MolBiol 08 Translation
description
Transcript of MolBiol 08 Translation
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TRANSLATION
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The flow of genetic information
Gene: The region of DNA that
controls a discrete hereditary
characteristic of an organism,
usually corresponding to a single
protein or RNA.
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Prokaryotes and eukaryotes handle their transcripts
somewhat differently
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Several types of RNA are produced in cells
Type of RNA Function
Messenger RNA
(mRNA)
Code for proteins
Ribosomal RNA
(rRNA)
Form part of the structure of the ribosome and participate
in protein synthesis
Transfer RNA
(tRNA)
Used in protein synthesis as adaptors between mRNA
and amino acids
Small RNA (snRNA) Used in pre-mRNA splicing and other cellular processes
Small nucleolar RNA
(snoRNA)
Used to process and chemically modify rRNAs
MicroRNA (miRNA) Regulate gene expression typically by blocking
translation of selective mRNAs
Small interfering
RNA (siRNA)
Turn off gene expression by directing degradation of
selective mRNAs and the establishment of compact
chromatin structures
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Messenger RNA (mRNA)
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The universal genetic code
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Deviations from the universal genetic code
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Open Reading Frame (ORF)
- The protein-coding region of each mRNA is composed of a contiguous, non-
overlapping string of codons called an open reading frame (ORF).
-The first and last codons of an ORF are known as the start and stop codons.
- In bacteria, the start codon is usually 5-AUG-3 (Met), but 5-GUG-3 and
sometimes even 5-UUG-3 are also used.
- Eukaryotic cells always use 5-AUG-3 as start codon.
- Start codon has two important functions: 1) it specifies the first amino acid
(Met) to be incorporated into the growing polypeptide chain; 2) it defines the
reading frame for all subsequent codons.
- Stop codons, of which there are three (UAA, UAG, UGA), defines the end of
ORF and signal termination of polypeptide synthesis.
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A mRNA molecule can be translated in three
possible reading frame
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Transfer RNA (tRNA)
- tRNA molecules are molecular adaptors, linking amino acids with codons.
- tRNAs share a common secondary structure that resembles a cloverleaf
- tRNAs have an L-shape three-dimensional structure.
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A subset of modified nucleosides found in tRNA
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Codon-anticodon pairing involves wobbling at
the third position
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Attachment of amino acids to tRNA
- tRNA molecules to which an amino acid is attached are said to be charged,
and tRNAs that lack an amino acid are said to be uncharged.
- Two steps of aminoacyl-tRNA charging: 1) Adenylation of amino acid; 2)
tRNA charging in which the adenylated amino acid react with tRNA.
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Acyl linkage between the carboxyl group of the
amino acid and 3-hydroxyl group of the adenosine
nucleotide that protrudes from the acceptor stem
This acyl linkage is cinsidered to be high-energy bond
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Specific enzymes couple tRNAs to the correct
amino acid
- Recognition and attachment of the correct amino acid depends on enzymes
called aminoacyl-tRNA synthetase, which covalently couple each amino acid
to its appropriate set of tRNA molecules.
- There is a different synthetase enzyme for each amino acid (that is, there are
20 synthetase in all)
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tRNA synthetase recognize unique structural
feature of cognate tRNAs
- The acceptor stem and the anticodon loop are the specificity determinants for
tRNA synthetase recognition.
- In some cases changing a single base pair in the acceptor stem (discriminator
base) is sufficient to convert the recognition specificity of a tRNA from one
synthetase to another.
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Some aminoacyl tRNA synthetase use an editing
pocket to charge tRNAs with high accuracy
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The RNA message is decoded on ribosomes
- The ribosome is macromolecular machine that directs the synthesis of
proteins.
- In prokaryotes, the transcription machinery and the translation machinery are
located in the same compartment.
- In eukaryotes, the translation is completely separate from transcription:
transcription occurs in the nucleus, whereas translation occurs in the cytoplasm.
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The ribosome is composed of a large and small subunit
- The ribosome is composed of two subassemblies of RNA and protein known as the large
and small subunits. The large subunit contains the peptidyl transferase center, which
responsible for the formation of peptide bonds. The small subunit contains the decoding
center in which charged tRNA read or decode the codon units of the mRNA.
