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TRANSLATION

M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.


THE LANGUAGE OF NUCLEOTIDE SEQUENCE ON m-
RNA ( GENTIC CODE ) IS TRANSLATED IN TO THE
LANGUAGE OF AMINIO ACID SEQUENCE.




t-RNA ( soluble RNA)
Transfer A.A from cytosol to the
ribosomal protein synthesising
machinery.
73 93 nucleotides in length
RIBOSOMES

INFRASTRUCTURE FOR THE m-RNA, t-RNA &
A.A TO INTERACT WITH EACH OTHER FOR
TRANSLATION.

80S IN EUKARYOTES & 60S IN PROKARYOTES.
LARGER 60S (50S) SUBUNIT
SMALLER 40S (30S) SUBUNIT
Requirements for TRANSLATION:
ribosomes
m-RNA
t-RNA
A.As
AMINOACYL t-RNA SYNTHETASES
ATP, GTP
ENZYMES OF TRANSLATION
PROTEIN FACTORS

TRANSLATION: Steps
1. ACTIVATION OF A.A
2.INITIATION
3.ELONGATION
4.TERMINATION
5.POST TRANSLATIONAL PROCESSING
ACTIVATION OF A.A

AMINOACYL t-RNA SYNTHETASE

A.A + t-RNA + ATP Aminoacyl t-RNA + AMP


ACTIVATION OF A.A
AMINOACYL t-RNA SYNTHETASE
D ARM OF tRNA RECOGNISED BY THE ENZYME
A.A + t-RNA + ATP Aminoacyl t-RNA + AMP
ENZYME IDENTIFIES THE SPECIFIC t-RNA
TRANSFERS AMINOACYL GROUP TO 3 END CCA
-COOH GROUP OF A.A ESTERIFIED WITH THE 3 OH
GROUP OF t-RNA.
AT LEAST 1 ENZYME FOR 1 A.A
mRNA is read in 5 to 3 direction

PROTEIN SYNTHESIS TAKES PLACE FROM N-
TERMINAL TO C-TERMENAL END
INITIATION:

Basic requirements for initiation

Ribosome
mRNA to be translated
The initiating Met-tRNA
A set of protein factors called initiation factors.
In Eukaryotes there are at least nine factors, designated
e If to indicate eukaryotic origin.
In prokaryotes, three initiation factors are known IF1, IF2
and IF3

Steps involved in Initiation:

1. Ribosomal dissociation
2. Formation of 43S pre-initiation complex
3. Formation of 48S initiation complex
4. Formation of 80S initiation complex
Ribosomal dissociation:

eIF3 and eIF1 A binds to 40S subunit and
prevents the reassociation with 60S causing
ribosomal subunit dissociation.
2. Formation of 43S pre-initiation complex

GTP + eIF2
This complex binds to met-tRNA-I
This GTP-eIF2-tRNA-I complex binds to the 40S
ribosomal subunit to form 43S pre-initiation
complex.


3. Formation of 48S initiation complex

Binding of m-RNA to 43S pre-initiation complex forms 48S
initiation complex.

5 end of m-RNA contains CAP which helps in binding of m-RNA
to 43S pre-initiation complex.

The association of mRNA with 43S initiation complex requires:
CAP binding protein, eIF4, ATP
KOZAK CONSENSUS (EUKARYOTES) on mRNA facilitates
binding of mRNA to pre-initiation complex
SHINE DALGARNO SEQUENCE on mRNA facilitates binding of
mRNA to pre-initiation complex




4. FORMATION OF 80S RIBOSOME ASSEMBLY

48S INITIATION COMPLEX + 60S RIBOSOME.
+ e-IF2 + e-IF5.

e-IF 5 HAS GTPase ACTIVITY.
GTP BOUND IN STEP 1 HYDROLYSED FOR
ENERGY.

ALL INITIATION FACTORS NOW RELEASED.

NEXT STAGE IS
ELONGATION.
80S ribosome
Ribosomal dissociation
40S subunit
Ternary complex
60S
43S Preinitiation complex
48S Inititation complex
80S Inititation complex
Initiating codon
Cap
eIF4
eIF
eIF
eIF
P & A SITES
RIBOSOME CONTAINS 2 RECEPTOR SITES FOR t-
RNA MOLECULES
P (PEPTIDYL) SITE
A (AMINOACYL) SITE
Met-t-RNA OCCUPIES P SITE.
ALL OTHER t-RNAs OCCUPY A SITE
t-RNA BINDS WITH RIBOSOME THROUGH THE
PSEUDOURIDINE ARM
ANTICODON OF t-RNA & CODON ON m-RNA BIND
A SITE IS FREE NOW.
ELONGATION:
BINDING OF NEW AMINOACYL t-RNA
CODON IN m-RNA DETERMINES THE INCOMING A.A
EF 1 & GTP REQD.


PEPTIDE BOND FORMATION:
-NH2 GROUP OF INCOMING A.A IN A SITE FORMS
PEPTIDE BOND WITH COOH GROUP OF A.A IN P SITE.
ENZYME IS PEPTIDYL TRANSFERASE
NO NEED FOR ENERGY AS A.A IS ACTIVATED.
TRANSLOCATION:
WHEN PEPTIDE BOND FORMED Met
FROM P SITE SHIFTED TO A SITE.
t RNA IS RELEASED FROM P SITE, NOW P SITE
FREE
RIBOSOME MOVES FORWARD BY 1 CODON
PEPTIDYL t-RNA TRANSLOCATED TO P
SITE.NOW A SITE EMPTY

NEW AMINOACYL t-RNA WILL COME ONLY TO A
SITE.
STEP REQUIRES EF 2 AND ENERGY FROM GTP.
NEW AMINOACYL t-RNA CAN COME IN
ELONGATION CONTINUES.

A P P
P
P P
P
A A
A A
A
ENERGY REQUIREMENTS:

ACTIVATION OF A.A 2 ATP
FOR PRE-INITIATION COMPLEX 1 GTP
FOR INITIATION COMPLEX 1 ATP
BINDING OF AMINOACYL t-RNA 1 GTP
TANSLOCATION PROCESS 1 GTP



TERMINATION;
SUCCESSIVE ADDITIONS OF A.A
TERMINATION CODONS REACHED.
NO t-RNA CORRESPONDING TO
TERMINATION CODON
A SITE REMAINS FREE
RELEASING FACTOR ENTERS A SITE
RELEASES THE PEPTIDE CHAIN WITH
HYDROLYSIS OF GTP.
RIBOSOME DISSOCIATES INTO 60 AND 40S
SUBUNITS.
P P P A A
A


POLYRIBOSOME (POLYSOME)


PROTEIN TARGETTING:
PROTEINS FOR EXTERNAL SECRETION:
SYNTHESISED ON RER
SIGNAL PEPTIDE (LEADER sequence)
THRO ER, GOLGI
REACHES CORRECT DESTINATION.

INTERNAL PROTEINS:
LACK SIGNAL PEPTIDE
FREE RIBOSOMES
DO NOT ENTER ER. ARE AVAILLABLE IN
CYTOPLASM.
THANK YOU