this slide provide a basic view on translational mechanisms in prokaryotes

1. Submitted to – Dr. Umesh Gautam
Submitted by- Jaskirat Singh ( 11715744)
Navdeep Singh (11700891)
Thounaojam Ronald (11715437)
Smita Bhattacharjee (11713639)
Section – B1724

2. Translation
 The genetic information contained within the order of
nucleotides in the messenger RNA (mRNA) is used to
generate the linear sequences of amino acids in
proteins.
 These linear chains of amino acids are further
processed and modified.

3.  Translation is highly conserved among all the
organisms and is energetically costly for the cells.
 The machinery responsible for translating the
lanaguage of messenger RNA into the language of
proteins
 It occur in cytoplasm where ribosomes arelocated
 It is a universal process

5. Machinery of Translation
 mRNA
 tRNA
 Aminoacyl tRNA synthetases
 Ribosome
The proteins coding region of mRNA consists of an
ordered series of three nucleotide long units called
Codons.

6. Steps of translation
 Activation of amino acids
 Charging of tRNA (loading of tRNA)
 TRANSLATION
1. Initiation of polypeptide chain
2. Elongation of polypeptide chain
3. Termination of polypeptide chain

7. Activation of amino acids
 20 types of amino acids partcipate in protein synthesis
 Amino acids reacts with ATP to form “amio acyl AMP
enzyme complex” which is also known as “activated
Amino acid”
 This reaction is catalyzed by specific ‘amino acyl
tRNA synthetse’ enzyme

8. Speific amino acyl tRNA synthetse’ enzyme for
each amino acid

9. Charging of t-RNA
 Specific amino acid is recognized by its specific tRNA
 Now amino acid attaches to the ‘amino acid
attachement site’ of its specific t-RNA and AMP and
Enzyme are separated from it.
 Amino acyl t-RNA complex is also called ‘charged t-
RNA’

10. Now amino acyl t-RNA moves to the ribosome for
protein synthesis,

11. Initiation of polypeptide chain
Elongation of polypeptide chain
Termination of polypeptide chain

13. Initiation of polypeptide chain
 In this step 30’s’ and 50’s’ sub units of ribosome, GTP,
Mg2+, charged t-RNA, mRNA and some initiation factors
are required
 In prokaryotes there are three initiation factors IF1, IF2,
IF3
 Initiation factors are specific protiens
 GTP and initiation factors promote the initiation process.

14.  Both subunits of ribosomes are separated with the
help of IF3 factor
 In prokaryotes with the help of “S D sequence mRNA
recognises the smaller subunit of the ribosome. A
sequence of 8 N2 base is present before the 4-12 N2
base of initiation codon og mRNA called SD sequence.
 In smaller subunit of ribosome, a comlementary
sequence of SD sequence is present on 16’s’ rRNA,
which is called as anti shine delgarno sequence.

15.  With the help of SD and ASD sequence of rRNA
mRNA recognises smaller unit of ribosomes
 This 30’s’ mRNA complex reacts with ‘formyl
methionyl tRNA complex’ and “30’s’ mRNA fromyl
metthionyl tRNA complex” is fromed . This tRNA
attaches with codon parrt of mRNA . A GTP molecule
is required.

16.  Now the larger subunit of ribosome (50s sub unit)
joins the complex. The initiation factor released and
complete 70s ribosome is formed.
 In larger sub unit of ribosome there are three sites for
tRNA
 ‘P site’ = peptidtyl site
 ‘A site’ = amino acyl site
 ‘E site’ = Exit site

19. (prokaryotes)

20. Elongation – It’s the addition of amino acids by the
formation of peptide bonds. Elongation is a chain of
amino acid eventually forming a polypeptide bond .
 Steps of elongation – Elongation mainly consists of
two steps :-
1) Binding of new Aminoacyl t-RNA : codon in m-
RNA determines the incoming AA. EF 1 & GTP
required.
2) PEPTIDE BOND FORMATION : alpha- NH2
group of incoming AA in A site forms peptide
bond with COOH group of AA in P site .
Enzyme is Peptidyl Transferase . No need for
energy as AA is activated.

21. Diagram

22. Step 1
 The second charged tRNA binds to the first ribosome at
the latter’s ‘A’ site with the help of the proteins ,called
elongation factors (EF-Tu). EF-Tu carries a molecule of
GTP. Correct hydrogen bonding with the mRNA
template dictates the selection of a new tRNA , and the
activity of ghe EF-Tu ensures the proper positions of the
tRNA in the A site. After performing its function , the
EF-Tu protein dissociates from the ribosome , and in the
cytoplasm is subsequently regenerated to its active form
by another elongation fator, the EF-Ts. At this point ,
both sites of the ribosome are occupied by the tRNA’s
,each of which carries an amino acid ,and each of which
is hydrogen bonded to the template mRNA.

24. Ist step

25. 2nd step
 The next step is the formation of a peptide bond
between the two amino acids. To accomplish this job ,
the first amino acid ( N- formylmethionine) is removed
from its attachment to its tRNA and transferred to the
free-NH2 terminus of the second amino acid . The first
amino acid is thus placed on the top of second amino
acid. The resulting compound is a dipeptide whose
carboxyl end is still bonded to the second tRNA, but
whose amino end ids free. The reaction is catalysed by
an enzyme associated with the 50S subunit and called
peptidyl transferase.
 The energy for peptide bond formation is supplied by
the dissolution of the aminoacyl bond between the first
amino acid and its carrier tRNA,this energy having
originally been donated by ATP .

27. 3rd step (translocation)
 Although a dipeptide has been generated ,continued
synthesis requires that the next codon be made available and
that the next tRNA be admitted to the A site on the ribosome :
this site being still occupied by dipeptide-carrying tRNA . The
problem is solved by a movement of the entire ribosome
relative to the mRNA strand. This is called translocation and
consists of following three steps-
 Ejection of discharged tRNAfmet from the P site .
 Movement or physical shifting of tRNA dipeptide from the ‘A’
site to the P site.
 Movement of the mRNA is such that the effect is the apparent
movement of the ribosome in 5’→3’ direction by the length of
one codon (3 nucleotide) . This step requires the presence of
an elongation factor EF-G called translocase and GTP.

29. Contd.
 The sequential formation of a polypeptide continues
in the manner described above. A tRNA in the p site
shifts its burden of growing polypeptide to the next
succeeding tRNA,followed by translocation ,exit of the
discharged tRNA ,and entrance of a new charged tRNA
to base pair with anew codon at A aite . Thus, the
growing polypeptide is adopted in turn by each tRNA
,with each successive amino acid being added in effect
, to the bottom of stack . As process continues , the
mRNA ia progressively translated from 5’→3’ end.

32. 1. This is the last phase of translation.
2. Termination occurs when one of the three termination
codons moves into the A site.
3. These codons are not recognized by any tRNAs. Instead,
they are recognized by proteins called release factors,
namely RF1 (recognizing the UAA and UAG stop codons)
or RF2 (recognizing the UAA and UGA stop codons).
4. These factors trigger the hydrolysis of the ester bond in
peptidyl-tRNA and the release of the newly synthesized
protein from the ribosome.
5. A third release factor RF-3 catalyzes the release of RF-1
and RF-2 at the end of the termination process.