Transcript Elongation factors

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Protein Synthesis
Miss Hradhaya C Bhatlawande
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Definitions and Keywords
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proteins by decoding mRNA produced in
transcription
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Translation-Translation is the production of
Ribosome-complexes of RNA and protein that are
found in all cells with nuclei.
transfer RNA (tRNA)-is a small RNA molecule (usually
about 74-95 nucleotides) that transfers a specific active
amino acid to a growing polypeptide chain at the
ribosomal site of protein synthesis during translation
Codon-The genetic code is the set of rules by which
information encoded in genetic material (DNA or RNA
sequences) is translated into proteins (amino acid
sequences) by living cells
Anticodon-base pair to the corresponding three base
codon region on mRNA.
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Shine-Dalgarno sequence-is a ribosomal binding site in the mRNA,
generally located 16 nucleotides upstream of the start codon AUG.
wobble hypothesis-a wobble base pair is a non-Watson-Crick
base pairing between two nucleotides in RNA molecules.
initiation factor-Initiation factors are proteins that bind to the small
subunit of the ribosome during the initiation of translation, a part of
protein biosynthesis
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elongation factor-Elongation factors are a set of proteins that
facilitate the events of translational elongation, the steps in protein
synthesis from the formation of the first peptide bond to the
formation of the last one
elongation factor Tu (EF-Tu)
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elongation factor Ts (EF-Ts)
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elongation factor G (EF-G)
molecular mimicry-Molecular mimicry is defined as the theoretical
possibility that sequence similarities between foreign and selfpeptides are sufficient to result in the cross-activation of
autoreactive T or B cells by pathogen-derived peptides
release factor-The release factor is a protein that recognizes the
termination codon or stop codon in a mRNA sequence on the
ribosome
Translation-An Introduction
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Translation is the whole process by which
the base sequence of an mRNA is used
to order and to join the amino acids in a
protein.
The three types of RNA participate in this
essential protein-synthesizing pathway in
all cells; in fact, the development of the
three distinct functions of RNA was
probably the molecular key to the origin of
life.
Translation An introduction(contd)
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Three kinds of RNA molecules perform different but cooperative functions
in protein synthesis
1. Messenger RNA (mRNA) carries the genetic information copied from
DNA in the form of a series of three-base code “words,” each of which
specifies a particular amino acid.
2. Transfer RNA (tRNA) is the key to deciphering the code words in
mRNA. Each type of amino acid has its own type of tRNA, which binds it
and carries it to the growing end of a polypeptide chain if the next code
word on mRNA calls for it. The correct tRNA with its attached amino acid
is selected at each step because each specific tRNA molecule contains a
three-base sequence that can base-pair with its complementary code
word in the mRNA
3. Ribosomal RNA (rRNA) associates with a set of proteins to form
ribosomes.
These complex structures, which physically move along an mRNA
molecule, catalyze the assembly of amino acids into protein chains.
They also bind tRNAs and various accessory molecules necessary for
protein synthesis. Ribosomes are composed of a large and small subunit,
each of which contains its own rRNA molecule or molecules
Step no.1
Text for Step no. 1
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Initiation of protein synthesis in eukaryotic cells, as in
bacteria, begins with formation of a preinitiation
complex prior to mRNA binding.
Cells can regulate protein synthesis by phosphorylating
a serine residue on the eIF2 bound to GDP; this
complex is then unable to bind Met-tRNAiMet, thus
inhibiting protein synthesis
The A preceding the AUG seems to be the most
important nucleotide affecting initiation efficiency.
Scanning of the mRNA by the preinitiation complex
eventually yields a initiation complex in which MettRNAiMet is correctly positioned at the translation start
site.
Step no. 2
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The correctly positioned bacterial 70S
ribosome Met-tRNAiMet complex is now
ready to begin the task of stepwise
addition of amino acids by the in-frame
translation of the mRNA.
Text for Step No 2
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Once the small ribosomal subunit with its
bound Met-tRNAiMet is correctly
positioned at the start codon, union with
the large ribosomal subunit completes
formation of the initiation complex .
Initiation of translation of most mRNAs by
theprokaryotic protein-synthesizing
machinery begins near the 5′ capped end
as just described.
