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					Protein Synthesis
 IB Biology HL
  Mr. McIntyre
Translation: From messenger RNA to protein:

  The information encoded in the DNA is transferred to
  messenger RNA and then decoded by the ribosome to
  produce proteins.
 5’-ATGCCTAGGTACCTATGA-3’                   DNA
5’-AUGCCUAGGUACCUAUGA-3’                    mRNA

               decoded as


 N-MET-PRO-ARG-TYR-LEU-C                    Protein
Alanine tRNA
Generalized tRNA
   Modified Bases
   Found in tRNAs

= UH2
tRNAs are activated by amino-acyl tRNA synthetases
Structure of an amino acyl-tRNA synthetase bound to a tRNA
One mechanism for maintaining high fidelity of protein
synthesis is the high fidelity of aa-tRNA synthetases
Amino-acyl tRNA synthetases:
One synthetase for each amino acid
      a single synthetase may recognize multiple tRNAs
      for the same amino acid

Two classes of synthetase.
        Different 3-dimensional structures
        Differ in which side of the tRNA they recognize
        and how they bind ATP
Class I - monomeric, acylates the 2’OH on the terminal ribose
        Arg, Cys , Gln, Glu, Ile, Leu, Met, Trp Tyr, Val

Class II - dimeric, acylate the 3’OH on the terminal ribose
        Ala, Asn, Asp, Gly, His, Lys, Phe, Ser, Pro, Thr
Two levels of control to ensure that the proper amino acid
is incorporated into protein: 1) Charging of the proper tRNA
2) Matching the
cognate tRNA to the
messenger RNA
Incorporation of amino acids into polypeptide chains I
Incorporation of amino acids into polypeptide chains II
Protein synthesis occurs on ribosomes
Protein synthesis occurs on ribosomes
and mitochondria
Ribosome Assembly

The proteins of each
ribosomal subunit
are organized around
rRNA molecules

                       16S rRNA
Ribosome Assembly: takes place largely in a specialized domain of
the nucleus, the nucleolus
  In the nucleolus, RNA polymerase I transcribes the rDNA repeats
  to produce a 45S RNA precursor

The 45S precursor
 is processed
and cleaved into
mature rRNAs and
ribosomal proteins
then bind to generate
the large and small
ribosomal subunits
23S rRNA secondary structure
3D organization of the eukaryotic large subunit rRNA
Ribosomal Proteins decorate the surface of the ribosome

   Large subunit. Grey = rRNA Gold = ribosomal proteins
Ribosomal proteins often have extensions that snake into
the core of the rRNA structure

Crystal structure of L19      L15 (yellow) positioned in a fragment
                              of the rRNA (red)
The ribosomal proteins are important for maintaining the
stability and integrity of the ribosome, but NOT for catalysis

ie. the ribosomal RNA acts as a ribozyme
 The large and small subunits come together to form the ribosome

or Prokaryotic
Eukaryotic       60S subunit     80S ribosome           40S subunit
The association of the large and small subunits creates the
structural features on the ribosome that are essential for
protein synthesis

                                             Three tRNA binding
                                             A site = amino-acyl
                                             tRNA binding site

                                             P site = peptidyl-tRNA
                                             binding site

                                             E site = exit site
In addition to the APE sites there is an mRNA binding groove
that holds onto the message being translated
There is a tunnel through the large subunit that allows the
growing polypeptide chain to pass out of the ribosome
Peptide bond formation is catalyzed by the large subunit rRNA
Peptide bond formation is catalyzed by the large subunit rRNA
Incorporation of the correct amino acyl-tRNA is determined
by base-pairing interactions between the anticodon of the
tRNA and the messenger RNA
=EF-1   Proper reading of the
        anticodon is the second
        important quality control
        step ensuring accurate
        protein synthesis

         Elongation factors
         Introduce a two-step
         “Kinetic proofreading”
A second elongation factor
EF-G or EF-2, drives the
translocation of the ribosome
along the mRNA

Together GTP hydrolysis
by EF-1 and EF-2 help drive
protein synthesis forward
Termination of translation
is triggered by stop codons

