Regulasi Ekspresi Gen

PROTEIN TRANSLATION AND
TRANSPORT
Andriani
Dept. Biokimia dan Biologi Molekuler
FK UNTAN
Translation
􀁺 Process of converting information stored in
nucleic acid sequences into proteins
􀁺 Sequences of mRNA (messenger RNA) are
translated into unique sequence of amino
acids in a polypeptide chain (linear order is
preserved throughout !)
Translation
􀁺 Takes place in the cytoplasm
􀁺 Exception are few proteins coded by
mitochondrial and chloroplastic DNA
􀁺 Performed on ribosomes
What is necessary?
􀁺 Template - mRNA
􀁺 tRNAs (transfer RNAs)
􀁺 Linked to amino acids
􀁺 Ribosomes
􀁺 Many accessory proteins
􀁺 Some energy (GTP hydrolysis)
mRNA
􀁺 Single stranded molecule of RNA that encodes
sequence of the polypeptide
􀁺 Transcribed and processed in the nucleus and
then exported into cytoplasm
􀁺 5’ end has binding sites for translation initiation
􀁺 Middle is a coding sequence
􀁺 3’ end regulates stability of mRNA
Genetic code
􀁺 Codon – specifies the sequence of amino
acids
􀁺 Initiation (start) codon
􀁺 AUG – methionine
􀁺 Every protein in a cell starts with methionine
􀁺 Termination (stop) codons
􀁺 UAA, UGA, UAG
Genetic code
􀁺 Universal
􀁺 Degenerate - some amino acids are specified
by more than one codon
􀁺 64 possible codons and only 20 amino acids
GENETIC CODE
Eukaryotic mRNA
􀁺 Codes for one protein
􀁺 Is capped on both 5’ and 3’
ends
􀁺 5’ cap - methylated base
􀁺 Protects from nucleases 3’ poly-A tail
50-200 adenines added post-
transcriptionally Protects mRNA from
degradation
tRNA
􀁺 Deliver amino acids to the
translational complex
􀁺 Serve as adapters between
codons in mRNA and amino
acid
􀁺 4 stems and 3 loops
􀁺 Anticodon – decoding triplet -
localized on the anticodon stem
Charging of tRNA
􀁺 Linking amino acids to correct t-RNAs
􀁺 Catalyzed by aminoacyl-tRNA synthetase (aa-
tRNA)
􀁺 Couples an amino acid to its cognate tRNA
􀁺 Fidelity of coupling – 20 different synthetases
􀁺 Two steps
Activation of amino acid
Transfer of amino acid to tRNA
Ribosomes
􀁺 RNA-protein complexes (ribonucleoproteins)
􀁺 Place of translation (protein synthesis)
􀁺 Abundant in cells that synthesize large
amounts of protein
􀁺 Structurally and functionally similar among
species (differ between prokaryotes and
eukaryotes)
Ribosomes
􀁺 Composed of small and large subunit
􀁺 Subunits bind together for
translation
􀁺 Ribosomes self-assemble without
additional
factors
Ribosomes
􀁺 Decoding and synthesis takes
place in the cavity between
subunits
􀁺 Ribosomes move along mRNA
chain during translation
􀁺 New peptide exits through the
tunnel in the large subunit
Ribosomes
􀁺 Contain rRNA molecules and
proteins
􀁺 In prokaryotes
􀁺 Large subunit contains rRNA
(5S and 23S) and 32 proteins
􀁺 Small subunit contains
Ribosomes
􀁺 In eukaryotes
􀁺 Large subunit contains rRNA
(5S, 5.8S and 28S) and 49
proteins
􀁺 Small subunit contains 18S
rRNA and 33 proteins
Ribosomes
􀁺 Cytosolic (free)
􀁺 Bound to ER
􀁺 Also located in mitochondria and chloroplasts
of eukaryotic cells
Free ribosomes
􀁺 Found in the cytosol
􀁺 May exist as a single ribosome or in groups
known as polysomes
􀁺 Occur in greater number than bound
ribosomes in cells that retain most of their