Step No 3
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With the initiating Met-tRNAiMet at the P
site and the second aminoacyl-tRNA
tightly bound at the A site, the α amino
group of the second amino acid reacts
with the “activated” (aminoacylated)
methionine on the initiator tRNA, forming
a peptide bond
Step No 4
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For example, the second aminoacyl-tRNA
is brought into the ribosome as a ternary
complex in association with an EF-Tu –
GTP in bacteria and becomes bound to
the A site on the ribosome.
Step No 5
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the initiating Met-tRNAiMet is
bound at the P site and basepaired with the AUG start codon. If
the anticodon of the incoming
(second) aminoacyl-tRNA correctly
matches the second codon of the
mRNA, a tight binding ensues at
the A site.
If this second codon does not
match the incoming aminoacyltRNA, it diffuses away. The choice
of the correct aminoacyl-tRNA and
its tight binding at the A site
requires energy that is supplied by
hydrolysis of the EF-Tu – GTP
complex
Step No 6
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The key steps in elongation are
entry of each succeeding
aminoacyl-tRNA, formation of a
peptide bond, and the
movement, or translocation, of
the ribosome with respect to the
mRNA
Step No 7
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During the process of peptide synthesis and tRNA
site changes, the ribosome is moved along the mRNA
a distance equal to one codon with the addition of
each amino acid.
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This translocation step is catalyzed by bacterial EFG – GTP, which is hydrolyzed to provide the
required energy.
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Referring again to , we can see that after peptide
linkage tRNAiMet, now without its activated
methionine, is moved to an exit (E) site on the
ribosome and is soon discharged. Concurrently,
another ternary complex, carrying the next amino
acid to be added, enters the ribosome, and the cycle
continues.Protein Synthesis Is Terminated by Release
Factors When a Stop Codon Is Reached
RRF is another protein that resembles tRNA. The α
helices of this protein mimic the tRNA structure. In
contrast, in EF-G, β strands are the mimics, revealing an
independent evolutionary origin.
Step No 8
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The final phase of protein synthesis, like
initiation and elongation, requires highly
specific molecular signals that decide the
fate of the mRNA-ribosome-tRNApeptidyl complex.
Step No 9
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RF3 acts in concert with the codonrecognizing factors to promote cleavage
of the peptidyl-tRNA, thus releasing the
completed protein chain.Folding of a
newly released protein into its native
three-dimensional conformation is
facilitated by other proteins called
chaperones
Questionnaire
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Q1)Ribosomes were isolated from bacteria grown in a “heavy” medium
(13C and 15N) and from bacteria grown in a “light” medium (12C and
14N). These 60S ribosomes were added to an in vitro system actively
engaged in protein synthesis. An aliquot removed several hours later
was analyzed by density-gradient centrifugation. How many bands of
70S ribosomes would you expect to see in the density gradient?
ANS:-Four bands: light, heavy, a hybrid of light 30S and heavy 50S,
and a hybrid of heavy 30S and light 50S .
Q2) Devise an experimental strategy for switching off the expression of
a specific mRNA without changing the gene encoding the protein or the
gene's control elements
Ans:-The translation of an mRNA molecule can be blocked by
antisense RNA, an RNA molecule with the complementary sequence.
The antisense-sense RNA duplex cannot serve as a template for
translation; single-stranded mRNA is required. Furthermore, the
antisense-sense duplex is degraded by nucleases. Antisense RNA
added to the external medium is spontaneously taken up by many
cells. A precise quantity can be delivered by microinjection.
Alternatively, a plasmid encoding the antisense RNA can be introduced
into target cells.
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Q3)Which protein in G-protein cascades plays a
role similar to that of elongation factor Ts?
ANS:-EF-Ts catalyzes the exchange of GTP for
GDP bound to EF-Tu. In G-protein cascades, an
activated 7TM receptor catalyzes GTP-GDP
exchange in a G protein
Q4)Eukaryotic elongation factor 2 is inhibited by
ADP ribosylation catalyzed by diphtheria toxin.
What other G proteins are sensitive to this mode of
inhibition?
ANS:-The α subunits of G proteins are inhibited by
a similar mechanism in cholera and whooping
cough
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Q5]Schematically describe initiation in
prokaryotes.
Ans
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Links for further reading
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-BIOCHEMISTRY by STRYER
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- BIOCHEMISTRY by Lehninger
-MOLECULAR CELL BIOLOGY
-Baltimore,Lodish
Thank you