TRANSLATION

The flow of genetic information

Gene: The region of DNA that

controls a discrete hereditary

characteristic of an organism,

usually corresponding to a single

protein or RNA.

Prokaryotes and eukaryotes handle their transcripts

somewhat differently

Several types of RNA are produced in cells

Type of RNA Function

Messenger RNA

(mRNA)

Code for proteins

Ribosomal RNA

(rRNA)

Form part of the structure of the ribosome and participate

in protein synthesis

Transfer RNA

(tRNA)

Used in protein synthesis as adaptors between mRNA

and amino acids

Small RNA (snRNA) Used in pre-mRNA splicing and other cellular processes

Small nucleolar RNA

(snoRNA)

Used to process and chemically modify rRNAs

MicroRNA (miRNA) Regulate gene expression typically by blocking

translation of selective mRNAs

Small interfering

RNA (siRNA)

Turn off gene expression by directing degradation of

selective mRNAs and the establishment of compact

chromatin structures

Messenger RNA (mRNA)

The universal genetic code

Deviations from the universal genetic code

Open Reading Frame (ORF)

- The protein-coding region of each mRNA is composed of a contiguous, non-

overlapping string of codons called an open reading frame (ORF).

-The first and last codons of an ORF are known as the start and stop codons.

- In bacteria, the start codon is usually 5-AUG-3 (Met), but 5-GUG-3 and

sometimes even 5-UUG-3 are also used.

- Eukaryotic cells always use 5-AUG-3 as start codon.

- Start codon has two important functions: 1) it specifies the first amino acid

(Met) to be incorporated into the growing polypeptide chain; 2) it defines the

reading frame for all subsequent codons.

- Stop codons, of which there are three (UAA, UAG, UGA), defines the end of

ORF and signal termination of polypeptide synthesis.

A mRNA molecule can be translated in three

possible reading frame

Transfer RNA (tRNA)

- tRNA molecules are molecular adaptors, linking amino acids with codons.

- tRNAs share a common secondary structure that resembles a cloverleaf

- tRNAs have an L-shape three-dimensional structure.

A subset of modified nucleosides found in tRNA

Codon-anticodon pairing involves wobbling at

the third position

Attachment of amino acids to tRNA

- tRNA molecules to which an amino acid is attached are said to be charged,

and tRNAs that lack an amino acid are said to be uncharged.

- Two steps of aminoacyl-tRNA charging: 1) Adenylation of amino acid; 2)

tRNA charging in which the adenylated amino acid react with tRNA.

Acyl linkage between the carboxyl group of the

amino acid and 3-hydroxyl group of the adenosine

nucleotide that protrudes from the acceptor stem

This acyl linkage is cinsidered to be high-energy bond

Specific enzymes couple tRNAs to the correct

amino acid

- Recognition and attachment of the correct amino acid depends on enzymes

called aminoacyl-tRNA synthetase, which covalently couple each amino acid

to its appropriate set of tRNA molecules.

- There is a different synthetase enzyme for each amino acid (that is, there are

20 synthetase in all)

tRNA synthetase recognize unique structural

feature of cognate tRNAs

- The acceptor stem and the anticodon loop are the specificity determinants for

tRNA synthetase recognition.

- In some cases changing a single base pair in the acceptor stem (discriminator

base) is sufficient to convert the recognition specificity of a tRNA from one

synthetase to another.

Some aminoacyl tRNA synthetase use an editing

pocket to charge tRNAs with high accuracy

The RNA message is decoded on ribosomes

- The ribosome is macromolecular machine that directs the synthesis of

proteins.

- In prokaryotes, the transcription machinery and the translation machinery are

located in the same compartment.

- In eukaryotes, the translation is completely separate from transcription:

transcription occurs in the nucleus, whereas translation occurs in the cytoplasm.

The ribosome is composed of a large and small subunit

- The ribosome is composed of two subassemblies of RNA and protein known as the large

and small subunits. The large subunit contains the peptidyl transferase center, which

responsible for the formation of peptide bonds. The small subunit contains the decoding

center in which charged tRNA read or decode the codon units of the mRNA.