    Release factor enters
    the A site and triggers
    hydrolysis the peptidyl-tRNA
    bond leading to release of
    the protein.
Release of the protein causes
the disassociation of the
ribosome into its constituent
Release Factor is a molecular mimic of a tRNA

    eRF1                      tRNA
   Initiation of Translation

   Initiation is controlled differently in prokaryotic and
   eukaryotic ribosomes

In prokaryotes a single transcript can give rise to multiple proteins
In prokaryotes, specific
sequences in the mRNA around
the AUG codon, called
Shine-Delgarno sequences,
are recognized by an intiation
complex consisting of a Met
amino-acyl tRNA, Initiation
Factors (IFs) and the small
ribosomal subunit
GTP hydrolysis by
IF2 coincident with
release of the IFs and
binding of the large
ribosomal subunit leads
to formation of a complete
ribosome,on the mRNA
and ready to translate.
Eukaryotic mRNAs have a distinct structure at the 5’ end
         Structure of the 7-methyl guanosine cap

The 7me-G cap is required
for an mRNA to be
In contrast, Eukaryotes
use a scanning mechanism
to intiate translation.

 Recognition of the AUG
 triggers GTP hydrolysis
 by eIF-2
GTP hydrolysis by
eIF2 is a signal for
binding of the large
subunit and beginning
of translation
Messenger RNAs are translated on polyribosomes
Protein synthesis is often regulated at the
level of translation initiation
An example of control of specific mRNAs: regulation by iron (Fe):

Ferritin is a cytosolic iron binding protein expressed when
iron is abundant in the cell.

Transferrin receptor is a plasma membrane receptor important
for the import of iron into the cytosol.

They are coordinately regulated, in opposite directions, by
control of protein synthesis.
Regulation by iron (Fe):
There is also general control of translational initiation.

ie. all transcripts of the cell are effected (though the relative
effect differs between specific mRNAs)

Global downregulation or upregulation can occur in response
to various stimuli the most common are
        1) Nutrient availability
               low nutrient (amino acids/carbohydrate)
               downregulates translation

        2) Growth factor signals.
               stimulation of cell division upregulates translation
General control of translational initiation is exerted through
two primary mechanisms.

Control of the phosphorylation of eIF2

Control of the phosphorylation of eIF4 binding proteins
Control of translation by eIF2 phosphorylation

Stimulated by
Amino acid deprivation
  Control of translation by eIF4E availability

The 7MEG cap binding subunit of eIF4, eIF4E, is sequestered
by eIF4E binding protiens (4E-BPs). The binding of these
proteins is regulated by their phosphorylation state

Growth                    Nutrient
Factors                   Limitation

Nutritional signals can control both the recognition of the mRNA
and loading of the 40S subunit.
Modification of the translation machinery is a common
feature of viral life cycles

 e.g. Picornaviruses
         Polio virus
         Encephalomyocarditis virus

 Picornaviruses have single stranded RNA genomes.
Poliovirus Life Cycle
The poliovirus genome is translated into a single,
large polyprotein that then auto-proteolyzes itself into
smaller proteins.

One of these proteins, viral protease 2A cleaves
the translation initiation factor eIF4G so that it
can no longer function as a bridge between the
methyl cap binding subunit and the 40S subunit
The consequence of this cleavage is that translation of
cellular mRNAs stops

 But…the viral RNA is still translated due to the presence of
 an internal ribosomal entry site (IRES). This acts like a
 bacterial initiation site to allow Cap-independent initiation
 from internal AUG codons.

                        What is X?
“X” is not a protein, as suggested
by the textbook model at right,
rather it is a structure in the mRNA
itself that can bind to the remaining
fragment of eIF4G
   Some cellular mRNAs are also translated using IRESs

During G2/M phase of the cell cycle, translation is generally
downregulated by activation of 4E-BPs. Many proteins expressed
during this period bypass this control by using IRES elements
Ribosomal Frameshifting

                Because translation
                uses a triplet code,
                there are three potential
                reading frames in
                each mRNA
As the ribosome translocates, it moves in three nucleotide
steps, ensuring that the frame defined by the AUG is used
throughout translation

 If the ribosome moves 1 or 2 (or 4 or 5) nucleotides
 this produces a frameshift
Many retroviruses induce ribosomal frameshifting in
the synthesis of viral proteins

   e.g. HIV
Translation Inhibitors are important antibiotics

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