manufactured protein in the cytosol
Bound ribosomes
􀁺 Bound to the exterior of the rough
endoplasmic reticulum
􀁺 Occur in greater number than free ribosomes
in cells that secrete their manufactured
proteins (e.g., pancreatic cells)
Protein synthesis
􀁺 Initiation
􀁺 Elongation
􀁺 Termination
Initiation
􀁺 Attachment of initiator
tRNA (Met-tRNA) to start
codon on mRNA and
assembly of
ribosomal subunits
Initiation
􀁺 Several initiation factors assist the process
􀁺 Strongly regulated process
􀁺 Uses energy form ATP or GTP hydrolysis
Initiation
􀁺 Cap recognition
􀁺 mRNA initiation factors
􀁺 Helicase (ATPase) unwinds
mRNA
Initiation
􀁺 Formation of preinitiation
complex
􀁺 Initiator tRNA (Met-tRNA)
binds to small ribosomal
subunit
􀁺 Initiation factors use GTP
hydrolysis
Initiation
􀁺 Unwinding and
scanning
􀁺 Complex scans and
binds to start codon
􀁺 Dissociation of
initiation factors
Initiation
􀁺 Association with large
ribosomal subunit
Elongation
􀁺 Repetitive cycles of codon
directed addition of
aa-tRNA
􀁺 Aa-tRNA binding
􀁺 Proofreading
􀁺 Peptidyl transfer
􀁺 Translocation
Elongation
􀁺 Aa-tRNA binding
􀁺 Elongation factor eEF-1
delivers amino acid-tRNA to
ribosome
􀁺 eEF-1 is bound to GTP
Elongation
􀁺 Proofreading
􀁺 Aa-tRNA is checked against codon
􀁺 GTP hydrolysis and dissociation of eEF-1 from
ribosome
􀁺 Correct amino acid-tRNA retained based on
codonanticodon pairing
􀁺 Incorrect amino acid-tRNA escape from the
ribosome
Elongation
Elongation
􀁺 Peptidyl transfer
􀁺 Large rRNA catalyzes formation of peptide
bond
􀁺 Precise orientation and stabilization of the
transition state
Elongation (steps) :
1. binding of an aminoacyl-tRNA to the “A” site
on the ribosome where it base-pairs with
the second codon on the mRNA;
2. formation of a peptide bond between the
first and second amino acids
3. translocation, movement of the mRNA
relative to the ribosome, so that the third
mRNA codon moves into the “A” site.
Elongation
􀁺 Translocation
􀁺 Binding EF-2-GTP
􀁺 Movement of peptidyl-tRNA to
P site
􀁺 Sliding of mRNA by 3 bases
􀁺 tRNA moved to the exit
􀁺 GTP hydrolysis and release of
EF-2
Termination
􀁺 Protein factor eTF binds to stop codon and
catalyzes hydrolysis of last amino acid-tRNA
􀁺 Peptide is released from the ribosome
􀁺 Ribosomal subunits dissociate
Polysomes
􀁺 In eukaryotes the same molecule of mRNA
can be simultaneously translated several times
􀁺 Each emerging peptide is synthesized on a
separate ribosome
􀁺 Many ribosomes on the same “string” of
mRNA are called polysomes
Protein Processing
• Folding to 3D structure :
- Chaperones
- Protein disulfide isomerase
POSTTRANSLATIONAL MODIFICATION
PROTEIN TARGETTING
Proteins synthesized on cytosolic ribosomes are
released into the cytosol or transported into
mitochondria, peroxisomes, and nucleus.
Proteins synthesized on ribosomes attached to the
rough endoplasmic reticulum (RER) are destined
for lysosomes, cell membranes, or secretion from
the cell. These proteins are transferred to the
Golgi complex, where they are modified and
targeted to their ultimate locations.

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PROTEIN TRANSLATION AND

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