TRANSLATION

M.Prasad NaiduMSc 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) SUBUNITSMALLER 40S (30S) SUBUNIT

Requirements for TRANSLATION: ribosomesm-RNAt-RNA A.AsAMINOACYL t-RNA SYNTHETASESATP, GTPENZYMES OF TRANSLATIONPROTEIN FACTORS

TRANSLATION: Steps 1. ACTIVATION OF A.A2.INITIATION 3.ELONGATION 4.TERMINATION5.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 dissociation2. Formation of 43S pre-initiation complex3. Formation of 48S initiation complex4. 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-IThis 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, ATPKOZAK CONSENSUS (EUKARYOTES) on mRNA

facilitates binding of mRNA to pre-initiation complexSHINE 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 codonCap

eIF4eIF

eIF

eIF

P & A SITESRIBOSOME CONTAINS 2 RECEPTOR SITES FOR t-RNA MOLECULESP (PEPTIDYL) SITEA (AMINOACYL) SITEMet-t-RNA OCCUPIES P SITE.ALL OTHER t-RNAs OCCUPY A SITEt-RNA BINDS WITH RIBOSOME THROUGH THE PSEUDOURIDINE ARMANTICODON 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 TRANSFERASENO 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 CODONPEPTIDYL 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 INELONGATION CONTINUES….

APPP

P P P

AA

A A A

ENERGY REQUIREMENTS:

ACTIVATION OF A.A 2 ATPFOR PRE-INITIATION COMPLEX 1 GTPFOR INITIATION COMPLEX 1 ATPBINDING OF AMINOACYL t-RNA 1 GTPTANSLOCATION PROCESS 1 GTP

TERMINATION;SUCCESSIVE ADDITIONS OF A.ATERMINATION CODONS REACHED.NO t-RNA CORRESPONDING TO TERMINATION CODONA SITE REMAINS FREERELEASING FACTOR ENTERS A SITERELEASES THE PEPTIDE CHAIN WITH HYDROLYSIS OF GTP. RIBOSOME DISSOCIATES INTO 60 AND 40S SUBUNITS.

P P PA A A

POLYRIBOSOME (POLYSOME)

PROTEIN TARGETTING:PROTEINS FOR EXTERNAL SECRETION:SYNTHESISED ON RERSIGNAL PEPTIDE (LEADER sequence)THRO ER, GOLGIREACHES CORRECT DESTINATION.

INTERNAL PROTEINS:LACK SIGNAL PEPTIDEFREE RIBOSOMESDO NOT ENTER ER. ARE AVAILLABLE IN CYTOPLASM.

THANK YOU