TRANSLATION IN PROKARYOTES

It is the process of synthesis of protein by encoding information on mRNA.

Protein synthesis requires mRNA, tRNA, aminoacids, ribosome and enzyme aminoacyl tRNA synthase

Steps in translation:

1. Activation of aminoacids:

The activation of aminoacids take place in cytosol.

The activation of aminoacids is catalyzed by their aminoacyl tRNA synthetases.

All the 20 aminoacids are activated and bound to 3’ end of their specific tRNA in the presence of ATP
and Mg++.

The N-formylated methionine is chain initiating aminoacid in bacteria whereas methionine is chain
initiating aminoacid in eukaryotes.

Methionine is activated by methionyl-tRNA synthetase. For N-formylmethionine two types of tRNA

Similarly, all 2o aminoacids are activated and then bound to their specific tRNA forming Aminoacyl
tRNA.

2. Initiation:

In the first step, initiation factor-3 (IF-3) binds to 30S ribosomal unit.

Then mRNA binds to 30S ribosomal subunit in such a way that AUG codon lie on the peptidyl (P) site
and the second codon lies on aminoacyl (A) site.

The tRNA carrying formylated methionine ie. FMet–tRNAFMet is palced at P-site. This specificity is
induced by IF-2.

Shine dalgarno sequence in the mRNA guide correct positioning of AUG codon at P-site of 30S
ribosome.

After binding of FMet–tRNAFMet on P-site, IF-3, IF-2 and IF-1 are released so that 50S ribosomal unit
bind with 30S forming 70S sibosome. The exit site is located in 50S.

3. Elongation

i. Binding of AA-tRNA at A-site:

The 2nd tRNA carrying next aminoacid comes into A-site and recognizes the codon on mRNA. This
binding is facilitated by EF-TU.

After binding, GTP is hydrolysed and EF-TU is releasd

ii. Peptide bond formation:

The aminoacid present in t-RNA of P-site ie Fmet is transferred to t-RNA of A-site forming peptide
bond.

Now, the t-RNA at P-site become uncharged

After peptide bond formation ribosome moves one codon ahead along 5’-3’ direction on mRNA, so
that dipeptide-tRNA appear on P-site and next codon appear on A-site.

The uncharged tRNA exit from ribosome and enter to cytosol..

The codon on A-site is now recognized by other aminoacyl-tRNA as in previous.

The dipeptide on P-site is transferred to A-site forming tripeptide.

This process continues giving long polypeptide chain of aminoacids.

4. Termination

The peptide bond formation and elongation of polypeptide continues until stop codon appear on A-
site.

The stop codon are recognized by next protein called release factor (Rf-1, RF-2 and RF-3) which
hydrolyses and cause release of all component ie 30s, 50S, mRNA and polypeptide separates.

RF-1 recognisaes UAA and UAG while RF-2 recognises UAA and UGA while RF-3 dissociate 30S and
50S subunits.

In case of eukaryotes only one release actor eRF causes dissociation.

– Initiation

1. During initiation, a group of proteins called initiation factors assist in assembling the
ribosome around the mRNA.

2. The initiation factors temporarily recognize specific sequences in the mRNA.

3. The small ribosomal subunit then recognizes the initiation factors, followed by the large
ribosomal subunit.

4. The ribosome is assembled around the mRNA, much like a series of toy plastic blocks.

5. Near the beginning of the mRNA is a codon called the start codon (AUG). This codes for an
amino acid called methionine.

6. Three regions are important as the ribosome is assembled around the mRNA. They are
commonly called the A, P, and E sites.

7. Each site will fit a single tRNA.

8. The only tRNA that can effectively enter the site is the one whose anticodon complements
the codon of the mRNA revealed within the site.

9. In initiation, the assembly of the ribosome occurs with the AUG start codon within the P site.
This
ends the initiation stage.

Elongation

1. The elongation stage involves the assembly of specified amino acids into a polypeptide chain.

2. The key to elongation are the E, P, and A sites within the ribosome.

3. Following initiation, the first tRNA (for methionine) is located within the P site.

4. A second codon in the mRNA is exposed in the A site.

5. Only a tRNA with an anticodon complementary to the mRNA codon exposed in the A site will
correctly fit.

6. At this point there are two tRNAs in the ribosome.

7. By an enzymatic reaction, the amino acids between the P and A chains are joined together by
a
peptide bond.

8. As the peptide bond forms, the amino acid is released from the tRNA in the P site. The
ribosome then moves one codon down the mRNA (in the 3′ direction).

9. As it does so, the tRNA that was in the P site enters into the E site and leaves the ribosome.

10. The tRNA that was in the A site, which still has the polypeptide chain attached, moves into
the
P site.

11. A new mRNA codon is then revealed in the A site.

13. The rate at which this reaction occurs is amazing.

14. In eukaryotic systems, the ribosome may read up to six codons per second.

Termination

1. The process of termination begins once the end of the mRNA is reached by the ribosome.

2. In place of tRNAs, proteins called release factors enter into the A site.

3. Since the release factors do not contain amino acids, the process of translation is stopped at
this point.

4. The release factors also promote the disassembly of the ribosome and its interaction with
the mRNA.

5. The end result of translation is a polypeptide chain. This polypeptide chain must undergo a
series of folds in order to produce a functional protein.

RNA SPLICING

RNA splicing is a form of RNA processing in which a newly made precursor messenger RNA (mRNA) is
transformed into a mature RNA by removing the non-coding sequences termed introns.

What are Introns?

Introns are non-coding DNA sequences present within a gene that are removed by the process of
RNA splicing

What are Exons?

Exons are protein-coding DNA sequences that contain the necessary codons or genetic information
essential for protein synthesis.
Spliceosome

A spliceosome is a large and complex molecule formed of RNAs and proteins that regulate the
process of RNA splicing.

RNA Splicing Process

The process of RNA splicing begins with the binding of the spliceosomes to the introns present on
the splice site.

The binding of the spliceosome results in a biochemical process called transesterification between
RNA nucleotides.

During this reaction, the 3’OH group of a specific nucleotide on the intron, which is defined during
spliceosome assembly, causes a nucleophilic attack on the first nucleotide of the intron at the 5’
splice site.

This causes the folding of the 5’ and 3’ ends, resulting in a loop. Meanwhile, the adjacent exons are
also brought together.

Finally, the looped intron is detached from the sequence by the spliceosomes.

Now, a second transesterification reaction occurs during the ligation of adjacent exon segments.

In this case, the 3’OH group of the released 5’ exon then performs an electrophilic attack on the first
nucleotide present just behind the last nucleotide of the intron at the 3’ splice site.

This causes the binding of the two exon segments along with the removal of the intron segment.

Earlier, the intron released during splicing is thought of as a junk unit. Still, it has been recently
observed that these introns are involved in other processes related to proteins after their removal.

Besides the spliceosomes, another group of protein/ enzymes termed ‘ribozymes’ are also involved
in the control and regulation of the splicing process.