- The large and small subunits are named according to the velocity of their sedimentation

when subjected to a centrifugal force. The unit used to measure sedimentation velocity is the

Svedberg (S).

- The prokaryotic ribosome composed of 50S and 30S subunits, which together form an 70S

ribosome. The eukaryotic ribosome composed of 60S and 40S subunits, which together form

an 80S ribosome.

Composition of prokaryotic ribosome

Composition of eukaryotic ribosome

The large and small subunits undergo association and

dissociation during each cycle of translation

- Translation begins with the binding of

the mRNA and an initiating tRNA to small

subunit of ribosome

- The small subunit-mRNA complex then

recruits a large subunit to create an intact

ribosome with the mRNA sandwiched

between two subunits.

- As the ribosome translocates from codon

to codon, one charged tRNA after another

is slotted into the decoding and peptidyl

transferase centers of the ribosome

- When the ribosome encounters a stop

codon, the completed peptide chain is

released, and the ribosome disassociates

from the mRNA as separate large and

small subunits.

- An mRNA can be translated

simultaneously by multiple ribosomes

called polyribosome or polysome.

The peptidyl transferase reaction

-The ribosome catalyzes a single chemical reaction: the formation of peptide

bond. This reaction occurs between the amino acid residue at the carboxyl-

terminal end of the growing polypeptide and the incoming amino acid to be

added to the chain.

- Both the growing chain and the incoming amino acid are attached to tRNAs:

the peptidyl-tRNA and the aminoacyl-tRNA.

Each ribosome has binding site for mRNA and

three binding sites for tRNA

(A) 3D-structure of bacterial ribosome

with small subunit in the front (dark

green) and the large subunit in the back

(light green). tRNAs are shown bound in

the E-site (red), the P-site (orange) and A-

site (yellow).

Large subunit Small subunit

Each ribosome has binding site for mRNA and

three binding sites for tRNA

The ribosome has three binding sites: the A-site is the binding site for

Aminoacylated-tRNA, the P-site is the binding site for the Peptidyl-tRNA, and

the E-site is the binding site for the tRNA that is released after the growing

peptide chain has been transferred to the aminoacyl-tRNA (E for exit)

Initiation of translation

Translation initiation requires:

- Ribosome brought to mRNA

- Ribosome properly aligned over start codon

- P site of ribosome containing the charged tRNA

In prokaryotes, the initiator

tRNA, which base-pairs with the

start codon AUG, is charged with

a modified form of methionin (N-

formyl methionine). The charged

initiator tRNA is referred to as

fMet-tRNAifMet

Translation Initiation in Prokaryotes (1)

-Prokaryotes mRNAs are initially recruited to small subunit by base-pairing to

16S rRNA

- Many prokaryotic ORFs contain a short sequence upstream (on the 5 side) of

the start codon called the ribosome binding site (RBS). This element is also

referred to as a Shine-Dalgarno sequence.

Translation Initiation in Prokaryotes (2)

- In prokaryotes, three initiation factors

direct the assembly of an initiation complex:

* IF1: prevents tRNAs from entering A site

* IF2: is a GTPase (a protein that binds and

hydrolyzes GTP). IF2 binds IF 1 and guides

the initiator tRNA (fMet-tRNAifMet) to P site

* IF3: prevents association of large subunit

- With all three IFs bound, the small subunit

is prepared to bind the mRNA and the

initiator tRNA.

-When start codon and fMet-tRNA base-pair,

the small subunit undergo a change in

conformation. This alter conformation

results in the release of IF3.

- In the absence of IF3, the large subunit can

bind to the small subunit complex to create

the 70S initiation complex.

Initiation Factors

Translation Initiation in Eukaryotes (1)

- In eukaryotes, the initiator tRNA is charged

with methionine (Met-tRNAiMet)

- Two GTP-binding proteins (eIF2 and eIF5B)

mediate the recruitment of the charged tRNA

- The small subunit is already associated with

the initiator tRNA when it is recruited to the

capped 5 end of the mRNA

- Together two GTP-binding proteins position

the Met-tRNA in the future P-site of small

subunit, resulting in the formation of the 43S

pre-initiation complex.

