CBCS BOTANY 4TH SEM requirements, steps factorsenzymes

1. Protein synthesis
Dr.Sujit Ghosh

2. Occurrence of protein synthesis
Prokaryotes
Cytoplasm
Eukaryotes
In cytoplasm by
free ribosomes
Cytoplasm
In cytoplasm by
free ribosomes
In ER by
attached
ribosomes
In the matrix of
mitochondria
and chloroplast

3. Requirement of protein
synthesis
Ribosomes
30S/40S
50S/60s
mRNA
tRNA
Amino acids
GTP
initiation
IF3
IF2
Alpha
Beta
Gamma
IF1
factors
EF-Tu
EF-Ts
factors
elongation EF-G
Enzymes
Peptidyl
transferase
Termination
RF-1
Signal recived
UAA AND UAG
RF-2
Signal received
UGA
Hydrolysis of
Peptidyl tRNA
Dissociation by
IF-1

7. Initiation
Initiation

8. 1. This phase of protein synthesis results in the assembly of a functionally
competent ribosome in which an mRNA has been positioned correctly so that its
start codon is positioned in the P (peptidyl) site and is paired with the initiator
tRNA
2. IF3 promotes the dissociation of the ribosome into its two component subunits.
The presence of IF3 permits the assembly of the initiation complex and prevents
binding opf the 50S subunit prematurely. IF3 assists the mRNA to bind with the
30S subunit of the ribosome so that the start codon is correctly positioned at
the peptidyl site of the ribosome.
3. The mRNA is positioned by means of base-pairing between the 3' end of the 16S
rRNA with the Shine-Dalgarno sequence immediately upstream of the start
codon.
4. IF2(GTP) assists the fMet-tRNAf
Met to bind to the 30S subunit in the correct site
- the P site.
- the P site.
5. It is not clear whether the mRNA or fMet-tRNAf
Met binds first. It may be that
either can bind first.
6. At this stage of assembly, the 30S initiation complex is complete and IF3 can
dissociate.
7. As IF3 is released, the 50S subunit of the ribosome binds to complete the
initiation complex. Simultaneously, GTP hydrolysis occurs on IF2. This
hydrolysis may be helped by the L7/L12 ribosomal proteins rather than by IF2
itself. Hydrolysis is required for dissociation of IF2. GTP hydrolysis probably
serves as a timing mechanism to ensure that the tRNA is correctly positioned
before IF3 dissociates
8. IF1 assists IF3 in some way, perhaps by increasing the dissociation rate of the
30S and 50S subunits of the ribosome.
9. Once IF2 and IF1 are both released, translation can proceed.

12. Elongation

16. Three special Elongation Factors are required for this phase of protein synthesis: EF-Tu (GTP), EF-Ts
and EF-G (GTP).
The Elongation phase of protein synthesis consists of a cyclic process whereby a new aminoacyl-tRNA
is positioned in the ribosome, the amino acid is transferred to the C-terminus of the growing
polypeptide chain, and the the whole assembly moves one position along the ribosome:
Binding of a new aminoacyl-tRNA at the A site
At the start of each cycle, the A (aminoacyl) site on the ribosome is empty, the
P (peptidyl) site contains a peptidyl-tRNA, and the E (exit) site contains an
uncharged tRNA.
The elongation factor, EF-Tu (GTP) binds with an aminoacyl-tRNA and brings it
to the ribosome. Once the correct aminoacyl-tRNA is positioned in the
ribosome, GTP is hydrolyzed and EF-Tu (GDP) dissociates away from the
ribosome.
There are two ways that EF-Tu functions to ensure that the correct aminoacyl-
There are two ways that EF-Tu functions to ensure that the correct aminoacyl-
tRNA is in place:
EF-Tu prevents the aminoacyl end of the charged tRNA from entering the A site
on the ribosome. This ensures that codon-anticodon pairing is checked first
before the charged tRNA is irreversibly bound in the A site and a new,
potentially incorrect, peptide bond is made.
GTP hydrolysis is SLOW and EF-Tu cannot dissociate from the ribosome until
it occurs. The amount of time prior to GTP hydrolysis allows the final fidelity
check to take place.
If the anticodon-codon interaction is incorrect, the aminoacyl-tRNA simply
dissociates and a new one is brought in. This check, however, can verify
nothing about the aminoacid -- it simply verifies that the correct pairing takes
place.