- The large and small subunits are named according to the velocity of their sedimentation
when subjected to a centrifugal force. The unit used to measure sedimentation velocity is the
Svedberg (S).
- The prokaryotic ribosome composed of 50S and 30S subunits, which together form an 70S
ribosome. The eukaryotic ribosome composed of 60S and 40S subunits, which together form
an 80S ribosome.
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Composition of prokaryotic ribosome
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Composition of eukaryotic ribosome
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The large and small subunits undergo association and
dissociation during each cycle of translation
- Translation begins with the binding of
the mRNA and an initiating tRNA to small
subunit of ribosome
- The small subunit-mRNA complex then
recruits a large subunit to create an intact
ribosome with the mRNA sandwiched
between two subunits.
- As the ribosome translocates from codon
to codon, one charged tRNA after another
is slotted into the decoding and peptidyl
transferase centers of the ribosome
- When the ribosome encounters a stop
codon, the completed peptide chain is
released, and the ribosome disassociates
from the mRNA as separate large and
small subunits.
- An mRNA can be translated
simultaneously by multiple ribosomes
called polyribosome or polysome.
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The peptidyl transferase reaction
-The ribosome catalyzes a single chemical reaction: the formation of peptide
bond. This reaction occurs between the amino acid residue at the carboxyl-
terminal end of the growing polypeptide and the incoming amino acid to be
added to the chain.
- Both the growing chain and the incoming amino acid are attached to tRNAs:
the peptidyl-tRNA and the aminoacyl-tRNA.
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Each ribosome has binding site for mRNA and
three binding sites for tRNA
(A) 3D-structure of bacterial ribosome
with small subunit in the front (dark
green) and the large subunit in the back
(light green). tRNAs are shown bound in
the E-site (red), the P-site (orange) and A-
site (yellow).
Large subunit Small subunit
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Each ribosome has binding site for mRNA and
three binding sites for tRNA
The ribosome has three binding sites: the A-site is the binding site for
Aminoacylated-tRNA, the P-site is the binding site for the Peptidyl-tRNA, and
the E-site is the binding site for the tRNA that is released after the growing
peptide chain has been transferred to the aminoacyl-tRNA (E for exit)
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Initiation of translation
Translation initiation requires:
- Ribosome brought to mRNA
- Ribosome properly aligned over start codon
- P site of ribosome containing the charged tRNA
In prokaryotes, the initiator
tRNA, which base-pairs with the
start codon AUG, is charged with
a modified form of methionin (N-
formyl methionine). The charged
initiator tRNA is referred to as
fMet-tRNAifMet
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Translation Initiation in Prokaryotes (1)
-Prokaryotes mRNAs are initially recruited to small subunit by base-pairing to
16S rRNA
- Many prokaryotic ORFs contain a short sequence upstream (on the 5 side) of
the start codon called the ribosome binding site (RBS). This element is also
referred to as a Shine-Dalgarno sequence.
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Translation Initiation in Prokaryotes (2)
- In prokaryotes, three initiation factors
direct the assembly of an initiation complex:
* IF1: prevents tRNAs from entering A site
* IF2: is a GTPase (a protein that binds and
hydrolyzes GTP). IF2 binds IF 1 and guides
the initiator tRNA (fMet-tRNAifMet) to P site
* IF3: prevents association of large subunit
- With all three IFs bound, the small subunit
is prepared to bind the mRNA and the
initiator tRNA.
-When start codon and fMet-tRNA base-pair,
the small subunit undergo a change in
conformation. This alter conformation
results in the release of IF3.
- In the absence of IF3, the large subunit can
bind to the small subunit complex to create
the 70S initiation complex.
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Initiation Factors
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Translation Initiation in Eukaryotes (1)
- In eukaryotes, the initiator tRNA is charged
with methionine (Met-tRNAiMet)
- Two GTP-binding proteins (eIF2 and eIF5B)
mediate the recruitment of the charged tRNA
- The small subunit is already associated with
the initiator tRNA when it is recruited to the
capped 5 end of the mRNA
- Together two GTP-binding proteins position
the Met-tRNA in the future P-site of small
subunit, resulting in the formation of the 43S
pre-initiation complex.