- The 43S pre-initiation complex recognize the

5 cap of mRNA. The recognition is mediated

by eIF-4E/ G

- Once assemble at the 5 end of the mRNA,

the small subunit and its associated factors

move along the mRNA in a 5-3 direction to

scan for the first start codon AUG

Translation Initiation in Eukaryotes (2)

- Correct base-pairing between initiator

tRNA and start codon triggers the release

of eIF2 and eIF3.

- Lost of eIF3 and eIF2 allow the large

subunit to bind to the small subunit

complex to create the 80S initiation

complex.

- With the start codon and Met-tRNA

placed in the P-site, the eukaryotic

ribosome is now poised to accept a

charged tRNA into its A-site and carry

out the formation of the first peptide bond.

Translation Elongation

- Step 1: an aminoacyl-tRNA binds to vacant A-site

on the ribosome

- Step 2: a new peptide bond is formed

- Step 3: the mRNA moves a distance of three

nucleotides (a codon) through the small subunit,

ejecting the spent tRNA molecule and resetting

the ribosome so that the next aminoacyl-tRNA

molecule can bind.

Translation elongation in prokaryotes

Translation elongation in eukaryotes

Prokaryotic

elongation factors

Eukaryotic

elongation factors

Function

EF-Tu eEF1 Escort aminoacyl-tRNA

to the A-site of ribosome

EF-G eEF2 Drive translocation of the

tRNA and the mRNA

Termination of translation

- The end of the protein-coding message is

signaled by the presence of one of the several

stop codon (UAA, UAG or UGA)

- The stop codon is recognized by proteins

called release factors.

- Release factors bind to any stop codon that

reaches to the A-site on the ribosome, and this

binding alters the activity of the peptidyl

transferase in the ribosome, causing it to

catalyze the additional water molecule instead

of an amino acid to the peptidyl-tRNA.

- This reaction frees the carboxyl end of the

growing polypeptide chain from its attachment

to a tRNA molecule.

- The ribosome release the mRNA and

disassociate into its two separate subunits

Release factors are an example of molecular mimicry:

the three-dimensional structure of release factors resembles the

shape and charge distribution of a tRNA molecule

Human eRF1 tRNA

Classification of release factors

Release factors (RFs)

class 1 RF class 2 RF

UAG

UAA

UGA

Eukaryotes

eRF1

Prokaryotes Eukaryotes

RF3 eRF3

Prokaryotes

RF1

RF2

Class 1 RF is responsible for stop codon

recognition and hydrolysis of the peptidyl-

tRNA linkage.

Class 2 RF is a GDP/ GTP-binding

protein, which stimulate class 1 RF

activity.

GDP GDP

Class 1 and 2 RFs form functional

complex in translation termination

Translation initiation factors hold eukaryotic

mRNA in circle

Circular polyribosome in eukaryotic cell

Proteins Fold into a Conformation of Lowest E nergy

Steps in the creation of a functional protein

Translation of an mRNA sequence

into an amino acid sequence on the

ribosome is not the end of the

process of forming a protein. To

function, the completed polypeptide

chain must fold correctly into its

three-dimensional conformation,

bind any cofactors required, and

assemble with its partner protein

chains (if any).

Some Proteins Begin to Fold While Still Being Synthesized

Molecular Chaperones Help Guide the Folding of Most Proteins

Chaperone (molecular chaperone):

Protein that helps guide the proper

folding of other proteins, or helps

them avoid misfolding. Includes

Heat shock proteins (Hsp).

The Hsp70 family of molecular chaperones

The Hsp70 machinery acts early in the life of many proteins, binding to a

string of about seven hydrophobic amino acids before the protein leaves the

ribosome

The structure and function of the Hsp60 family of

molecular chaperones

Hsp60-like proteins form a large barrel shaped

structure that acts after a protein has been fully

synthesized.

This type of chaperone, sometimes called a

chaperonin, forms an isolation chamber into

which misfolded proteins are fed, preventing their

aggregation and providing them with a favorable

environment in which to attempt to refold

Release factors

RF1, RF2, RF3

Release factors

eRF1, eRF3

Comparison of protein synthesis