                                     3’ end
 5’ end                      ( 1 coding region )
               5’ UTR                               3’ UTR   poly A

    cap                           only 1                     AAA
                                                               ~150
7-MeGpppGXY
                                        Cistron = coding region =
                                        open reading frame (ORF)



  Prokaryotic: Polycistronic
                             ( >1 coding region )
          5’                                                        3’
   ppp                  #1             #2             #3

                                                                             13
     3. tRNA
Translational Adaptor




                        14
                       4. Amino Acids
       tRNAs carry “activated” amino acids:
                         aaRS
 (1) AA + tRNA + ATP             AA ~tRNA + AMP + PPi
                                              DG°¢ ~0 Kcal/mole
                         PPase
 (2)       PPi + H2O               2 Pi
                                              DG°¢   =   -6.6 Kcal/mole


Overall free energy change for aminoacylation of tRNA
                                           DG°¢ ~ -6.6 Kcal/mole




aaRS = aminoacyl-tRNA synthetase
PPase = pyrophosphatase
                                                                          15
Formation of
aminoacyl-tRNA

     The amino acid is first
     activated by
     reacting with ATP




     The activated amino
     acid is transferred
     from aminoacyl-AMP
     to tRNA

 These enzymes are vital for the fidelity of
 protein synthesis: 2 steps allow “proofreading”
                                                   16
   Genetic Code                   Translation Machinery
   20 AA’s                        20 AA – tRNA synthases
                                             ( i.e., 1 per AA )
   61     Codons for AA’s         ~50        tRNA species
                                             (at least 1 per AA,
                                             but
                                             less than 1 per codon)


“WOBBLE” Pairing
                                                  Wobble Position
         anti-codon stem-loop
         of tRNA                                  e.g. CUU 1 anti–codon
                                                       GAA
                                tRNA                   GAG        2 codons
                            3’         5’
                                            ANTI-CODON
                             3 2 1

    5’                       1 2 3
                                                                         17
 mRNA                       CODON
                    2 tRNAs for AUG / Methionine:
                         2 different functions
  N-formyl            Met                        Met
in bacteria:
   F-Met
                     CCA                         CCA
Met – tRNA F or I               Met – tRNA M




                3’         5’              3’         5’
                     UAC                        UAC
        5’                                                 3’
                     AUG                        AUG
                      1

                Initiation                 Internal
                 Codon                    Met Codon

                                                                18
                 5. Translation Factors
Translation Step                      Enzymes
                              Prokaryotes   Eukaryotes

    Charging of tRNA           Aminoacyl – tRNA synthetases


1. Initiation                  IF1- IF3     eIF1- eIF5 (multiple)


2. Elongation                  EF1, EF2     eEF1, eEF2


3. Termination                 RF1- RF3     eRF1, eRF3


   Modifications, cleavage, etc.
                                                                    19
I.   INTRODUCTION

II. TRANSLATIONAL MACHINERY

III. MECHANISM OF TRANSLATION AND INHIBITORS OF
     PROTEIN SYNTHESIS
     Initiation
     Elongation
     Termination
     Antibiotics
     Toxins

IV. ENERGETICS AND REGULATION OF TRANSLATION

                                          20   20
HOW RIBOSOMES FIND THEIR INITIATION SITES
               eukaryotes                                      prokaryotes
 1. Cap - dependent scanning

  40S                                                                         30S
                                                                   16S rRNA


 cap                                            AUG...                 S-D           AUG..


                                                              Shine - Dalgarno box
 2. Internal ribosome entry
                                 40S


                                        AUG..

                                                         Next step: large subunit
                                                         50S/60S subunit joining
       ---------------IRES-----------                                                        21
30S ribosomal subunit initiation at S-D sequence




                                                   22
HOW RIBOSOMES FIND THEIR INITIATION SITES
               eukaryotes                                      prokaryotes
 1. Cap - dependent scanning                             STREPTOMYCIN

  40S                                                                          30S
                                                                    16S rRNA


 cap                                             AUG..                  S-D           AUG..


                                                               Shine - Dalgarno box
 2. Internal ribosome entry
                                                              Streptomycin, Gentamycin,
                                 40S                          Tobramycin, Amikacin, etc.
                                                                 are aminoglycosides.
                                                                   They also cause
                                        AUG...
                                                              miscoding during elongation


       ---------------IRES-----------                                                         23
ELONGATION                     A Site



                P Site
                                    TETRACYCLINES
                E Site
                                    SPECTINOMYCIN
   AA – tRNA
                                 EF 1A, 1B (EF-Tu, Ts)
    binding
                                   [eEF 1α, eEF1βγ ]



                               PUROMYCIN
                               CHLORAMPHENICOL
     Peptidyl                    Peptidyl
     Transfer                  transferase
  CLINDAMYCIN                   (50S / 60S)
  Macrolides e.g.
 ERYTHROMYCIN
                         GTP         EF2
    Translocation                  [eEF2]

                               DIPHTHERIA
         RICIN                    TOXIN                  24
       a-SARCIN
          Puromycin imitates AA-tRNA


   Puromycin




Tyrosinyl-tRNA



                                       25
Inhibition of ribosome translocation


  1) Diphtheria toxin inactivates eEF2
  2) Erythromycin inhibits EF2




                                         26   26
TERMINATION       stop codons
                     UAG
                     UAA
                     UGA


