Molecular biology translation mb11

Translation : The Process

Molecular Biology

Dr. Aga Syed SameerCSIR Lecturer Department of Biochemistry,Medical College,Sher-I-Kashmir Institute of Medical Sciences, Bemina, Srinagar, Kashmir, 190018. India.

mRNA is read in the 53→׳ direction ׳

Ribosomes began translating the 5׳- end of the mRNA before the transcription is complete (in prokaryotes)

Thus in other words protein synthesis begins at the amino-terminal

The AUG initiator codon specifies the amino-terminal methionine residue

In both pro & eukaryotes there are two different tRNAs that code for the AUG codon

Translation The First Codon

One is used when AUG is a initiator codon where it incorporates N-formyl methionine (in prokaryotes) and methionine (in eukaryotes)

Other is used to incorporate methionine in response to the AUG located in the internal position in mRNA

They are respectively designated as tRNAfMet & tRNAMet

Both the tRNAs are charged by same enzyme Met-tRNA synthetase

But methionine bound to tRNAfMet is then modified by addition of formyl group by transformylase to give N-formylmethionyl-tRNAfMet

Translation The First Codon

A reading frame without a termination codon among 50 or more codons is referred to as an open reading frame (ORF)

In a random sequence of nucleotides, 1 in every 20 codons in each reading frame is, on average, a termination codon

Translation ORFs

Initiation

Elongation

Termination

Translation

The initiation of polypeptide synthesis in bacteria requires

(1) the 30S ribosomal subunit,

(2) the mRNA coding for the polypeptide to be made,

(3) the initiating fMet-tRNAfMet,

(4) a set of three proteins called initiation factors (IF-1, IF-2, and IF-3),

(5) GTP,

(6) the 50S ribosomal subunit, and

(7) Mg2+.

TranslationInitiation

The 30S ribosomal subunit binds two initiation factors, IF-1and IF-3.

IF-1 prevents premature binding of tRNA to A site.

IF-3 prevents the 30S and 50S subunits from combining prematurely.

The mRNA then binds to the 30S subunit.


Step 1

GTP bound IF-2 and the initiating fMet-tRNAfMet

bind to mRNA.

The initiator tRNAhowever unlike other tRNAs to follow, binds to P site and forms correct base pairing with the mRNA‘s initiating codon.

After this IF-3 is released


Step 2

At the end 50S subunit binds with the simultaneous hydrolysis of GTP bound to IF-2 to GDP and their consequent release from the complex.

All the three initiating factors depart from the ribosome at this point thus completing the initiation phase of translation.

Thus this phase utilizes one GTP molecule.


Step 3

Thus; a functional 70S ribosome called the initiation complex, containing the mRNA and the initiating fMet-tRNAfMet is assembled at the end of initiation process.

The correct binding of the fMet-tRNAfMet to the P site in the complete 70S initiation complex is assured by at least three points of recognition and attachment:

Interaction between the Shine-Dalgarno Sequence in the mRNA and the 16S rRNA of ribosome;

The codon-anticodon interaction involving the initiation AUG fixed in the P site; and

Binding interactions between the ribosomal P site and the fMet-tRNAfMet


Prior to formation of initiation complex there is circularization of the mRNA to be translated which is facilitated by poly (A) binding protein (PAB) and eIF4A/4G/4E.

PAB binds to 3‘ end and eIF4E to 5‘ end of mRNA .

Both are bridged by eIF4G and whole complex in turn is bounded by eIF3 located in the 40S subunit.


The elongation phase, is essentially identical in bacteria and eukaryotes,

It occurs in three stages:

Recruitment,

Transpeptidation,

Translocation.

TranslationElongation

The appropriate incoming aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu.

The resulting aminoacyltRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex.

The GTP is hydrolyzed and an EF-Tu–GDP complex is released from the 70S ribosome.

The EF-Tu–GTP complex is regenerated in a process involving EF-Ts and GTP.


Recruitment

The GTP associated with EF-Tu (or eEF1α) is hydrolyzed and the elongation factor is ejected.

EF-Tu/GDP is inactive, and the nucleotide is displaced by a second EF-Ts (eEF1βγ in eukaryotes), which is itself displaced by GTP.

EF-Tu can now be reused.


Recruitment

The peptide bond joining the initiator tRNA in the P-site to methionine is then broken and a new peptide bond is formed between methionine and the amino acid occupying the A-site.

The initiator tRNA is discharged.