- The 43S pre-initiation complex recognize the
5 cap of mRNA. The recognition is mediated
by eIF-4E/ G
- Once assemble at the 5 end of the mRNA,
the small subunit and its associated factors
move along the mRNA in a 5-3 direction to
scan for the first start codon AUG
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Translation Initiation in Eukaryotes (2)
- Correct base-pairing between initiator
tRNA and start codon triggers the release
of eIF2 and eIF3.
- Lost of eIF3 and eIF2 allow the large
subunit to bind to the small subunit
complex to create the 80S initiation
complex.
- With the start codon and Met-tRNA
placed in the P-site, the eukaryotic
ribosome is now poised to accept a
charged tRNA into its A-site and carry
out the formation of the first peptide bond.
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Translation Elongation
- Step 1: an aminoacyl-tRNA binds to vacant A-site
on the ribosome
- Step 2: a new peptide bond is formed
- Step 3: the mRNA moves a distance of three
nucleotides (a codon) through the small subunit,
ejecting the spent tRNA molecule and resetting
the ribosome so that the next aminoacyl-tRNA
molecule can bind.
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Translation elongation in prokaryotes
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Translation elongation in eukaryotes
Prokaryotic
elongation factors
Eukaryotic
elongation factors
Function
EF-Tu eEF1 Escort aminoacyl-tRNA
to the A-site of ribosome
EF-G eEF2 Drive translocation of the
tRNA and the mRNA
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Termination of translation
- The end of the protein-coding message is
signaled by the presence of one of the several
stop codon (UAA, UAG or UGA)
- The stop codon is recognized by proteins
called release factors.
- Release factors bind to any stop codon that
reaches to the A-site on the ribosome, and this
binding alters the activity of the peptidyl
transferase in the ribosome, causing it to
catalyze the additional water molecule instead
of an amino acid to the peptidyl-tRNA.
- This reaction frees the carboxyl end of the
growing polypeptide chain from its attachment
to a tRNA molecule.
- The ribosome release the mRNA and
disassociate into its two separate subunits
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Release factors are an example of molecular mimicry:
the three-dimensional structure of release factors resembles the
shape and charge distribution of a tRNA molecule
Human eRF1 tRNA
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Classification of release factors
Release factors (RFs)
class 1 RF class 2 RF
UAG
UAA
UGA
Eukaryotes
eRF1
Prokaryotes Eukaryotes
RF3 eRF3
Prokaryotes
RF1
RF2
Class 1 RF is responsible for stop codon
recognition and hydrolysis of the peptidyl-
tRNA linkage.
Class 2 RF is a GDP/ GTP-binding
protein, which stimulate class 1 RF
activity.
GDP GDP
Class 1 and 2 RFs form functional
complex in translation termination
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Translation initiation factors hold eukaryotic
mRNA in circle
Circular polyribosome in eukaryotic cell
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Proteins Fold into a Conformation of Lowest E nergy
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Steps in the creation of a functional protein
Translation of an mRNA sequence
into an amino acid sequence on the
ribosome is not the end of the
process of forming a protein. To
function, the completed polypeptide
chain must fold correctly into its
three-dimensional conformation,
bind any cofactors required, and
assemble with its partner protein
chains (if any).
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Some Proteins Begin to Fold While Still Being Synthesized
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Molecular Chaperones Help Guide the Folding of Most Proteins
Chaperone (molecular chaperone):
Protein that helps guide the proper
folding of other proteins, or helps
them avoid misfolding. Includes
Heat shock proteins (Hsp).
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The Hsp70 family of molecular chaperones
The Hsp70 machinery acts early in the life of many proteins, binding to a
string of about seven hydrophobic amino acids before the protein leaves the
ribosome
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The structure and function of the Hsp60 family of
molecular chaperones
Hsp60-like proteins form a large barrel shaped
structure that acts after a protein has been fully
synthesized.
This type of chaperone, sometimes called a
chaperonin, forms an isolation chamber into
which misfolded proteins are fed, preventing their
aggregation and providing them with a favorable
environment in which to attempt to refold
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Release factors
RF1, RF2, RF3
Release factors
eRF1, eRF3
Comparison of protein synthesis