17. EF-Tu is the most abundant protein in the E. coli cell.
There are approximately 70-100,000 molecules/cell
which is 5% of the total cell protein. There are also
approximately 70-100,000 tRNA molecules/cell. Nearly
all of the aminoacyl-tRNA in the cell is bound by EF-Tu.
EF-Tu cannot bind with tRNAf
Met. This tRNA has a slight
difference in its structure compared with that of tRNAMet
which means that it is not bound by EF-Tu.
EF-Tu (GDP) is inactive and cannot function to bind
EF-Tu (GDP) is inactive and cannot function to bind
aminoacylated tRNAs. However, EF-Tu has a higher
affinity for GDP (Ka = 10-8M) than for GTP (Ka = 10-6M).
In order to recycle EF-Tu, the elongation factor EF-Ts
binds to the EF-Tu (GDP) complex to displace the GDP.
GTP then, in turn, displaces EF-Ts. Many other G-
proteins require a guanine nucleotide release protein
(GNRP) to release GDP; EF-Ts is the GNRP for EF-Tu.

18. Formation of the new peptide bond (Transpeptidation)
Peptide bond formation occurs as a result of nucleophilic attack by
the lone pair of electrons on the amino nitrogen of the aminoacyl-
tRNA on the carbonyl carbon that attaches the growing
polypeptide chain to a tRNA molecule in the P site of the
ribosome. As a result, the peptide chain is attached to the tRNA
which is paired with the codon in the A site. The new amino acid
is, therefore, added to the C-terminal end of the polypeptide chain.
Older illustrations show this reaction as a transfer of the entire
polypeptide chain from the tRNA in the P site to the tRNA in the A
polypeptide chain from the tRNA in the P site to the tRNA in the A
site. This is not an accurate representation. It is more likely that
the aminoacyl arm of the tRNA in the A site extends to join with
the polypeptide chain in the P site.
The peptidyltransferase activity of the ribosome which catalyzes
this reaction is located on the 23S rRNA though it will be assisted
by some of the ribosomal protein subunits. In other words,
peptidyl transferase is a ribozyme - another example of a
catalytic RNA.

19. Translocation of the Ribosome
Finally, the ribosome translocates along the mRNA thereby
moving the new peptidyl-tRNA to the P site and the old (now
uncharged) tRNA, which has just lost its peptidyl chain, to the E
site. This step requires the elongation factor, EF-G(GTP). There
are 20,000 molecules/cell of EF-G which is the same as the
number of ribosomes.
GTP is hydrolyzed during translocation and, once again, GTP
hydrolysis is required for dissociation of EF-G not for binding.
hydrolysis is required for dissociation of EF-G not for binding.
EF-G blocks the binding of aminoacyl tRNAs to the A site as well
as blocking the binding of Release Factors. It effectively makes
sure that translocation must take place before the cycle continues.
EF-G and the tRNA-EF-Tu complex are mutually exclusive. The
structures of these two are remarkably similar and demonstrate
very nicely why these two cannot bind to the ribosome
simultaneously:

20. The following diagram summarizes the movement of tRNA through the ribosome during the
elongation phase of protein synthesis

22. Termination

23. The final phase of protein synthesis requires that the finished polypeptide chain be
detached from a tRNA. This can only happen in response to the signal that a stop codon
has been reached. After hydrolysis, the ribosome subunits dissociate.
Binding of Release factors
There are no tRNAs that recognize the stop codons. Rather they are recognized by release
factor RF1 (which recognizes the UAA and UAG stop codons) or RF2 (which recognizes
.
factor RF1 (which recognizes the UAA and UAG stop codons) or RF2 (which recognizes
the UAA and UGA stop codons). These release factors act at the A site of the ribosome. A
third release factor, RF3 (GTP), stimulates the binding of RF1 and RF2.
Hydrolysis of the peptidyl-tRNA
Binding of the release factors alters the peptidyltransferase activity so that
water is now the nucleophilic attack agent. The result is hydrolysis of the
peptidyl-tRNA and release of the completed polypeptide chain. The uncharged
tRNA then dissociates as do the release factors. GTP is hydrolyzed
Dissociation
Finally, the ribosome dissociates into its 30S and 50S subunits and the mRNA
is released. IF3 may help this process.

24. GENE 4 BENJAMIN LEWIN
Reference