This transpeptidationreaction is mediated by the peptidyl-transferaseactivity of large ribosomal subunit.


Transpeptidation

The next stage is translocation.

The initiator tRNA occupying the P-site is ejected (in bacteria it is moved to a further ribosomal domain termed the E site, whereas in eukaryotes the tRNAdissociates from the ribosome).

The, methionyl residue is thus tethered to the amino acid occupying the A site by a peptide bond, and the tRNA in the A site is now referred to as the peptidyl-tRNA.


Translocation

The ribosome then translocates three bases along the mRNA while the peptidyl-tRNA remains associated with its codon.

The peptidyl-tRNA is thus transferred to the P-site and the mRNA moves through the ribosome so that the next codon is aligned with the A site.

Translocation requires a second elongation factor (EF-G in bacteria, eEF-2 in eukaryotes, also associated with GTP).


Translocation

The elongation cycle in eukaryotes is quite similar to that

in prokaryotes

Three eukaryotic elongation factors (eEF1α, eEF1β, and

eEF2) have functions analogous to those of the bacterial

elongation factors (EF-Tu, EF-Ts, and EF-G, respectively)

Eukaryotic ribosomes do not have an E site; uncharged

tRNAs are expelled directly from the P site


As there are no tRNA that will recognize the termination codon, but the termination of protein synthesis takes place with the help of termination factors called as release factors.

Prokaryotes contain three of them.

RF1 recognises the codon UAA & UAG, and RF2 recognizes UAA & UGA.

RF3 assists the two in their function.

In eukaryotes GTP dependent factor (eRF) recognizes all the three termination codons.

TranslationTermination

RFs enter the A site of the ribosomes and catalyze the transfer of the polypeptide to the water molecule hence releasing it from the ribosome.

For the release of the uncharged tRNA from the P site of the ribosome EF-G together with the ribosome release factor work together and cause the final dissociation of the complex.

At the end IF-3 binds to the small subunit to prevent the formation of 70S ribosome.

TranslationTermination

Translation – Quick Steps

Nuclearmembrane

Transcription

RNA Processing

Translation

DNA

Pre-mRNA

mRNA

Ribosome

Protein

Eukaryotic Cell

Translation - Eukaryotes

PSite

ASite

Largesubunit

Small subunit

mRNA

A U G C U A C U U C G

Ribosomes

Initiation

mRNA


2-tRNA

G

aa2

A U

A

1-tRNA

U A C

aa1

anticodon

hydrogenbonds codon

mRNA


1-tRNA 2-tRNA

U A C G

aa1 aa2

A U

A

anticodon

hydrogenbonds codon

peptide bond

3-tRNA

G A A

aa3

Elongation

mRNA


1-tRNA

2-tRNA

U A C

G

aa1

aa2

A U

A

peptide bond

3-tRNA

G A A

aa3

Ribosomes move over one codon

(leaves)

mRNA


2-tRNA

G

aa1

aa2

A U

A

peptide bonds

3-tRNA

G A A

aa3

4-tRNA

G C U

aa4

A C U

mRNA


2-tRNA

G

aa1

aa2

A U

A

peptide bonds

3-tRNA

G A A

aa3

4-tRNA

G C U

aa4

A C U

(leaves)


mRNA

G C U A C U U C G

aa1

aa2

A

peptide bonds

3-tRNA

G A A

aa3

4-tRNA

G C U

aa4

A C U

U G A

5-tRNA

aa5

mRNA

G C U A C U U C G

aa1

aa2

A

peptide bonds

3-tRNA

G A A

aa3

4-tRNA

G C U

aa4

A C U

U G A

5-tRNA

aa5


mRNA

A C A U G U

aa1

aa2

U

primarystructureof a protein

aa3

200-tRNA

aa4

U A G

aa5

C U

aa200

aa199

terminatoror stopcodon

Termination

End Product

The end products of protein synthesis is a primary structure of a protein.

A sequence of amino acid bonded together by peptide bonds.

aa1

aa2aa3

aa4

aa5

aa200

aa199

Question:

The anticodon UAC belongs to a tRNA that recognizes and binds to a particular amino acid.

What would be the DNA base code for this amino acid?

Answer:

tRNA - UAC (anticodon)

mRNA - AUG (codon)

DNA - TAC

Examination

NEXT THURSDAY::

MOLECULAR BIOLOGY (MB01-MB06)

Genome and Genomics

Replication