    Termination
                  RF 1,2,3
         &
                  [eRF1,3]
      Release




                                27
      ENERGETICS OF PROTEIN SYNTHESIS

1.   Charging                     ATP, 2~

2.   Initiation
     Unwinding and                ATP (several), 1~
     scanning                     GTP, 1~
     Met-tRNAi binding

3.   Elongation                   GTP, 1~ (see later)
     AA-tRNA binding              GTP, 1~
     Translocation
                                  GTP (number unknown), 1~
4.   Termination



 TOTAL: 4~ per AA polymerized + initiation + termination

          > 1200~ for an average protein

 Compared to 36-38 ATP’s generated by Glucose              CO2   28
Down-regulation of the supply of initiator Met-tRNAi via eIF2



                            eIF2 • GDP

                                  eIF2B

                            eIF2 • GTP



                         eIF2 • GTP • Met-tRNAi




                           PROTEIN
                          SYNTHESIS



               eIF2 supplies Met- tRNAi to 40S subunit


                                                            29
Control : Down-regulation of the supply of initiator                                       Met
                   -tRNAi via eIF2 kinases

                                                                          eIF2 kinases
                                  kinases
                                                                    HRI: reticulocytes minus heme
                    eIF2 • GDP               eIF2   P               PKR: interferon plus virus-
                                                                          infection (dsRNA)
                          eIF2B
                                                                    PERK: ER stress
                                                                    GCN2: amino acid starvation
                    eIF2 • GTP
                                            eIF2B    eIF2   P
                                                    Trapped eIF2B

                 eIF2 • GTP • Met-tRNAi             INITIATION INHIBITED




                   PROTEIN
                  SYNTHESIS



                eIF2 supplies Met- tRNAi to 40S subunit
                eIF2 phosphorylation inhibits initiation
                                                                                                30
GTP/GDP exchange during elongation by (e)EF1 (aka EF-Tu)


                                                           Terminology
                 EF-Tu • GDP
                                                          PROK.        EUK.
                       EF-Ts                              Old New

                 EF-Tu • GTP          aa-tRNA complex Tu 1A              1α

                                                    GEF Ts 1B          1βγ
              EF-Tu • GTP • aa-tRNA




                PROTEIN
               SYNTHESIS




             This factor supplies aa- tRNA to ribosome during elongation.

                                                                              31
                              membrane-bound
nuclear                       polysome on         endoplasmic
membrane                      “rough” ER          reticulum
                                                  lumen




                                               secreted
                                               protein

            “free” polysome




                     cytosolic
CYTOPLASM            protein

                                           cell membrane



                                                                32
                       Inhibitors of Protein Synthesis:
                           Antibiotics and Toxins
      Inhibitor                   Class          Target              Action
STREPTOMYCIN, Gentamicin,      Aminoglycosides   30S                 (1) Inhibits initiation
 Kanamycin, Neomycin, etc.                                           (2) Causes misreading
TETRACYCLINE, doxycycline      Tetracylines      30S                 Inhibits binding of AA-tRNA to A-site
CHLORAMPHENICOL                                  50S                 Inhibits peptidyl transferase
ERYTHROMYCIN,                  Macrolides        50S                 Inhibit translocation
Clarithromycin,
 Azithromycin
Clindamycin, Lincomycin        Lincosamides      50S                 Inhibit translocation
Mupirocin (pseudomonic acid)                     Ile-tRNA synthase   Inhibits isoleucine tRNA charging

PUROMYCIN                                        50S, 60S            Premature release of nascent
                                                                     polypeptide

Cycloheximide                                    80S                 Inhibits translocation
DIPHTHERIA TOXIN                                 eEF2                Inhibits translocation¤
RICIN (castor beans)                             60S                 Inhibits binding of AA-tRNA to A-site♦
a-Sarcin (fungus)                                60S                 Inhibits binding of AA-tRNA to A-site
                                                                            & translocation#



                                                                     Catalytic activities of toxins
                                                                     ¤ ADP ribosylation
                                                                     ♦ 28S rRNA depurination (A)
  CAPITALIZED: most important                                        # 28S rRNA cleavage



                                                                                                             33
                           PROKARYOTES               EUKARYOTES

Nucleus                     No                       Yes
Transcription & translation Coupled                  Separated

mRNA                      Polycistronic              Monocistronic, Capped
                                                         & Polyadenylated
Ribosomes                 70S (50S, 30S)             80S (60S, 40S)

Initiator                 f Met – tRNAi              Met – tRNAi

Site selection            Shine-Dalgarno mediated    1) Scanning
                            internal initiation      2) IRES mediated internal
                                                          entry

Initiation factors        3                          >12

Order of events           1) mRNA binding            1) Met – tRNAi binding
                          2) f Met – tRNAi binding   2) mRNA binding

Antibiotics               Sensitive                  Resistant

Toxins                    Resistant                  Sensitive
                                                                              34
Protein Modifications

•Phosphorylation - (Tyr, Ser,Threo) Metabolic Regulation, Signal
transduction, etc
•Hydroxylation - (Proline) in collagen, Endoplasmic Reticulum

•Glycosylation – (O-linked as with Ser/Threo- OH or N-Linked as in
lysine)
•Other - biotinilation, farnesyl, etc

Protein Degradation - Mostly thru specific proteases and
ubiquitin-proteosome system



                                                                     35

				
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